專利名稱：活疫苗及生產方法
技術領域：
本發明屬于病毒學和疫苗發展的領域，涉及生產病毒疫苗的改良方法以及用該方法可獲得的疫苗，所述病毒疫苗尤其是完整病毒疫苗，最好是減毒活疫苗。
背景技術：
流感血凝素(HA)抗原是宿主對病毒的保護性免疫反應的主要靶。
回收新病毒分離物的一般實踐涉及從鼻拭子和喉拭子或從類似來源回收，然后在含胚的雞蛋中培養所述分離物。病毒適應其雞蛋宿主，因此可以在雞蛋中進行所述病毒的大規模生產。然而，所述涉及用含胚雞蛋生產流感疫苗的常規方法異常麻煩，涉及每周處理成千上萬個雞蛋，以及大規模純化從尿囊液獲得的病毒懸浮液以保證不含雞蛋蛋白。
使用雞胚生產病毒的另一個缺點是該支持物非常傾向于選擇在抗原特異性上與野生型病毒不同的病毒變異體，并且有時導致病毒由于表型改變而不適用于生產疫苗，所述表型改變包括，例如，免疫原性顯著降低。
因此，本領域內已經進行許多努力，利用標準組織培養技術，用已經建立的哺乳動物細胞系如MDCK(Madin-Darby犬腎)細胞或Vero(非洲綠猴腎)細胞進行病毒生產，尤其是流感病毒的生產。
在組織細胞培養物中培養流感株的困難之一是需要對宿主中的流感血凝素進行蛋白水解切割。切割病毒HA前體成為HA1和HA2亞片段雖然對于裝配病毒元件形成完整病毒粒不是必要的，但卻是賦予病毒粒感染性(即使其能夠感染新的細胞)所必需的。
已經報道(如Lazarowitz等，“通過蛋白水解切割血凝素多肽增強B型流感病毒的感染性”，Virology，68440-454，1975)通過在組織培養物的培養基中加入蛋白酶如胰蛋白酶，可以克服幾個A型流感病毒株在標準細胞培養物中的有限復制。然而，在某些情況下，例如在使用Vero細胞的情況下，這仍然是有困難的。
Kaverian和Webster(J Virol 69/42700-2703，1995)報道在Vero細胞培養物中，培養基中的胰蛋白酶活性從溫育開始就快速降低，導致由于無法產生足夠數量的感染性病毒粒而不能在培養基中積累病毒，但這種現象在MDCK、豬腎或恒河猴腎細胞培養物中不是那么明顯。他們得出結論從Vero細胞中釋放出一種胰蛋白酶抑制因子。他們進一步顯示通過重復加入胰蛋白酶，可以恢復病毒復制，并使病毒復制維持多個循環，導致更好的病毒產量。
US5,753,489報道另一種有效生產疫苗的方法，其中使用無血清培養基在多種不同哺乳動物細胞系(包括MDCK細胞和Vero細胞)中增殖病毒。該文獻公開的方法包括在無血清培養基中培養脊椎動物細胞、用病毒感染所述細胞培養物、溫育病毒感染的所述細胞培養物、取出包含病毒的一部分培養基并用蛋白酶接觸該部分、然后在該部分中加入蛋白酶抑制劑并將該部分返回所述細胞培養物。在該文獻中，優選在第一個容器中提供培養、感染和溫育的步驟，在第二個容器中進行接觸胰蛋白酶和加入抑制劑的步驟，所述第二個容器與第一個容器形成循環，以便所述步驟能夠在閉合循環內進行。該系統允許在培養物中以比細胞所能夠通常承受的濃度更高的濃度使用胰蛋白酶或其它蛋白水解酶。
EP 0870508報道一種生產病毒抗原疫苗的方法，該方法包括用病毒感染一種動物細胞系(可選地是Vero細胞系)，在所述細胞培養物中繁殖病毒，在病毒增殖結束前不久在所述細胞培養物中加入核酸酶消化從裂解的宿主細胞釋放到培養基中的核酸物質，然后收獲所述病毒，通過提取獲取病毒抗原，以制造病毒抗原疫苗。該方法沒有提到用于增殖病毒的營養培養基種類以及蛋白酶的加入，而加入蛋白酶是最后加工流感病毒血凝素獲得感染性病毒所需的。該方法還要求各種純化步驟以提供現成的疫苗制備物。
然而，已知宿主支持物的性質和用于增殖病毒的營養培養基的組成可以顯著影響由此獲得的病毒后代的免疫原性和抗原性。尤其是含血清的培養基不僅降低病毒后代的抗原性，而且還降低培養基中的蛋白酶活性，由此抑制病毒成熟，并隨后要求昂貴的純化步驟。
發明簡述本發明克服在先技術的缺點。本發明涉及一種簡單有效的方法，該方法用于從各種來源分離病毒，并在最小化或完全防止由于適應性選擇而引起的所述病毒表面抗原改變的條件下，生產用作疫苗(尤其是活減毒流感疫苗)的病毒后代。
本發明的另一個目標是提供生產病毒(尤其是流感病毒)的方法，所述方法產生選擇性凝集人紅細胞而不凝集雞紅細胞的病毒后代，并且所述病毒后代的抗原特性最好與最初接種的病毒株(如初級臨床野生型分離物)的抗原特性相同。
在一個優選實施方案中，所繁殖病毒的HA基因以及可選NA基因的核酸序列與最初接種的株(如流行株、感染患者的初級臨床分離物)的所述核酸序列相同。
本發明的再一個目標是提供在單一步驟方法中有效生產完整病毒疫苗(尤其是活減毒疫苗)的方法，所述方法在通過離心從細胞培養物上清液收獲病毒懸浮液后不需要任何層析或其它純化步驟，尤其不需要蛋白分離或純化步驟。
本發明的又一個目標是提供減毒、冷適應和溫度敏感的A型和B型流感病毒株以及用它們制備的疫苗。
附圖簡述

圖1是一個示意圖，說明在Vero細胞培養物上清液中胰蛋白酶失活隨時間變化的過程。
圖2是一個示意圖，說明在MDCK細胞培養物上清液中胰蛋白酶失活隨時間變化的過程。
發明詳述使用含胚卵、MDCK和Vero細胞的比較試驗清楚地證明最初接種的病毒有可能在這些支持物中任何一種上生長時發生抗原性改變。
我們的試驗證實在無血清培養基內Vero細胞上培養的流感病毒株發生最少改變或不發生改變。此外，證實當A型流感病毒(至少H3N2亞型株)在無血清和無蛋白培養基內的Vero細胞上生長時表現選擇性凝集人紅細胞，但不凝集雞紅細胞。此外，它們不能在蛋內生長。首次表明與在MDCK細胞或蛋上培養的病毒相比，這些Vero培養的病毒可能與對應臨床分離物的野生型病毒更相似。
事實上，從鼻拭子獲取的野生型分離物的HA基因和NA基因與分別在Vero細胞和MDCK細胞上培養的同樣病毒相比，揭示MDCK培養的病毒的HA或NA相對于拭子分離物的HA或NA發生改變，或相對于Vero培養的病毒發生改變，或相對于拭子分離物和Vero培養的病毒都發生改變。
此外，用Vero或MDCK培養的野生型病毒免疫雪貂的試驗數據指出Vero培養的病毒比MDCK培養的病毒的毒力強得多。另外，對獼猴進行動物試驗測試的Vero培養的病毒的免疫原性顯著優于在MDCK細胞或卵上培養的病毒的免疫原性。
這些發現綜合在一起為下面的假說提供強有力的證據根據下文更詳細描述的本發明用于擴增和增殖病毒的方法產生與最初接種(如野生型)病毒相比未改變或僅發生很小程度修飾的病毒。
本發明的病毒擴增方法不僅避免抗原改變，而且異常簡單，使其非常適于大規模工業化疫苗生產。
其它試驗已經顯示胰蛋白酶(或胰蛋白酶原)的來源可能是影響感染性病毒粒總產量的另外一個因素。事實上，雖然本領域內已知的方法(如Kaverin和Webster，J Virol 69/42700-2703，1995；或US5,753,489)或者重復加入胰蛋白酶(Kaverin和Webster)，或者使用高胰蛋白酶濃度(US5,753,489)，但依照本發明的方法僅使用在先技術中報道的一半或更低胰蛋白酶濃度。此外，在溫育感染宿主細胞開始前或開始時，在細胞培養物培養基中一次性加入低至0.5-10μg/ml、優選2-5μg/ml的胰蛋白酶足以達到最佳感染性病毒滴度。失活試驗揭示豬或人重組胰蛋白酶與牛胰蛋白酶相比，對Vero細胞或MDCK細胞失活的敏感性低得多。由于在本領域內最廣泛使用牛胰蛋白酶，因此很有可能在先技術文獻暗示牛胰蛋白酶，明確指出其他胰蛋白酶來源的除外。這也能幫助解釋在例如Kaverin等和US5,753,489中陳述的應用胰蛋白酶的模式和濃度。
因此，根據本發明，在開始就使用豬或人重組胰蛋白酶或胰蛋白酶原補充用于Vero細胞培養物的無血清培養基，使得可以使用極低的胰蛋白酶或胰蛋白酶原濃度，并因此避免在收獲含病毒的上清液后需要勞動密集型和昂貴的純化步驟。
另一有利于使本方法簡單并因此對于疫苗生產商有吸引力的步驟是在溫育感染Vero細胞用于病毒增殖開始前或開始時，在細胞培養物培養基中一次性加入高活性內切核酸酶。在培養基中一次性加入該內切核酸酶(最好是BenzonaseTM)的起始濃度非常低，為2-30單位/ml、優選5-15單位/ml，該內切核酸酶從細胞培養物培養基中有效清除主要由正在裂解或已裂解的宿主細胞產生的游離DNA和RNA。由離心后上清液獲得的現成疫苗制備物的殘余Benzonase酶濃度保持在每劑5ng或更低。
BenzonaseTM是Nycomed Pharma A/S Denmark的商標，指從粘質沙雷氏菌(Serratia marcescens)獲得的一種細胞外非特異性內切核酸酶。Benzonase是一種遺傳工程化的內切核酸酶，分解多種形式的DNA和RNA成為小寡核苷酸。它促進快速降低細胞裂解液的粘度，便利超速離心。它降低蛋白水解，并增加靶蛋白的產量，從例如重組蛋白中完全清除核酸。它具有400,000U/mg的異常高的活性。
本方法第三個重要優點是時間因子，這因此降低方法的費用。由于使用不含動物來源蛋白并且最好不含抗生素的無血清培養基，因此可以減少昂貴和耗時的純化程序到最少或甚至完全避免。此外，因為外源酶例如蛋白酶(如胰蛋白酶或胰蛋白酶原)和核酸酶(如Benzonase)的添加一次性發生在病毒增殖期開始時，因此這節省了本領域目前使用的方法花費在含病毒的培養物上清液的溫育后處理(如HA激活、RNA/DNA消化、蛋白純化等)上的大量時間。
驚人的是，證實在病毒感染的Vero細胞培養物中早期加入蛋白酶(如胰蛋白酶或胰蛋白酶原)和核酸酶(如Benzonase)中的一種或兩種對于病毒產量沒有負面影響，這可能是由于本發明方法中應用的酶濃度非常低。
本發明的病毒增殖方法可用于擴增各種病毒，尤其是H3N2亞型A型流感病毒，但也適于分離和繁殖任何流行性或實驗室流感病毒株，不論病毒接種物的種類如何(如血清樣品、鼻洗液、鼻拭子、咽拭子、唾液等)。使用本發明方法的原理，已經產生多種A型流感和B型流感疫苗，這些疫苗構成本發明的一部分，并且在下面的實施例中更詳細表征。
此外，已經證實根據本發明制造的疫苗的保護性功效和疫苗安全性，這將在實施例中得到證實。
本文使用的與按照本發明的病毒擴增或增殖方法有關的術語“不含蛋白”或“不含非血清蛋白”是指不含任何有功能活性的蛋白。然而，該術語應當不排除可能來自蛋白水解物例如酵母提取物或大豆提取物的非功能性肽。除另有陳述外，術語“不含蛋白”也不排除以本文公開和要求保護的濃度存在的蛋白酶和核酸酶。
在一個優選實施方案中，本發明涉及一種生產完整病毒疫苗的簡單、可靠和高度經濟的方法，所述完整病毒疫苗最好是一種減毒活疫苗，該方法包括下面步驟a)用所需病毒感染非洲綠猴腎(Vero)細胞，其中所述Vero細胞在無血清培養基中培養并從中分離，所述無血清培養基也不含非血清蛋白；b)將所述感染細胞與合適的不含非血清蛋白(除蛋白酶和核酸酶外)的無血清細胞培養基混合；然后c)在所述蛋白酶和所述核酸酶存在下溫育所述細胞，產生感染性病毒，同時消化釋放到細胞培養基中的核酸物質；d)離心所述細胞培養物，收集包含病毒的上清液，從而收獲感染性病毒；然后e)如下制備其疫苗用選自以下的至少一種加工步驟處理所述含病毒的上清液過濾、濃縮、冷凍、凍干以及通過加入穩定劑穩定。
優選用于增殖的所述病毒未曾接觸過除Vero細胞系外的宿主支持物。這將保證獲得與最初病毒(如鼻拭子分離物)的最佳的免疫原性和抗原性同一性，以及增殖后獲得最佳的病毒后代。
還優選用于增殖的病毒是選自以下的流感病毒A/Sing/1/57ca株、A/Sing/1/57ca/ΔNS 87株、A/Sing/1/57ca/ΔNSPR8株、A/Sing/1/57ca/NS124PR8株、B/Vienna/1/99ca株、B/Vienna/99/ca37株以及來自這些病毒株中任一種的減毒變異體和重排列體，所述病毒尤其用于生產完整病毒疫苗，最好是一種流感減毒活疫苗。下面實施例詳細地公開優選病毒株如原株(master strain)的遺傳特征。
在另一實施方案中，本發明涉及完整病毒疫苗本身，最好涉及減毒活疫苗，所述疫苗的現成形式包含用于生產所述病毒的無血清和無蛋白Vero細胞培養物的含病毒上清液，所述上清液基本未調整處理、可選地經過過濾和/或濃縮。尤其優選所述疫苗根據本文公開和要求保護的本發明方法生產。
所述“一步”疫苗不需要進一步的加工，如除離心和/或常規過濾(即非凝膠過濾)外的純化步驟，符合FDA批準的要求。
本文在按照本發明的疫苗制備中關于含病毒的上清液所使用的術語“基本未改變”應當指收獲增殖病毒時上清液的組成，即在所述上清的液相中存在的可溶性組分和成分的組成。由于對所述含病毒的上清液進行例如過濾、無菌過濾、離心、濃縮、干燥或凍干等步驟而發生的成分組成的微小變化應當視為在“基本未改變”的范圍內。此外，該術語應當不排除本領域內一般用于疫苗制備物的防腐劑和/或穩定劑的存在。
本發明的完整病毒疫苗可用于預防性或治療性治療病毒感染，尤其是治療流感病毒感染。可以使用本領域內已知的途徑給予，例如靜脈內途徑、皮下途徑、肌內途徑或最優選鼻內途徑。然而，本文公開的病毒株以及用其制造的疫苗也可用作呈遞異源抗原到免疫系統的載體或穿梭系統，例如呈遞病毒被膜蛋白的抗原如HIV-1或肝炎抗原。
在從屬權利要求中定義其它優選實施方案。
為更充分理解本文描述的本發明，陳述下面的實施例。它們僅用于舉例說明的目的，不應當認為是在任何方面限制本發明。
實施例1生產病毒培養Vero/SF(＝無血清)細胞SF-培養基DMEM(Biochrom F0435)，Ham’s F12(BiochromF0815)，5mM L-Gln，0.1％SF添加劑(a)或(b)；抗生素(僅用于病毒分離物的第一次傳代)。
SF-添加劑非動物來源的蛋白水解物，不含功能蛋白如胰島素、轉鐵蛋白或生長因子
a)62.5g hy-soy/UF，Quest 5X59100，加入500g HQ水，用PES0.2μm濾器過濾；b)12.5g hy-pep 1510，Quest，加入100g HQ水，用PES0.2μm濾器過濾。
融化一支消毒過的(70％乙醇)深度冰凍(液氮)的盛裝WCB Vero細胞的安瓿的內容物，將其加入10ml管內的9ml冷的無血清(SF)培養基，在1000rpm(170g)離心10分鐘。將沉淀重懸浮于SF培養基中到總共30ml，然后轉移到一個80cm2的Roux瓶，在37℃和7％CO2下溫育至少15分鐘。然后，移走所述培養基，用約0.1ml/cm2PBS def.(＝不含Ca+和Mg2+的PBS)洗滌細胞。加入胰蛋白酶/EDTA溶液(8-10μl/cm2；0.1％胰蛋白酶/0.02％EDTA溶液)，在室溫下溫育約3分鐘。用手掌溫和推動所述Roux瓶，使細胞解吸附，然后加入約1/5胰蛋白酶/EDTA溶液體積的SF培養基和胰蛋白酶抑制劑(Sigma，T6522)。將所述細胞懸浮液重新分配到Roux瓶或滾瓶，在37℃和9％CO2下溫育。
在DMEM/Ham’s F12+2％FCS(熱失活)中培養MDCK細胞；含胚的雞蛋11-12天大，SPF(不含特定病原體)來源。
繁殖病毒株從含Vero細胞的滾瓶中取出舊培養基，在每個滾瓶的SF培養基中加入5ml病毒懸浮液，使病毒感染細胞，MOI(感染復數)約0.01。在33℃溫育45分鐘后，用吸液管取出病毒接種物。在每個滾瓶中加入90ml補充0.5-10、優選2-5、最優選2μg/ml豬胰蛋白酶(供應商AvP)或人重組胰蛋白酶或胰蛋白酶原(自產)以及0.5g/l碳酸氫鈉的SF培養基，所述滾瓶在33℃和5％CO2下溫育。為產生用于動物試驗以及人體臨床試驗的減毒活疫苗樣品，在所述SF培養基中補充胰蛋白酶以及BenzonaseTM，BenzonaseTM使用濃度為每ml培養基2-30、優選5-15、最優選10單位。感染后64小時，培養物上清液在10℃下50ml管中以4000rpm(3000g)離心5分鐘，收獲病毒。匯集每個病毒株的上清液，貯存在+4℃。其等分試樣用于疫苗測試。
為貯存疫苗，可以凍干所述病毒制備物，或者可以加入穩定劑如海藻糖和HEPES緩沖液中的乳清蛋白酶促水解物。可以用無菌水進行重建。
實施例2比較各細胞培養物中的胰蛋白酶失活表1Vero細胞與MDCK細胞培養物中胰蛋白酶失活的比較

測試從未感染的Vero細胞培養物(如實施例1所述在SF培養基中培養)和MDCK細胞培養物(在如實施例所述在補充FCS的培養基中培養)中它們失活不同來源胰蛋白酶的能力，所述胰蛋白酶在時間0h以同等濃度加入上清液中。應用兩種不同質量(從不同生產商獲得)的豬胰蛋白酶，即高活性或低活性豬胰蛋白酶。結果顯示于表1和圖1和圖2。
數據明白地顯示牛胰蛋白酶在Vero細胞培養物上清液中快速失活，在MDCK細胞培養物上清液中失活較慢。豬和人重組胰蛋白酶(本實驗室生產)在MDCK上清液中保持完全活性，但它們在Vero上清液中逐漸失活，其速率是牛胰蛋白酶失活速率的約一半或更低。所測試的兩種豬胰蛋白酶的差別僅是在247nm處的開始OD水平，而兩批豬胰蛋白酶的失活特性基本相同。
實施例3在不同宿主細胞支持物上培養后各種病毒特性的比較如實施例1所述進行在不同宿主細胞支持物上的病毒增殖。從Vero細胞回收的七個分離物的每一個都與人紅細胞反應，但不與雞紅細胞反應，并且沒有一種在含胚卵中積累。另一方面，所述從MDCK細胞回收的分離物都與雞和人紅細胞反應，并且能夠在蛋中生長。雖然這些差異沒有在H1N1亞型A型流感病毒或B型流感病毒分離物中出現(見下面的表3和表4)，但仍然可以假設在Vero細胞上培養流感病毒將比在其它支持物上培養更好地維持抗原性特性。
表2從臨床材料分離的H3N2型病毒在Vero/SF細胞上的特征

由表3的數據，看起來H1N1流感病毒對于適應性選擇較不易感，因為Vero和MDCK培養的分離物在它們的血細胞凝集特性以及它們的HA序列上不顯示顯著差異。對于表4中列出的B分離物可以得出相似結論。
用于分離和復制病毒的臨床起始材料(如血清樣品、拭子)主要來自1.Institute of Virology，Vienna，Austria(Prof.F.Heinz)1995/96，1996/972.Unite de Genetique Moleculaire des Virus Respiratoires，Institute Pasteur，Paris，France(Prof.S.van der Werf)1996/973.Public Health Laboratory Service，London，UK(Dr.M.Zambon)1996/974.Laboratorie Central de Virologie，Hopitaux Universitaires deGeneve，Geneva，Switzerland(Dr.W.Wunderli)1996/97，1997/985.Virus Unit，Queen Mary Hospital，Hong Kong(Dr.W.L.Lim)1997/98
表3分離自臨床材料的H1N1病毒在Vero/SF細胞上的特性

表4分離自臨床材料的B型病毒在Vero/SF細胞上的特征

表5分離自不同宿主系統的H3N2流感病毒的HA、NA和M蛋白的氨基酸改變

結果顯示對于一些分離物，Vero或MDCK增殖的病毒的HA序列與通過PCR擴增直接得自棉拭材料的HA序列相比沒有改變。在一些在MDCK細胞上生長的其它分離物中，HA和/或NA序列與從Vero細胞獲得的對應序列不同。然而，從Vero得到的病毒在HA序列上并不顯示與已經測定的拭子分離物的HA序列有任何差異。
表6從Vero分離的病毒、從MDCK分離的病毒和從蛋分離的病毒對獼猴的免疫原性

在不使用麻醉的情況下用1ml病毒懸浮液鼻內免疫獼猴V-從Vero分離的病毒M-從MDCK分離的病毒E-從蛋分離的病毒表7從Vero和MDCK分離的A/Vienna/47/96野生型病毒變異體對雪貂的毒力

動物在用乙醚麻醉的情況下用1ml病毒懸浮液鼻內接種。
N-正常溫度，從38.1℃到39.9℃F-發熱，超過40.0℃.
表6中最驚人但重要的結果是從MDCK分離的A/Vienna/47/96病毒與從Vero分離的對應病毒相比免疫原性非常低。并不特別令人驚訝的是從蛋分離的病毒僅顯示弱的免疫原性。
相似地，表7中的結果指出從Vero分離的病毒與從MDCK分離的病毒相比，在它們的宿主支持物上受到的適應性選擇改變(如果有的話)更少。這意味著與從MDCK分離的病毒相比，從Vero分離的病毒保持原始病毒更多或甚至全部的免疫相關特性，尤其是抗原性特性。
實施例4用優選株生產疫苗實施例1中描述的方法也用于生產用于動物測試和人類臨床研究的疫苗樣品。應當理解，其中描述的病毒增殖方法也包括本領域內一般技術人員不需創造性投入就可應用的變化，只要所述變化不改變本文和權利要求中所述的本發明的含義。
使用連續的Vero細胞系作為宿主細胞系統(除非為比較目的使用得自其它宿主支持物的樣品)，在無血清培養基中專門生產包含一種或多種優選A型或B型野生型株、原株或重排列株(隨后更詳細描述)的疫苗樣品，其中所述無血清培養基中另外補充實施例1中所述的營養成分和酶。
一些適于修飾野生型病毒的方法是本領域內已知的，包括減毒方法(如溫度敏感性)、冷適應方法和重排列方法，這些方法的廣泛綜述見，例如，WO 99/64068。
下面表8-13給出用于生產減毒活疫苗的兩個最優選的A型和B型流感原株候選物的其它特征，所述生產例如通過WHO推薦的對實際流行性流感病毒的HA和NA基因進行6/2重排列。
表8活流感疫苗的原株候選物的特征

表98個基因組區段和A/Singapore/1/57/ca株的10種對應蛋白的完整序列

ca-冷適應然而，應當注意到第4和第6基因組區段(即HA基因和NA基因)對于表征A型流感原株候選物不是必需的，因為這些基因可以與實際流行性流感病毒(如上文所述)的對應基因互換。對于疫苗安全性重要的特性(如溫度敏感性)或允許鼻內給予疫苗的特性(既冷適應)(因為鼻內的平均溫度低于實際體溫)主要是由于其余6個基因組區段中的突變引起的。
下面表10列出A/Singapore/1/57/ca與對應野生型株A/Singapore/1/57wt相比在基因組區段中的突變。
表10減毒、溫度敏感性、冷適應流感株A/Singapore/1/57/ca與A/Singapore/1/57wt相比在基因組區段中的突變

突變總數-13(8編碼)*編碼的突變優選的A/Sing/1/57/ca的變異體包含下面表11列出的那些突變，其中“Δ”指“del”或“delta”，表示在其NS基因區段中包含至少一個“缺失”的突變異體。
表11A/Sing/1/57/ca的優選變異體 *源自A/Singapore/1/57/ca的基因組區段**源自流感A/PR8/34的基因組區段ca-冷適應；ts-溫度敏感性；aa-氨基酸IFN-induct.-在能夠生產IFN的宿主支持物中以及在給予動物或人免疫系統時引起干擾素釋放的株。
IFN-sensit.-對干擾素敏感的株；在生產干擾素的細胞中的復制減少或停止。
Sing ca/ΔNS 87-包含在NS1基因位置36-123缺失87個氨基酸的A/Singapore/1/57/ca株。
Sing ca/ΔNSPR8-包含來自A/PR8/34(本文也縮寫為“PR8”)的NS基因區段的A/Singapore/1/57/ca株，所述區段缺失整個NS1基因。
Sing ca/NS124PR8-包含來自A/PR8/34的NS基因區段的A/Singapore/1/57/ca株，所述區段在NS1基因的氨基酸位置124包含一個終止密碼子。
+/-指該表型需要進一步澄清，并且還不能明確地定義。
下表12、13和13A分別指出優選的B型流感原株候選物以及其變異和重排列體。
表12B/Vienna/1/99/ca株的8個基因組區段和11個對應蛋白的完整序列

ca-冷適應最初的B/Vienna/1/99株于1999年2月分離自用無血清培養基培養的Vero細胞培養物，來自奧地利維也納一名患有急性流感的12歲女子。疾病控制中心(CDC)，Atlanta，USA將它評級為類似于B/Beijing/184/93。野生型株(名為B/Vienna/1/99wt)在33℃下再傳一代后，通過在25℃下使用同樣細胞培養系統進行22次連續傳代而減毒。第一輪在25℃下進行噬菌斑純化，隨后在33℃下進行四輪純化。擴增所獲得的噬菌斑純化克隆，貯存于-70℃，該克隆名為B/Vienna/1/99 ca或簡稱為BV22。通過用來自NIBSC的標準抗血清進行的HI測定確認B/Beijing/184/93樣病毒的身份。
表13B/Vienna/1/99/ca(＝BV22)與B/Vienna/1/99/wt(Bvie)相比的突變，在Vero/SF中第一代

表26根據Los Alamos National Library流感病毒庫(db)(網址www.flu.lanl.gov)，B/Vienna/1/99wt的表征

此外，B/Vienna/1/99 ca繼續傳代另外15次后(即在無血清Vero細胞培養物上總共傳代37次)導致突變異體B/Vienna/1/99 ca37(簡稱為BV37)，該株具有甚至由于某些BV22的特性。該突變異體包含與BV22相比數量增加的突變，并且目前看起來是生產基于非重組流感病毒突變異體的完整病毒疫苗(尤其是減毒流感活疫苗)最有希望的候選物。其它突變列于下面表13A
表13ABV22和BV37與B/Vienna/1/99wt相比的突變，在Vero/SF上第一次傳代

與流感序列數據庫13.2.2001(www.flu-lanl.gov)的比較a)獨特突變用粗體下劃線表示；b)共有突變*B/Lee/40、B/Osaka/70、B/Kadoma/1076/99(獲得的氨基酸1)+B/Lee/40、B/Osaka/70#常常是B/Lee/40、B/Ann Arbor/1/66 ca & wt、B/Singapore/222/79、B/North Dakota/83、B/Norway/1/84、B/Ibaraki/2/85、B/Ann Arbor/1/86、B/Victoria/2/87、B/Aichi/5/88*B/Kanagawa/73
應當理解，根據本發明的A型和B型流感原株應當不限于在本文的表中明確列出的特點和遺傳特征，而且應當還包括它的小變異，只要所述變異在本發明的意義內并且不顯著改變該病毒的任何一種功能特征。所述變異可以例如由于病毒擴增或增殖的額外步驟而發生(如為獲得進行序列分析的材料)。
此外，本文列出的基因序列包括與本發明一起使用的引物序列(位于每個基因組區段的開始或結束)，所述引物序列可以與野生型病毒株或減毒病毒株中一種或二者的病毒基因組區段的對應真序列不同。
實施例5疫苗安全性和效能下面數據證實按照本發明生產(如按照實施例1所述)的流感疫苗的溫度敏感性和疫苗安全性。
表14不麻醉的情況下進行一次鼻內免疫后小鼠的抗體反應

1-陽性HI滴度＞1∶4的動物數量2-在肺中未檢測到病毒的動物數量3-血清中抗體的幾何平均滴度PR8wt-流感株A/PR/8/34野生型(H1N1)，對小鼠致病PR8/Sing ca-2/6-是減毒流感株A/Sing/1/57 ca和PR8wt之間重排列體，包含來自PR8wt的2個基因(HA和NA)以及來自A/Sing/1/57ca的所有其它基因。
PR8/Sing-ΔNS包含來自PR8wt的HA和NA基因、來自A/Sing/1/57ca的五個基因和來自PR8但缺乏NS1編碼序列的NS基因(NS1缺失或剔除)。
表15用A/Singapore/1/57病毒的不同變異體鼻內免疫(麻醉)后小鼠的抗體反應和保護

1-陽性HI滴度＞1∶4的動物數量2-va-Vero適應的3-ca-冷適應的4-在肺中未檢測到病毒的動物數量表16A/Singapore/1/57的wt、va和ca變異體在小鼠肺中的增殖a

a小鼠鼻內感染50μl滴度為1.0×106PFU/ml的病毒液。
b每ml組織懸浮液的PFU，在MDCK細胞上滴定。
表17A/Singapore/1/57病毒的wt和ca變異體對雪貂的毒力

每日兩次記錄動物的直腸溫度，特征如下N-正常溫度，從38.1℃到39.9℃F-發熱，超過40℃。
每組包括3只動物，用乙醚麻醉動物，用1ml滴度為2×106PFU/ml的病毒液鼻內免疫動物。
表18A/Hong Kong/1035/98wt和A/Singapore/1/57/ca的2/6重排列體在小鼠肺中的增殖a

a用乙醚麻醉小鼠，鼻內感染50μl病毒液。
b每ml 10％組織懸浮液的PFU/ml，在Vero/SF細胞上滴定，數據是6只小鼠的平均值(分別處理每只動物的肺)。
所述重排列體包含來自A/Hong Kong/1035/98wt野生型的HA和NA基因以及來自A/Singapore/1/57/ca的其它6個基因。
表19A/Vienna/47/96wt和A/Singapore/1/57/ca的6/2重排列體對雪貂的毒力

用乙醚麻醉動物，用1ml滴度為2×106PFU/ml的病毒進行鼻內免疫。
N-正常溫度，從38.1℃到39.9℃F-發熱，超過40℃。
b+++-重度鼻炎±無鼻炎表16到19的結果清楚地證明包含所述減毒溫度敏感性原株的疫苗的安全性，以及在重排列體的情況下，基于重排列病毒的疫苗的安全性，所述重排列病毒包含所述減毒溫度敏感性原株的“骨架”(6個基因)以及來自如致病野生型株A/Hong Kong/1035/98wt的HA基因和NA基因。
表20B/Vienna/1/99的Ts和ca表型

表21B/Vienna/1/99 ca的ts表型的遺傳穩定性

BV22株以高MOI在Vero細胞上傳代五次。然后再次控制ts表型。如表21所示，該株保持溫度敏感性。
表22B/Vienna/1/99ca和wt在小鼠肺中的毒力

*每株免疫9只OF1小鼠，用乙醚麻醉小鼠，以105PFU經鼻免疫。在感染后指定的天數，每組處死3只小鼠。勻漿肺和鼻甲，在PBS def.中制成10％(w/v)懸浮液。進行懸浮液的噬菌斑測定。
數據顯示ca原株候選物BV22在鼻粘膜中的溫和復制是可能的，而該病毒的ts特性防止其在肺中增殖。
表23B型流感株的重排列體的Ts和ca表型

建立一種6/2重排列體，該重排列體包含野生型流感株B/USSR/69wt的HA和NA以及來自B/Vienna/1/99ca(BV22)的其它6個基因組區段。通過HI測定測試血凝素的來源，使用本領域內已知方法通過RT-PCT和限制分析測試所有其它基因組區段。
表24B型流感株重排列體在小鼠肺中的毒力

*每株免疫9只OF1小鼠，用乙醚麻醉小鼠，以105PFU經鼻免疫。在感染后指定的天數，每組處死3只小鼠。勻漿肺和鼻甲，在PBS def.中制成10％(w/v)懸浮液。進行懸浮液的噬菌斑測定。
實施例6臨床研究根據本發明(如按照實施例1所述)生產下面疫苗(采用鼻噴霧劑的形式)用于經鼻傳遞。每ml的組成(在重建凍干材料后)(1)安慰劑2×SF培養基，40mM HEPES緩沖液，8％乳清蛋白酶促水解物，4％海藻糖；(2)Vero-Vac H1A/Beijing/262/95(H1N1)樣制備物，該制備物包含4.3×107TCID50的A/Singapore/1/57/ca與A/HongKong/1035/98的6/2重排列體；2x培養物上清液，40mMHEPES緩沖液，8％乳清蛋白酶促水解物，4％海藻糖；(3)Vero-Vac H3A/Syndey/5/97(H3N2)樣制備物，該制備物包含2.1×107TCID50的A/Singapore/1/57/ca與A/SW/7729/98的6/2重排列體；2x培養物上清液，40mM HEPES緩沖液，8％乳清蛋白酶促水解物，4％海藻糖；(4)俄羅斯三價疫苗(用于成人的活流感疫苗)A/17/Beijing/95/25(H1N1)1.1×108EID50A/17/Sidney/97/76(H3N2) 2.3×107EID50B/60/Petersburg/95/20 1.1×107EID50(5)單價Vero疫苗BV22B/Beijing/184/93-樣制備物，該制備物包含2×106TCID50的原株候選物B/Vienna/1/99/ca(＝BV22)；2x培養物上清液，40mM HEPES緩沖液，8％乳清蛋白酶促水解物，4％海藻糖；將所述疫苗給予每個接種組13名志愿者。將550μl重建的疫苗(或安慰劑，分別地)在第0天和在第22±1天第二次經鼻給予每名患者。結果在下面表25中綜述。
安全性結果第一次和第二次接種后五天內不良反應(AE)的總數是14，其中包括9名輕度AE，4名中度AE。只有一名患者顯示重度AE，包括體溫在第一次接種后3小時內升到38.8℃，沒有局部或系統性癥狀。隨后四小時內他的體溫再次回復正常。第一次接種后觀察到7例AE。其中一例是局部的，六例是系統性的。第二次接種后觀察到2例局部AE和5例系統性AE。
就安全性而言，揭示在研究的各組包括安慰劑組間沒有顯著差異。除上面提到的一例外，沒有觀察到與接種相關的重度AE。其中兩例中度AE發生在H3N2組(一名志愿者在第三天體溫升高到37.6℃并出現急性咽炎；另一名志愿者在第22-24天出現鼻塞、咽喉不適，并且體溫升到37.5℃)，一例出現在H1N1組(從22-26天咽喉疼痛、鼻炎，體溫在22-24天之間升到37-37.8℃)。
表25血清反應陰性的志愿者對Vero Vac疫苗以及對三價俄羅斯冷適應的從蛋產生的疫苗的反應

(8)患者在研究期間發展出自發感染。
因此，從所述臨床研究得出的結果證實根據本發明生產的疫苗以及使用本發明優選的A型和B型流感原株候選物具有非常好的安全性。
序列表<110>Polymun Scientific Immunbiologische Forschung GmbHKatinger，HermannKatinger，DietmarRomanova，JuliaEgorov，AndrejFerko，Boris<120>流感疫苗和生產方法<130>BEP-4847-PC(流感疫苗)<140>
<141>
<160>37<170>PatentIn Ver.2.1<210>1<211>2341<212>DNA<213>流感病毒A/Singapore/1/57/ca<400>1agcaaaagca ggtcaattat attcaatatg gaaagaataa aagaactacg gaatctgatg 60tcgcagtctc gcactcgcga gatactaaca aaaaccacag tggaccatat ggccataatt 120aagaagtaca catcagggag acaggaaaag aacccgtcac ttaggatgaa atggatgatg 180gcaatgaaat atccgattac agctgacaag aggataacag aaatgattcc tgagagaaat 240gagcaagggc agactctatg gagtaaaatg aatgatgccg gatcggatcg agtgatggta 300tcacctctgg ctgtgacatg gtggaataga aatggaccaa tgacaagtac ggttcattat 360ccaaaaatct acaaaactta ttttgagaaa gtcgaaaggt taaaacatgg aacctttggc 420cctgtccatt ttagaaacca agtcaaaata cgccgaagag ttgacataaa tcctggtcat 480gcagacctca gtgccaagga ggcacaggat gtaatcatgg aagttgtttt ccctaacgaa 540gtgggggcca ggatactaac gtcggaatcg caattaacaa caaccaaaga gaaaaaagaa 600gaactccagg attgcaaaat ttctcctttg atggttgcgt acatgttaga gagagaactt 660gtccgaaaaa cgagatttct cccagttgct ggtggaacaa gcagtgtgta cattgaagtg 720ttgcacttaa ctcaaggaac atgctgggaa cagatgtaca ctccaggtgg agaagtgagg 780aatgatgatg ttgatcaaag tctaattatt gcagccagga acatagtgag aagagcagca 840gtatcagcag atccactagc atctttattg gagatgtgcc acagcacaca gattggcggg 900acaaggatgg tggacattct taggcagaac ccaacggaag agcaagctgt ggatatatgc 960aaggctgcaa tgggactgag aatcagctca tccttcagtt ttggcgggtt cacatttaag 1020agaacaagcg gatcatcagt caagatagag gaagaagtgc ttacgggcaa tcttcaaaca 1080ttgaaaataa gggtgcatga gggatacgag gagttcacaa tggttgggaa aagggcaaca 1140gctatactca gaaaagcaac caggagattg attcagctga tagtgagtgg aagagacgaa 1200
cagtcgatag ccgaagcaat aattgtggcc atggtatttt cacaagaaga ttgtatgata 1260aaagcagtta gaggtgatct gaatttcgtt aatagggcaa atcagcgatt gaatcccatg 1320catcaacttt taagacattt tcagaaggat gcgaaagtgc tttttcaaaa ttggggaatt 1380gaacatatcg acaatgtgat gggaatgatt ggggtattac cagacatgac tccaagcaca 1440gagatgtcaa tgagaggggt aagagtcagc aaaatgggcg tagatgaata ctccagcgcg 1500gagagagtag tggtgagcat tgaccggttt ttgagagttc gagaccaacg aggaaatgta 1560ctactatctc ctgaggaggt cagtgaaaca cagggaacag agaaactgac aataacttac 1620tcatcgtcaa tgatgtggga gattaatggc cctgagtcag tgttggtcaa tacctatcag 1680tggatcatca gaaactggga aactgttaaa attcagtggt ctcagaatcc tacaatgcta 1740tacaataaaa tggaatttga gccatttcag tctttagttc ctaaggccat tagaggccaa 1800tacagtgggt ttgttaggac tctattccaa caaatgaggg atgtacttgg gacatttgat 1860accacccaga taataaaact tcttcccttt gcagccgccc caccaaagca aagtagaatg 1920cagttctctt cattgactgt gaatgtgagg ggatcaggaa tgagaatact tgtaaggggc 1980aattctcctg tattcaacta caacaagacc actaagagac taacaattct cggaaaggat 2040gctggcactt taactgaaga cccagatgaa ggcacatctg gagtggagtc cgctgttctg 2100agaggattcc tcattctggg caaagaagat aggagatatg gaccagcatt aagcatcaat 2160gaactgagta accttgcgaa aggagaaaag gctaatgtac taattgggca aggagacgtg 2220gtgttggtaa tgaaacgaaa acgggactct agcatactta ctgacagcca gacagcgacc 2280aaaagaattc ggatggccat caattaatgt tgaatagttt aaaaacgacc ttgtttctac 2340t 2341<210>2<211>2341<212>DNA<213>流感病毒A/Singapore/1/57/ca<400>2agcaaaagca ggcaaaccat ttgaatggat gtcaatccga ccttactttt cttgaaagtt 60ccagcgcaaa atgccataag tactacattc ccttatactg gagatcctcc atacagccat 120ggaacaggaa caggatacac catggacaca gtcaacagaa cacatcaata ttcagaaaag 180gggaagtgga caacaaacac ggaaactgga gcgccccaac ttaacccaat tgatggacca 240ctacctgagg acaatgaacc aagtggatat gcacaaacag actgcgtcct ggaagcaatg 300gctttccttg aagaatccca cccgggaatc tttgaaaact cgtgtcttga aacgatggaa 360gttattcaac aaacaagagt ggacaaactg acccaaggtc gtcagaccta tgattggaca 420ttgaacagaa atcagccggc tgcaactgcg ctagccaaca ctatagaggt cttcagatcg 480aatggtctga cagctaatga atcgggaagg ctaatagatt tcctcaagga tgtgatagaa 540tcaatggata aagaggagat ggaaataaca acacacttcc aaagaaaaag aagagtaaga 600gacaacatga ccaagaaaat ggtcacacaa cgaacaatag gaaaaaagaa gcaaagattg 660aacaagagaa gctatctaat aagagcactg acattgaaca caatgactaa agatgcagag 720agaggtaaat taaagagaag agcaattgca acacccggta tgcagatcag agggttcgtg 780tactttgtcg aaacactagc gagaagtatt tgtgagaagc ttgaacagtc tgggcttccg 840gttggaggta atgaaaagaa ggctaaactg gcaaatgttg tgagaaaaat gatgactaat 900tcacaagaca cagagctctc tttcacaatt actggagaca ataccaaatg gaatgagaat 960caaaatcctc ggatgttcct ggcgatgata acatacatca caagaaatca acctgaatgg 1020tttagaaacg tcctgagcat cgcacctata atgttctcaa ataaaatggc aagactaggg 1080aaaggataca tgttcgaaag caagagcatg aagctccgaa cacaaatacc agcagaaatg 1140ctagcaagta ttgacctgaa atactttaat gaatcaacaa gaaagaaaat cgagaaaata 1200aggcctctcc taatagatgg cacagtctca ttgagtcctg gaatgatgat gggcatgttc 1260aacatgctaa gtacagtcat aggagtctca atcctgaatc ttggacaaaa gaagtacacc 1320
aaaacaacat actggtggga cggactccaa tcctctgatg acttcgccct catagtgaat 1380gcaccaaatc atgagggaat acaagcagga gtggatagat tctacagaac ctgcaagcta 1440gtcggaatca atatgagcaa aaagaagtcc tacataaata ggacagggac atttgaattc 1500acaagctttt tctatcgcta tggatttgta gccaatttta gcatggagct gcccagtttt 1560ggagtgtctg gaattaatga atcggctgat atgagcattg gggtaacagt gataaagaac 1620aacatgataa acaatgacct tgggccagca acagcccaaa tggctcttca actattcatc 1680aaagactaca gatatacgta ccggtgccac agaggagaca cacaaattca gacaaggaga 1740tcattcgagc taaagaagct gtgggagcaa acccgctcaa aggcaggact tttggtttcg 1800gatggaggac caaacttata caatatccgg aatctccaca ttccagaagt ctgcttgaag 1860tgggagctaa tggatgaaga ctatcagggg aggctttgta atcccctgaa tccatttgtc 1920agtcataagg agattgagtc tgtaaacaat gctgtggtaa tgcccgctca cggtccagcc 1980aagagcatgg aatatgatgc tgttgctact acacactcct ggatccctaa gaggaaccgc 2040tccattctca acacaagcca aaggggaatt cttgaggatg aacagatgta tcagaagtgt 2100tgcaatctat tcgagaaatt cttccctagc agttcgtaca ggagaccagt tggaatttcc 2160agcatggtgg aggccatggt gtctagggcc cggattgatg cacggattga cttcgagtct 2220ggacggatta agaaagagga gttcgctgag atcatgaaga tctgttccac cattgaagag 2280ctcagacggc aaaaatagtg aatttagctt gtccttcatg aaaaaatgcc ttgtttctac 2340t 2341<210>3<211>2233<212>DNA<213>流感病毒A/Singapore/1/57/ca<400>3agcaaaagca ggtactgatc cgaaatggaa gattttgtgc gacaatgctt caatccgatg 60attgtcgagc ttgcggaaag ggcaatgaaa gagtatggag aggatctgaa aatcgaaaca 120aacaaatttg cagcaatatg cactcacttg gaagtatgct tcatgtattc agattttcat 180ttcatcaatg agcaaggcga gtcaataata gtagagcttg atgatccaaa tgcacttttg 240aagcacagat ttgaaataat agagggaaga gatcgcacaa tggcctggac agtagtaaac 300agtatttgca acactacagg agctgagaaa ccgaagtttc tgccagattt gtatgattac 360aaggagaata gattcatcga gattggagtg acaaggaggg aagtccacat atactatctt 420gaaaaggcca ataaaattaa atctgagaag acacacatcc acattttctc attcactggg 480gaagaaatgg ccacaaaggc cgactacact ctcgatgagg aaagcagggc taggatcaaa 540accagactat tcaccataag acaagaaatg gctagcagag gcctctggga ttcctttcgt 600cagtccgaaa gaggcgaaga aacaattgaa gaaagatttg aaatcacagg gacaatgcgc 660aggctcgccg accaaagtct cccgccgaac ttctcctgcc ttgagatttt tagagcctat 720gtggatggat tcgaaccgaa cggctacatt gagggcaagc tttctcaaat gtccaaagaa 780gtaaatgcta aaattgaacc ttttctgaaa acaacaccaa gaccaattag acttccggat 840gggcctcctt gttctcagcg gtccaaattc ctgctgatgg atgctttaaa attaagcatt 900gaggacccaa gtcacgaagg agagggaata ccactatatg atgcgatcaa gtgtatgaga 960acattctttg gatggaaaga accctatgtt gttaaaccac acgaaaaggg aataaatcca 1020aattatctgc tgtcatggaa gcaagtactg gcagaactgc aggacattga gaatgaggag 1080aagattccaa gaaccaaaaa catgaagaaa acgagtcagc taaagtgggc acttggtgag 1140aacatggcac cagagaaggt agactttgac gactgtagag atataagcga tttgaagcaa 1200tatgatagtg atgaacctga attaaggtca ctttcaagct ggatccagaa tgagttcaac 1260aaggcatgcg agctgaccaa ttcaatctgg atagagctcg atgagattgg agaagatgtg 1320gctccaattg aacacattgc aagcatgaga aggaattact tcacagcaga ggtgtctcat 1380tgcagagcca cagaatatat aatgaagggg gtatacatta atacagcctt gcttaatgca 1440
tcctgtgcag caatggacga tttccaacta attcccatga taagcaaatg tagaactaaa 1500gagggaaggc gaaagaccaa tttatatggt ttcatcgtaa aaggaagatc tcacttaagg 1560aatgacaccg acgtggtaaa ctttgtgagc atggagtttt ctctcactga cccaagactt 1620gagccacaca aatgggagaa gtactgtgtt cttgagatag gagatatgct actaagaagt 1680gccataggcc aggtgtcaag gcccatgttc ttgtatgtga ggacaaatgg aacatcaaag 1740attaaaatga aatggggaat ggagatgagg cgttgcctcc ttcagtcact ccaacaaatc 1800gagagcatga ttgaagccca gtcctctgtc aaggagaaag acatgaccaa agagtttttc 1860gagaataaat cagaaacatg gcccattgga gagtccccta aaggagtgga agaaggttcc 1920attgggaagg tctgcaggac tttattagcc aagtcggtat tcaatagcct gtatgcatct 1980ccacaattag aaggattttc agctgaatca agaaaactgc tccttgtcgt tcaggctctt 2040agggacaatc ttgaacctgg gacctttgat cttggggggc tatatgaagc aattgaggag 2100tgcctgatta atgatccctg ggttttgctt aatgcgtctt ggttcaactc cttcctaaca 2160catgcattaa gatagttgtg gcaatgctac tatttgctat ccatactgtc caaaaaagta 2220ccttgtttct act2233<210>4<211>1773<212>DNA<213>流感病毒A/Singapore/1/57/ca<400>4agcaaaagca ggggttatac catagacaac cagaagcaaa acaatggcca tcatttatct 60cattctcctg ttcacagcag tgagagggga ccagatatgc attggatacc atgccaataa 120ttccacagag aaggtcgaca caattctaga gcagaacgtc actgtgactc atgccaagga 180cattcttgag aagacccata acggaaagtt atgcaaacta aacggaatcc ctccacttga 240actaggggac tgtagcattg ccggatggct ccttggaaat ccagaatgtg ataggcttct 300aagtgtgcca gaatggtcct atataatgga gaaagaaaac ccgagagacg gtttgtgtta 360tccaggcagc ttcaatgatt atgaagaatt gaaacatctc ctcagcagcg tgaaacattt 420cgagaaagta aagattctgc ccaaagatag atggacacag catacaacaa ctggaggttc 480acgggcctgc gcggtgtctg gtaatccatc attcttcagg aacatggtct ggctgacaaa 540gaaagaatca aattatccgg ttgccaaagg atcgtacaac aatacaagcg gagaacaaat 600gctaataat ttggggggtgc accatcccaa tgatgagaca gaacaaagaa cattgtacca 660gaatgtggga acctatgttt ccgtaggcac atcaacattg aacaaaaggt caaccccaga 720catagcaaca aggcctaaag tgaatggact aggaagtaga atggaattct cttggaccct 780attggatatg tgggacacca taaattttga gagtactggt aatctaattg caccagagta 840tggattcaaa atatcgaaaa gaggtaattc agggatcatg aaaacagaag gaacacttga 900gaactgtgag accaaatgcc aaactccttt gggagcaata aatacaacat tgccttttca 960caatgtccac ccactgacaa taggtgagtg ccccaaatat gtaaaatcgg agaagttggt 1020cttagcaaca ggaccaagga atgttcccca gattgaatca agaggattgt ttggggcaat 1080agctggtttt atagaaggag gatggcaagg aatggttgat ggttggtatg gataccatca 1140cagcaatgac cagggatcag ggtatgcagc agacaaagaa tccactcaaa aggcatttga 1200tggaatcacc aacaaggtaa attctgtgat tgaaaagatg aacacccaat ttgaagctgt 1260tgggaaagaa ttcagtaact tagagagaag actggagaac ttgaacaaaa agatggaaga 1320cgggtttcta gatgtgtgga catacaatgc tgagcttcta gttctgatgg aaaatgagag 1380gacacttgac tttcatgatt ctaatgtcaa gaatctgtat gataaagtca gaatgcagct 1440gagagacaac gtcaaagaac taggaaatgg atgttttgaa ttttatcaca aatgtgatga 1500tgaatgcatg aatagtgtga aaaacgggac gtatgattat cccaagtatg aagaagagtc 1560taaactaaat agaaatgaaa tcaaaggggt aaaattgagc agcatggggg tttatcaaat 1620ccttgccatt tatgctacag tagcaggttc tctgtcactg gcaatcatga tggctgggat 1680
ctctttctgg atgtgctcca acgggtctct gcagtgcagg atctgcatat gattataagt 1740cattttataa ttaaaaacac ccttgtttct act 1773<210>5<211>1565<212>DNA<213>流感病毒A/Singapore/1/57/ca<400>5agcaaaagca gggtagataa tcactcactg agtgacatca aaatcatggc gtcccaaggc 60accaaacggt cttatgaaca gatggaaact gatggggaac gccagaatgc aactgaaatc 120agagcatccg tcgggaagat gattgatgga attggacgat tctacatcca aatgtgcacc 180gaacttaaac tcagtgatta tgaggggcga ctgatccaga acagcttaac aatagagaga 240atggtgctct ctgcttttga cgagaggagg aataaatatc tggaagaaca tcccagcgcg 300gggaaggatc ctaagaaaac tggaggaccc atatacaaga gagtaaatgg aaagtggatg 360agggaactcg tcctttatga caaagaagaa ataaggcgaa tctggcgcca agctaataat 420ggtgatgatg caacagctgg tctgactcac atgatgatct ggcattccaa tttgaatgat 480acaacatacc agaggacaag agctcttgtt cgcaccggaa tggatcccag gatgtgctct 540ttgatgcagg gttcgactct ccctaggagg tctggagccg caggcgctgc agtcaaagga 600gttgggacaa tggtgatgga gttgatcagg atgatcaaac gtgggatcaa tgatcggaac 660ttctggagag gtgagaatgg gcggaaaaca aggattgctt atgagagaat gtgcaacatt 720ctcaaaggaa aatttcaaac agctgcacaa agagcaatga tggatcaagt gagagaaagc 780cggaacccag gaaatgctga gatcgaagat ctcatctttc tggcacggtc tgcactcata 840ttgagagggt cagttgctca caaatcttgt ctgcctgcct gtgtgtatgg aactgccgta 900gccagtgggt acgacttcga aaaagaggga tactctttag tagggataga ccctttcaaa 960ctgcttcaaa acagccaagt atacagccta atcagaccga acgagaatcc agcacacaag 1020agtcagctgg tgtggatggc atgcaattct gctgcatttg aagatctaag agtatcaagc 1080ttcatcagag ggaccaaagtaatcccaagg gggaaacttt ccactagagg agtacaaatt 1140gcttcaaatg aaaacatgga tactatggaa tcaagtactc ttgaactgag aagcaggtac 1200tgggccataa ggaccagaag tggaggaaac actaatcaac agagggcctc tgcaggtcaa 1260atcagtgtac aacctacgtt ttctgtgcaa agaaacctcc catttgacaa aacaaccatc 1320atggcagcat tcactgggaa tgcagaggga agaacatcag acatgagggc agaaatcata 1380aggatgatgg aaggtgcaaa accagaagaa gtgtccttcc aggggcgggg agtcttcgag 1440ctctcggacg aaaaggcaac gaacccgatc gtgccctctt ttgacatgag taatgaagga 1500tcttatttct tcggagacaa tgcagaggag tacgacaatt aaggaaaaat acccttgttt 1560ctact 1565<210>6<211>1466<212>DNA<213>流感病毒A/Singapore/1/57/ca<400>6agcaaaagca ggagtgaaga tgaatccaaa tcaaaagata ataacaattg gctctgtctc 60tctcaccatt gcaacagtat gcttcctcat gcagattgcc atcctggcaa ctactgtgac 120attgcatttt aaacaacatg agtgcgactc ccccgcgagc aaccaagtaa tgccatgtga 180accaataata atagaaagga acataacaga gatagtgtat ttgaataaca ccaccataga 240gaaagagatt tgccccgaag tagtggaata cagaaattgg tcaaagccgc aatgtcaaat 300tacaggattt gcaccttttt ctaaggacaa ttcaatccgg ctttctgctg gtggggacat 360
ttgggtgacg agagaacctt atgtgtcatg cgatcctggc aagtgttatc aatttgcact 420cgggcagggg accacactat acaacaaaca ttcaaatggc acaatacatg atagaatccc 480tcatcgaacc ctattaatga atgagttggg tgttccattt catttaggaa ccaaacaagt 540gtgtgtagca tggtccagct caagttgtca cgatggaaaa gcatggttgc atgtttgtgt 600cactggggat gatagaaatg cgactgctag cttcatttat gacgggaggc ttgtggacag 660tattggttca tggtctcaaa atatcctcag gacccaggag tcggaatgcg tttgtatcaa 720tgggacttgc acagtagtaa tgactgatgg aagtgcatca ggaagagccg atactagaat 780actattcatt aaagagggga aaattgtccg tattagccca ttgtcaggaa gtgctcagca 840tatagaggag tgttcctgtt accctcgata tcctgacgtc agatgtatct gcagagacaa 900ctggaaaggc tctaataggc ccgttataga cataaatatg gaagattata gcattgattc 960cagttatgtg tgctcagggc ttgttggcga cacacccagg aacgacgaca gctctagcaa 1020tagcaattgc agggatccta acaatgagag agggaatcca ggagtgaaag gctgggcctt 1080tgacaatgga gatgatgtat ggatgggaag aacaatcaac aaagattcac gctcaggtta 1140tgaaactttc aaagtcattg gtggttggtc cacacctaat tccaaatcgc aggtcaatag 1200acaggtcata gttgacaaca ataattggtc tggttactct ggtattttct ctgttgaggg 1260caaaagctgc atcaataggt gcttttatgt ggagttgata aggggaaggc cacaggagac 1320tagagtatgg tggacctcaa acagtattgt tgtgttttgt ggcacttcag gtacttatgg 1380aacaggctca tggcctgatg gggcgaacat caatttcatg cctatataag ctttcgcaat 1440tttagaaaaa actccttgtt tctact 1466<210>7<211>1027<212>DNA<213>流感病毒A/Singapore/1/57/ca<400>7agcaaaagca ggtagatatt gaaagatgag tcttctaacc gaggtcgaaa cgtatgttct 60ctctatcgtc ccgtcaggcc ccctcaaagc cgagatcgca cagagacttg aagatgtctt 120tgctgggaag aacaccgatc ttgaggctct catggaatgg ctaaagacaa gaccaatcct 180gtcacctctg actaagggga ttttgggatt tgtattcacg ctcaccgtgc ccagtgagcg 240aggactgcag cgtagacgct ttgtccaaaa tgccctcaat gggaatgggg atccaaataa 300catggacaga gcagttaaac tgtataaaaa gcttaagagg gagataacat tccatggggc 360caaagaaata gcgctcagtt attctgctgg tgcacttgcc agttgtatgg gcctcatata 420caacaggatg ggggctgtga ccactgaagt ggcctttggc ctggtatgtg caacctgtga 480acagattgct gactcccacc ataggtctca taggcaaatg gtgacaacaa ccaatccact 540aataagacat gagaacagaa tggttctggc cagcactaca gctaaggcta tggagcaaat 600ggctggatcg agtgagcaag cagcagaggc catggaggtt gctagtcagg ccaggcaaat 660ggtgcaggca atgagagcca ttgggactca tcctagctcc agtgctggtc taaaagatga 720tcttcttgaa aatttgcagg cctatcagaa acgaatgggg gtgcagatgc aacgattcaa 780gtgaccctct tgttgttgcc gcgagtatca ttgggatctt gcacttgata ttgtggattc 840ttgatcgtct ttttttcaaa tgcatttatc gcttctttaa acacggtctg aaaagagggc 900cttctacgga aggagtacca gagtctatga gggaagaata tcgaaaggaa cagcagagtg 960ctgtggatgc tgacgatagt cattttgtca gcatagagct ggagtaaaaa actaccttgt 1020ttctact 1027<210>8<211>890<212>DNA<213>流感病毒A/Singapore/1/57/ca
<400>8agcaaaagca gggtgacaaa gacataatgg atcctaacac tgtgtcaagc tttcaggtag 60attgcttcct ttggcatgtc cgcaaacaag ttgcagacca agaactaggt gatgccccat 120tccttgatcg gcttcgccga gatcagaagt ccctaagggg aagaggcagc actctcggtc 180tgaacatcga aacagccacc cgtgttggaa agcagatagt ggagaggatt ctgaaggaag 240aatccgatga ggcacttaaa atgaccatgg cctccgcacc tgcttcgcga tacctaactg 300acatgactat tgaggaaatg tcaagggact ggttcatgct aatgcccaag cagaaagtgt 360caggccctct ttgtatcaga atggaccagg caatcatgga taagaacatc atattgaaag 420cgaatttcag tgtgattttt gaccggctag agaccctaat attactaagg gctttcaccg 480aagagggagc aattgttggc gaaatttcac cattgccttc tcttccagga catactaatg 540aggatgtcaa aaatgcaatt ggggtcctca tcggaggact tgaatggaat gataacacag 600ttcgagtctc taaaactcta cagagattcg cttggagaaa cagtaatgag aatgggagac 660ctccactcac tccaaaacag aaacggaaaa tggcgagaac aattaggtca aaagttcgaa 720gaaataagat ggctgattga agaagtgaga cacaaattga agataacaga gaatagtttt 780gagcaaataa catttatgca agccttacag ctgctatttg aagtggaaca agagataaga 840actttctcgt ttcagcttat ttaatgataa aaaacaccct tgtttctact890<210>9<211>771<212>PRT<213>流感病毒A/Singapore/1/57/ca<400>9Met Glu Arg Ile Lys Glu Leu Arg Asn Leu Met Ser Gln Ser Arg Thr1 5 10 15Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys20 25 30Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ser Leu Arg Met Lys35 40 45Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Thr50 55 60Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys65 70 75 80Met Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val85 90 95Thr Trp Trp Asn Arg Asn Gly Pro Met Thr Ser Thr Val His Tyr Pro100 105 110Lys Ile Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly115 120 125Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg
130 135 140Val Asp Ile Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln145 150 155 160Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile165 170 175Leu Thr Ser Glu Ser Gln Leu Thr Thr Thr Lys Glu Lys Lys Glu Glu180 185 190Leu Gln Asp Cys Lys Ile Ser Pro Leu Met Val Ala Tyr Met Leu Glu195 200 205Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr210 215 220Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp225 230 235 240Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Val Asp245 250 255Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Ala Val260 265 270Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln275 280 285Ile Gly Gly Thr Arg Met Val Asp Ile Leu Arg Gln Asn Pro Thr Glu290 295 300Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser305 310 315 320Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser325 330 335Ser Val Lys Ile Glu Glu Glu Val Leu Thr Gly Asn Leu Gln Thr Leu340 345 350Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Lys355 360 365Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu370 375 380Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val385 390 395 400
Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly405 410 415Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His420 425 430Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn435 440 445Trp Gly Ile Glu His Ile Asp Asn Val Met Gly Met Ile Gly Val Leu450 455 460Pro Asp Met Thr Pro Ser Thr Glu Met Ser Met Arg Gly Val Arg Val465 470 475 480Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Ala Glu Arg Val Val Val485 490 495Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Val Leu500 505 510Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Thr515 520 525Ile Thr Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser530 535 540Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Thr Val545 550 555 560Lys Ile Gln Trp Ser Gln Asn Pro Thr Met Leu Tyr Asn Lys Met Glu565 570 575Phe Glu Pro Phe Gln Ser Leu Val Pro Lys Ala Ile Arg Gly Gln Tyr580 585 590Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly595 600 605Thr Phe Asp Thr Thr Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala610 615 620Pro Pro Lys Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val625 630 635 640Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe645 650 655
Asn Tyr Asn Lys Thr Thr Lys Arg Leu Thr Ile Leu Gly Lys Asp Ala660 665 670Gly Thr Leu Thr Glu Asp Pro Asp Glu Gly Thr Ser Gly Val Glu Ser675 680 685Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asp Arg Arg Tyr690 695 700Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Asn Leu Ala Lys Gly Glu705 710 715 720Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys725 730 735Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys740 745 750Arg Ile Arg Met Ala Ile Asn Xaa Cys Xaa Ile Val Xaa Lys Arg Pro755 760 765Cys Phe Tyr770<210>10<211>757<212>PRT<213>流感病毒A/Singapore/1/57/ca<400>10Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn1 5 10 15Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His20 25 30Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln35 40 45Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Thr Gly Ala Pro50 55 60Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser65 70 75 80Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu85 90 95
Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu100 105 110Val Ile Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr115 120 125Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala130 135 140Asn Thr Ile Glu Val Phe Arg Ser Asn Gly Leu Thr Ala Asn Glu Ser145 150 155 160Gly Arg Leu Ile Asp Phe Leu Lys Asp Val Ile Glu Ser Met Asp Lys165 170 175Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg180 185 190Asp Asn Met Thr Lys Lys Met Val Thr Gln Arg Thr Ile Gly Lys Lys195 200 205Lys Gln Arg Leu Asn Lys Arg Ser Tyr Leu Ile Arg Ala Leu Thr Leu210 215 220Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala225 230 235 240Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu245 250 255Thr Leu Ala Arg Ser Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro260 265 270Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys275 280 285Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly290 295 300Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala305 310 315 320Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val325 330 335Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly340 345 350Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile
355 360 365Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Glu Ser370 375 380Thr Arg Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Ile Asp Gly Thr385 390 395 400Val Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser405 4l0 415Thr Val Ile Gly Val Ser Ile Leu Asn Leu Gly Gln Lys Lys Tyr Thr420 425 430Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala435 440 445Leu Ile Val Asn Ala Pro Asn His Glu Gly Ile Gln Ala Gly Val Asp450 455 460Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys465 470 475 480Lys Ser Tyr Ile Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe485 490 495Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe500 505 510Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr515 520 525Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala530 535 540Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg545 550 555 560Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu565 570 575Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Ala Gly Leu Leu Val Ser580 585 590Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu595 600 605Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Gln Gly Arg Leu6l0 615 620
Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Val625 630 635 640Asn Asn Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu645 650 655Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg660 665 670Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu Gln Met675 680 685Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser690 695 700Tyr Arg Arg Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser705 710 715 720Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys725 730 735Lys Glu Glu Phe Ala Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu740 745 750Leu Arg Arg Gln Lys755<210>11<211>716<212>PRT<213>流感病毒A/Singapore/1/57/ca<400>11Met Glu Asp Phe Val Arg Gln Cys Phe Asn Pro Met Ile Val Glu Leu1 5 10 15Ala Glu Arg Ala Met Lys Glu Tyr Gly Glu Asp Leu Lys Ile Glu Thr20 25 30Asn Lys Phe Ala Ala Ile Cys Thr His Leu Glu Val Cys Phe Met Tyr35 40 45Ser Asp Phe His Phe Ile Asn Glu Gln Gly Glu Ser Ile Ile Val Glu50 55 60Leu Asp Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu Ile Ile Glu65 70 75 80
Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser Ile Cys Asn85 90 95Thr Thr Gly Ala Glu Lys Pro Lys Phe Leu Pro Asp Leu Tyr Asp Tyr100 105 110Lys Glu Asn Arg Phe Ile Glu Ile Gly Val Thr Arg Arg Glu Val His115 120 125Ile Tyr Tyr Leu Glu Lys Ala Asn Lys Ile Lys Ser Glu Lys Thr His130 135 140Ile His Ile Phe Ser Phe Thr Gly Glu Glu Met Ala Thr Lys Ala Asp145 150 155 160Tyr Thr Leu Asp Glu Glu Ser Arg Ala Arg Ile Lys Thr Arg Leu Phe165 170 175Thr Ile Arg Gln Glu Met Ala Ser Arg Gly Leu Trp Asp Ser Phe Arg180 185 190Gln Ser Glu Arg GIy Glu Glu Thr Ile Glu Glu Arg Phe Glu Ile Thr195 200 205Gly Thr Met Arg Arg Leu Ala Asp Gln Ser Leu Pro Pro Asn Phe Ser210 215 220Cys Leu Glu Ile Phe Arg Ala Tyr Val Asp Gly Phe Glu Pro Asn Gly225 230 235 240Tyr Ile Glu Gly Lys Leu Ser Gln Met Ser Lys Glu Val Asn Ala Lys245 250 255Ile Glu Pro Phe Leu Lys Thr Thr Pro Arg Pro Ile Arg Leu Pro Asp260 265 270Gly Pro Pro Cys Ser Gln Arg Ser Lys Phe Leu Leu Met Asp Ala Leu275 280 285Lys Leu Ser Ile Glu Asp Pro Ser His Glu Gly Glu Gly Ile Pro Leu290 295 300Tyr Asp Ala Ile Lys Cys Met Arg Thr Phe Phe Gly Trp Lys Glu Pro305 310 315 320Tyr Val Val Lys Pro His Glu Lys Gly Ile Asn Pro Asn Tyr Leu Leu325 330 335
Ser Trp Lys Gln Val Leu Ala Glu Leu Gln Asp Ile Glu Asn Glu Glu340 345 350Lys Ile Pro Arg Thr Lys Asn Met Lys Lys Thr Ser Gln Leu Lys Trp355 360 365Ala Leu Gly Glu Asn Met Ala Pro Glu Lys Val Asp Phe Asp Asp Cys370 375 380Arg Asp Ile Ser Asp Leu Lys Gln Tyr Asp Ser Asp Glu Pro Glu Leu385 390 395 400Arg Ser Leu Ser Ser Trp Ile Gln Asn Glu Phe Asn Lys Ala Cys Glu405 410 415Leu Thr Asn Ser Ile Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Val420 425 430Ala Pro Ile Glu His Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala435 440 445Glu Val Ser His Cys Arg Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr450 455 460Ile Asn Thr Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp Asp Phe465 470 475 480Gln Leu Ile Pro Met Ile Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg485 490 495Lys Thr Asn Leu Tyr Gly Phe Ile Val Lys Gly Arg Ser His Leu Arg500 505 510Asn Asp Thr Asp Val Val Asn Phe Val Ser Met Glu Phe Ser Leu Thr515 520 525Asp Pro Arg Leu Glu Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu530 535 540Ile Gly Asp Met Leu Leu Arg Ser Ala Ile Gly Gln Val Ser Arg Pro545 550 555 560Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys565 570 575Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile580 585 590Glu Ser Met Ile Glu Ala Gln Ser Ser Val Lys Glu Lys Asp Met Thr
595 600 605Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser610 615 620Pro Lys Gly Val Glu Glu Gly Ser Ile Gly Lys Val Cys Arg Thr Leu625 630 635 640Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu645 650 655Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu Val Val Gln Ala Leu660 665 670Arg Asp Asn Leu Glu Pro Gly Thr Phe Asp Leu Gly Gly Leu Tyr Glu675 680 685Ala Ile Glu Glu Cys Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala690 695 700Ser Trp Phe Asn Ser Phe Leu Thr His Ala Leu Arg705 710 715<210>12<211>562<212>PRT<213>流感病毒A/Singapore/1/57/ca<400>12Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly Asp1 5 l0 15Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Lys Val Asp20 25 30Thr Ile Leu Glu Gln Asn Val Thr Val Thr His Ala Lys Asp Ile Leu35 40 45Glu Lys Thr His Asn Gly Lys Leu Cys Lys Leu Asn Gly Ile Pro Pro50 55 60Leu Glu Leu Gly Asp Cys Ser Ile Ala Gly Trp Leu Leu Gly Asn Pro65 70 75 80Glu Cys Asp Arg Leu Leu Ser Val Pro Glu Trp Ser Tyr Ile Met Glu85 90 95Lys Glu Asn Pro Arg Asp Gly Leu Cys Tyr Pro Gly Ser Phe Asn Asp
100 105 110Tyr Glu Glu Leu Lys His Leu Leu Ser Ser Val Lys His Phe Glu Lys115 120 125Val Lys Ile Leu Pro Lys Asp Arg Trp Thr Gln His Thr Thr Thr Gly130 135 140Gly Ser Arg Ala Cys Ala Val Ser Gly Asn Pro Ser Phe Phe Arg Asn145 150 155 160Met Val Trp Leu Thr Lys Lys Glu Ser Asn Tyr Pro Val Ala Lys Gly165 170 175Ser Tyr Asn Asn Thr Ser Gly Glu Gln Met Leu Ile Ile Trp Gly Val180 185 190His His Pro Asn Asp Glu Thr Glu Gln Arg Thr Leu Tyr Gln Asn Val195 200 205Gly Thr Tyr Val Ser Val Gly Thr Ser Thr Leu Asn Lys Arg Ser Thr210 215 220Pro Asp Ile Ala Thr Arg Pro Lys Val Asn Gly Leu Gly Ser Arg Met225 230 235 240Glu Phe Ser Trp Thr Leu Leu Asp Met Trp Asp Thr Ile Asn Phe Glu245 250 255Ser Thr Gly Asn Leu Ile Ala Pro Glu Tyr Gly Phe Lys Ile Ser Lys260 265 270Arg Gly Asn Ser Gly Ile Met Lys Thr Glu Gly Thr Leu Glu Asn Cys275 280 285Glu Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Thr Thr Leu Pro290 295 300Phe His Asn Val His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val305 310 315 320Lys Ser Glu Lys Leu Val Leu Ala Thr Gly Pro Arg Asn Val Pro Gln325 330 335Ile Glu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly340 345 350Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn355 360 365
Asp Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala370 375 380Phe Asp Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn385 390 395 400Thr Gln Phe Glu Ala Val Gly Lys Glu Phe Ser Asn Leu Glu Arg Arg405 410 415Leu Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp420 425 430Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu435 440 445Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Met450 455 460Gln Leu Arg Asp Asn Val Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe465 470 475 480Tyr His Lys Cys Asp Asp Glu Cys Met Asn Ser Val Lys Asn Gly Thr485 490 495Tyr Asp Tyr Pro Lys Tyr Glu Glu Glu Ser Lys Leu Asn Arg Asn Glu500 505 510Ile Lys Gly Val Lys Leu Ser Ser Met Gly Val Tyr Gln Ile Leu Ala515 520 525Ile Tyr Ala Thr Val Ala Gly Ser Leu Ser Leu Ala Ile Met Met Ala530 535 540Gly Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile545 550 555 560Cys Ile<210>13<21l>506<212>PRT<213>流感病毒A/Singapore/1/57/ca<400>13Met Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Asp1 5 10 15
Gly Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Lys Met20 25 30Ile Asp Gly Ile Gly Arg Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys35 40 45Leu Ser Asp Tyr Glu Gly Arg Leu Ile Gln Asn Ser Leu Thr Ile Glu50 55 60Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu65 70 75 80Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile85 90 95Tyr Lys Arg Val Asn Gly Lys Trp Met Arg Glu Leu Val Leu Tyr Asp100 105 110Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln Ala Asn Asn Gly Asp Asp115 120 125Ala Thr Ala Gly Leu Thr His Met Met Ile Trp His Ser Asn Leu Asn130 135 140Asp Thr Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp145150 155 160Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser165 170 175Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu180 185 190Leu Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg195 200 205Gly Glu Asn Gly Arg Lys Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn210 215 220Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp225 230 235 240Gln Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu245 250 255Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His260 265 270
Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Thr Ala Val Ala Ser Gly275 280 285Tyr Asp Phe Glu Lys Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe290 295 300Lys Leu Leu Gln Asn Ser Gln Val Tyr Ser Leu Ile Arg Pro Asn Glu305 310 315 320Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys Asn Ser Ala325 330 335Ala Phe Glu Asp Leu Arg Val Ser Ser Phe Ile Arg Gly Thr Lys Val340 345 350Ile Pro Arg Gly Lys Leu Ser Thr Arg Gly Val Gln Ile Ala Ser Asn355 360 365Glu Asn Met Asp Thr Met Glu Ser Ser Thr Leu Glu Leu Arg Ser Arg370 375 380Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Asn Gln Gln Arg385 390 395 400Ala Ser Ala Gly Gln Ile Ser Val Gln Pro Thr Phe Ser Val Gln Arg405 410 415Asn Leu Pro Phe Asp Lys Thr Thr Ile Met Ala Ala Phe Thr Gly Asn420 425 430Ala Glu Gly Arg Thr Ser Asp Met Arg Ala Glu Ile Ile Arg Met Met435 440 445Glu Gly Ala Lys Pro Glu Glu Val Ser Phe Gln Gly Arg Gly Val Phe450 455 460Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp465 470 475 480Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr485 490 495Asp Asn Xaa Gly Lys Ile Pro Leu Phe Leu500 505<210>14<211>469<212>PRT
<213>流感病毒A/Singapore/1/57/ca<400>14Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Ser Leu Thr1 5 10 15Ile Ala Thr Val Cys Phe Leu Met Gln Ile Ala Ile Leu Ala Thr Thr20 25 30Val Thr Leu His Phe Lys Gln His Glu Cys Asp Ser Pro Ala Ser Asn35 40 45Gln Val Met Pro Cys Glu Pro Ile Ile Ile Glu Arg Asn Ile Thr Glu50 55 60Ile Val Tyr Leu Asn Asn Thr Thr Ile Glu Lys Glu Ile Cys Pro Glu65 70 75 80Val Val Glu Tyr Arg Asn Trp Ser Lys Pro Gln Cys Gln Ile Thr Gly85 90 95Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly100 105 110Asp Ile Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Gly Lys115 120 125Cys Tyr Gln Phe Ala Leu Gly Gln Gly Thr Thr Leu Tyr Asn Lys His130 135 140Ser Asn Gly Thr Ile His Asp Arg Ile Pro His Arg Thr Leu Leu Met145 150 155 160Asn Glu Leu Gly Val Pro Phe His Leu Gly Thr Lys Gln Val Cys Val165 170 175Ala Trp Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val180 185 190Cys Val Thr Gly Asp Asp Arg Asn Ala Thr Ala Ser Phe Ile Tyr Asp195 200 205Gly Arg Leu Val Asp Ser Ile Gly Ser Trp Ser Gln Asn Ile Leu Arg210 215 220Thr Gln Glu Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val225 230 235 240Met Thr Asp Gly Ser Ala Ser Gly Arg Ala Asp Thr Arg Ile Leu Phe
245 250 255Ile Lys Glu Gly Lys Ile Val Arg Ile Ser Pro Leu Ser Gly Ser Ala260 265 270Gln His Ile Glu Glu Cys Ser Gys Tyr Pro Arg Tyr Pro Asp Val Arg275 280 285Cys Ile Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Val Ile Asp290 295 300Ile Asn Met Glu Asp Tyr Ser Ile Asp Ser Ser Tyr Val Cys Ser Gly305 310 315 320Leu Val Gly Asp Thr Pro Arg Asn Asp Asp Ser Ser Ser Asn Ser Asn325 330 335Cys Arg Asp Pro Asn Asn Glu Arg Gly Asn Pro Gly Val Lys Gly Trp340 345 350Ala Phe Asp Asn Gly Asp Asp Val Trp Met Gly Arg Thr Ile Asn Lys355 360 365Asp Ser Arg Ser Gly Tyr Glu Thr Phe Lys Val Ile Gly Gly Trp Ser370 375 380Thr Pro Asn Ser Lys Ser Gln Val Asn Arg Gln Val Ile Val Asp Asn385 390 395 400Asn Asn Trp Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Ser405 410 415Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro Gln420 425 430Glu Thr Arg Val Trp Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly435 440 445Thr Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asn Ile450 455 460Asn Phe Met Pro Ile465<210>15<211>252<212>PRT<213>流感病毒A/Singapore/1/57/ca
<400>15Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Val Pro1 5 10 15Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe20 25 30Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr35 40 45Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe50 55 60Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val65 70 75 80Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala85 90 95Val Lys Leu Tyr Lys Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala100 105 110Lys Glu Ile Ala Leu Ser Tyr Ser Ala Gly Ala Leu Ala Ser Cys Met115 120 125Gly Leu Ile Tyr Asn Arg Met Gly Ala Val Thr Thr Glu Val Ala Phe130 135 140Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser His His Arg145 150 155 160Ser His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu165 170 175Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met180 185 190Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Gln195 200 205Ala Arg Gln Met Val Gln Ala Met Arg Ala Ile Gly Thr His Pro Ser210 215 220Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr225 230 235 240Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys245 250
<210>16<211>97<212>PRT<213>流感病毒A/Singapore/1/57/ca<400>16Met Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly1 5 10 15Cys Arg Cys Asn Asp Ser Ser Asp Pro Leu Val Val Ala Ala Ser Ile20 25 30Ile Gly Ile Leu His Leu Ile Leu Trp Ile Leu Asp Arg Leu Phe Phe35 40 45Lys Cys Ile Tyr Arg Phe Phe Lys His Gly Leu Lys Arg Gly Pro Ser50 55 60Thr Glu Gly Val Pro Glu Ser Met Arg Glu Glu Tyr Arg Lys Glu Gln65 70 75 80Gln Ser Ala Val Asp Ala Asp Asp Ser His Phe Val Ser Ile Glu Leu85 90 95Glu<210>17<211>237<212>PRT<213>流感病毒A/Singapore/1/57/ca<400>17Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp1 5 10 15His Val Arg Lys Gln Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe20 25 30Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser35 40 45Thr Leu Gly Leu Asn Ile Glu Thr Ala Thr Arg Val Gly Lys Gln Ile50 55 60Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr
65 70 75 80Met Ala Ser Ala Pro Ala Ser Arg Tyr Leu Thr Asp Met Thr Ile Glu85 90 95Glu Met Ser Arg Asp Trp Phe Met Leu Met Pro Lys Gln Lys Val Ser100 105 110Gly Pro Leu Cys Ile Arg Met Asp Gln Ala Ile Met Asp Lys Asn Ile115 120 125Ile Leu Lys Ala Asn Phe Ser Val Ile Phe Asp Arg Leu Glu Thr Leu130 135 140Ile Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Ile Val Gly Glu Ile145 150 155 160Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn165 170 175Ala Ile Gly Val Leu Ile Gly Gly Leu Glu Trp Asn Asp Asn Thr Val180 185 190Arg Val Ser Lys Thr Leu Gln Arg Phe Ala Trp Arg Asn Ser Asn Glu195 200 205Asn Gly Arg Pro Pro Leu Thr Pro Lys Gln Lys Arg Lys Met Ala Arg210 215 220Thr Ile Arg Ser Lys Val Arg Arg Asn Lys Met Ala Asp225 230 235<210>18<211>121<212>PRT<213>流感病毒A/Singapore/1/57/ca<400>18Met Asp Pro Asn Thr Val Ser Ser Phe Gln Asp Ile Leu Met Arg Met1 5 10 15Ser Lys Met Gln Leu Gly Ser Ser Ser Glu Asp Leu Asn Gly Met Ile20 25 30Thr Gln Phe Glu Ser Leu Lys Leu Tyr Arg Asp Ser Leu Gly Glu Thr35 40 45Val Met Arg Met Gly Asp Leu His Ser Leu Gln Asn Arg Asn Gly Lys
50 55 60Trp Arg Glu Gln Leu Gly Gln Lys Phe Glu Glu Ile Arg Trp Leu Ile65 70 75 80Glu Glu Val Arg His Lys Leu Lys Ile Thr Glu Asn Ser Phe Glu Gln85 90 95Ile Thr Phe Met Gln Ala Leu Gln Leu Leu Phe Glu Val Glu Gln Glu100 105 110Ile Arg Thr Phe Ser Phe Gln Leu Ile115 120<210>19<211>2396<212>DNA<213>流感B/Vienna/1/99/ca<400>19agcagaagcg gagcgttttc aagatgacat tggctaaaat tgaattgtta aaacaactgt 60taagggacaa tgaagccaaa acagtattga aacaaacaac agtagatcaa tataacataa 120taagaaaatt caatacatca agaattgaaa agaacccttc attaaggatg aagtgggcaa 180tgtgttctaa ttttcccttg gctttgacca agggtgacat ggcaaacaga atccccttgg 240aatacaaggg aatacaactt aaaacaaatg ctgaagacat aggaaccaaa ggccaaatgt 300gctcaatagc agcagttacc tggtggaata catatggacc aataggagat actgaaggtt 360tcgaaaaagt ctacgaaagc ttttttctca gaaagatgag acttgacaat gccacttggg 420gccgaataac ttttggccca gttgaaagag taagaaaaag ggtactgcta aaccctctca 480ccaaggaaat gcctccagat gaagcaagta atgtgataat ggaaatattg ttccctaagg 540aagcaggaat accaagagaa tctacttgga tacataggga actgataaaa gaaaaaagag 600aaaaattgaa aggaacgatg ataactccca ttgtactggc atacatgctt gagagggaat 660tggttgccag gagaaggttc ctgccggtag caggagcaac atcagctgag ttcatagaaa 720tgctacactg cttacaaggt gaaaattgga gacaaatata tcacccggga gggaataaac 780taactgaatc taggtctcaa tcgatgattg tggcttgtag aaagataatc agaagatcaa 840tagtcgcatc aaacccattg gagctagctg tagaaattgc aaacaagact gtaatagata 900ctgaaccttt aaaatcatgt ctgacagcca tagacggagg tgatgtcgcc tgtgacataa 960taagggctgc attaggacta aagatcagac aaagacaaag atttggacga cttgaactaa 1020agagaatatc aggaagagga ttcaaaaatg atgaagaaat attaatcggg aacggaacaa 1080tacagaagat tggaatatgg gacggagaag aggagttcca tgtaagatgt ggtgaatgca 1140ggggaatatt aaaaaagagc aaaatgagaa tggaaaaact actaataaat tcagctaaaa 1200aggaagacat gaaagattta ataatcttgt gcatggtatt ttctcaagac actaggatgt 1260tccaaggagt gaggggagaa ataaattttc ttaatagagc aggccaactt ttatctccaa 1320tgtatcaact ccaaagatat tttttgaata gaagtaatga tctctttgat caatgggggt 1380atgaggaatc acccaaagca agtgagctac atgggataaa tgaattaatg aatgcatctg 1440actacacttt gaaaggggtt gtagtaacaa aaaatgtgat tgatgatttt agttctactg 1500aaacagaaaa agtatctata acaaaaaatc ttagtttaat aaaaaggact ggggaagtca 1560taatgggagc caatgacgta agtgaattag aatcacaagc tcagctaatg ataacatatg 1620atacacctaa gatgtgggag atgggaacaa ccaaagaact ggtgcaaaac acctatcaat 1680
gggtgctgaa aaatttggta acactgaagg ctcagtttct tctaggaaaa gaagacatgt 1740tccaatggga tgcatttgaa gcatttgaaa gcataatccc ccagaagatg gctggccaat 1800acagtggatt tgcaagagca gtgctcaaac aaatgagaga ccaagaggtc atgaaaactg 1860accagttcat aaagttgttg cccttttgtt tctcaccacc aaagttaagg agcaatgggg 1920agccttatca gttcttgagg cttgtattga agggaggagg agaaaatttc atcgaagtaa 1980ggaaagggtc tcctctattc tcttacaatc cacaaacaga agtcctaact atatgcggca 2040gaatgatgtc attaaaaggg aaaattgaag atgaagaaag gaatagatca atggggaatg 2100cagtgttggc gggttttctt gttagtggca agtatgaccc agatcttgga gatttcaaaa 2160ctattgaaga gcttgaaaag ctaaaaccgg gggagaaagc aaacatctta ctttatcaag 2220gaaagcccgt taaagtagtt aaaaggaaaa gatatagtgc tttatccaat gacatttcac 2280aaggaattaa gagacaaaga atgacagttg agtccatggg gtgggccttg agctaatata 2340aatttatcca ttaattcaat gaatgcaatt gagtgaaaaa tgctcgtgtt tctcat 2396<210>20<211>2369<212>DNA<213>流感B/Vienna/1/99/ca<400>20agcagaagcg gagcctttaa gatgaatata aatccttatt ttctcttcat agatgtaccc 60atacaggcag caatttcaac aacattccca tacaccggtg ttccccctta ttcccatgga 120acgggaacag gccacacaat agacaccgtg atcagaacac atgagtactc gaacaaagga 180aaacagtatg tttctgacat cacaggatgt acaatggtag atccaacaaa tggaccatta 240cccgaagaca atgagccaag tgcctatgca caattagatt gcgttctgga ggctttggat 300agaatggatg aggaacatcc aggtctgttt caagcagcct cacagaatgc catggaggca 360ctaatggtca caactgtaga caaattaacc caggggagac agactttcga ttggacagta 420tgcagaaatc agcctgctgc aacggcacta aacacaacaa taacctcctt taggttgaat 480gatttgaatg gagctgacaa gggtggattg gtaccctttt gccaagatat cattgattca 540ttagacaagc ctgaaatgac tttcttctca gtaaagaata taaagaaaaa attccctgct 600aaaaacagaa agggtttcct cataaagaga ataccaatga aagtaaaaga caggatatcc 660agagtggaat acatcaaaag agcattgtca ttaaacacaa tgacaaaaga tgctgaaagg 720ggcaaactaa aaagaagagc gattgcaacc gctggaatac aaatcagagg gtttgtatta 780gtagttgaaa acttggctaa aaatatctgt gaaaatctag aacaaagtgg tttgcccgta 840ggtggaaatg aaaagaaggc caaactgtca aatgcagtgg ccaaaatgct cagtaactgc 900ccaccaggag ggatcagcat gacagtaaca ggagacaata ctaaatggaa tgaatgctta 960aatccaagag tctttttggc tatgactgaa agaataacca gagacagccc aatttggttc 1020cgggattttt gtagtatagc accggtcttg ttctccaata aaatagccag attgggaaaa 1080ggatttatga taacaagtaa aacaaaaaga ctgaaggctc aaataccttg tcctgatctg 1140tttagcatac cattagaaag atataatgaa gaaacaaggg caaaattaaa aaagctgaaa 1200ccattcttca atgaagaagg aacggcatct ttgtcgcctg gaatgatgat gggaatgttt 1260aatatgctat ctaccgtgtt gggagtagca gcactaggta tcaaaaacat tggaaacaag 1320gaatacttat gggatggact gcaatcttct gatgattttg ctctgtttgt taatgcaaaa 1380gatgaagaga catgtatgga aggaataaac gatttttacc gaacatgtaa attattggga 1440ataaacatga gcaaaaagaa aagttactgt aacgaaactg gaatgtttga atttacaagc 1500atgttctata gagatggatt tgtatctaac tttgcaatgg aaattccttc atttggagtt 1560gctggagtaa atgaatcagc agatatggca ataggaatga caataataaa gaacaatatg 1620attaacaatg ggatgggtcc agcaacagca caaacagcca tacaattgtt catagctgat 1680tataggtaca cctacaaatg ccacagagga gattccaaag tggaaggaaa aagaatgaaa 1740attataaagg agctatggga aaacactaaa ggaagagatg gtctgttagt ggcagatggt 1800
gggcccaaca tttacaattt gagaaactta catatcccag aaatagtatt gaagtacaat 1860ctaatggacc ctgaatacaa agggcggtta cttcaccctc aaaatccctt tgtaggacat 1920ttgtctattg aaggcatcaa agaagcagat ataaccccag cacatggtcc tgtgaagaaa 1980atggattatg atgcagtgtc tggaactcat agttggagaa ccaaaaggaa cagatctata 2040ctaaatactg atcagaggaa catgattctt gaggaacaat gctacgctaa atgttgcaac 2100ctttttgagg cctgttttaa cagtgcatca tacaggaaac cagtagggca gcacagcatg 2160cttgaggcta tggcccatag attaagaatg gatgcacgac tggattatga atcaggaaga 2220atgtcaaagg atgattttga gaaagcaatg gctcaccttg gtgagattgg gtacacataa 2280gctccgaaga tgtccatggg gttattggtc atcattggat acatgtgata aacaaatgat 2340taaaatgaaa aaaggctcgt gtttctact 2369<210>21<211>2305<212>DNA<213>流感B/Vienna/1/99/ca<400>21agcagaagcg gtgcgtttga tttgtcataa tggatacttt tattacaaga aacttccaga 60ctacaataat acaaaaggcc aaaaacacaa tggcagaatt tagtgaagat cctgaattac 120aaccagcaat gctattcaac atctgcgtcc atctagaggt ttgctatgta ataagtgaca 180tgaattttct tgacgaagaa ggaaaagcat atacagcatt agaaggacaa gggaaagaac 240aaaatttgag accacaatat gaagtaattg agggaatgcc aagaaccata gcatggatgg 300tccaaagatc cttagctcaa gagcatggaa tagagactcc caagtatctg gctgatttgt 360ttgattataa aaccaagaga tttatagaag ttggaataac aaaaggattg gctgatgatt 420acttttggaa aaagaaagaa aagctgggaa atagcatgga actgatgata ttcagctaca 480atcaagacta ttcgttaagt aatgaatcct cattggatga ggaagggaaa gggagagtgc 540taagcagact cacagaactt caggctgaat taagtctgaa aaacctatgg caagttctca 600taggagaaga agatgttgaa aagggaattg actttaaact tggacaaaca atatctagac 660taagggatat atctgttcca gctggtttct ccaattttga aggaatgagg agctacatag 720acaatataga cccgaaagga gcaatagaga gaaatctagc aaggatgtct cccttagtat 780cagtcacacc taaaaagttg aaatgggagg acctaagacc aatagggcct cacatttaca 840accatgagct accagaagtt ccatataatg cctttcttct aatgtctgat gaactggggc 900tggccaatat gactgaggga aagtccaaaa aaccgaagac attagccaaa gaatgtctag 960aaaagtactc aacactacgg gatcaaactg acccaatatt aataatgaaa agcgaaaaag 1020ctaacgaaaa tttcctatgg aagctttgga gagactgtgt aaatacaata agtaatgagg 1080aaatgagtaa cgagttacag aaaaccaatt atgccaagtg ggccacaggg gatggattaa 1140cataccagaa aataatgaaa gaagtagcaa tagatgacga aacaatgtgc caagaagagc 1200ctaaaatccc taacaaatgt agagtggctg cttgggttca aacagagatg aatctattga 1260gcactctgac aagtaaaaaa gctctggacc taccagaaat agggccagac gtagcacccg 1320tggagcatgt agggagtgaa agaaggaaat actttgttaa tgaaatcaac tactgtaagg 1380cctctacagt tatgatgaag tatgtgcttt ttcacacttc attgttgaat gaaagcaatg 1440ccagcatggg aaaatacaaa gtaataccaa taaccaatag agtagtaaat gaaaaaggag 1500aaagtttcga catgctctat ggtctggcgg ttaaaggaca atctcatctg aggggagata 1560ctgatgttgt aacagttgta actttcgaat ttagtagtac agaccccaga gtggactcag 1620gaaagtggcc aaaatatact gtgtttagga ttggctccct atttgtgagt gggagggaaa 1680agtctgtgta cctatattgc cgagtgaatg gcacaaataa gatccaaatg aaatggggaa 1740tggaagctag aagatgtctg cttcaatcaa tgcaacaaat ggaagcaatt gttgaacaag 1800aatcatcgat acaaggatat gacatgacca aagcttgttt caagggagac agagtaaata 1860
gccccaaaac tttcagtatt ggaactcaag aaggaaaact agtaaaagga tcctttggaa 1920aagcactaag agtaatattt actaaatgtt tgatgcacta tgtatttgga aatgcccaat 1980tggaggggtt tagtgccgag tctaggagac ttctactgtt gattcaagca ttaaaggaca 2040gaaagggccc ttgggtgttc gacttagagg gaatgtattc tggaatagaa gaatgtatta 2100gtaacaaccc ttgggtaata cagagtgcat actggttcaa tgaatggttg ggctttgaaa 2160aggaggggag taaagtatta gaatcagtag atgaaataat ggatgaataa aaggacatag 2220tactcaattt agtactattt tgttcattat gtatctaaac atccaataaa aaggacaaag 2280aattaaaaat gcacgtgttt ctact 2305<210>22<211>1882<212>DNA<213>流感B/Vienna/1/99/ca<400>22agcagaagca gagcattttc taatatccac aaaatgaagg caataattgt actactcatg 60gtagtaacat ccaatgcaga tcgaatctgc actgggataa catcgtcaaa ctcacctcat 120gtggtcaaaa cagctactca aggggaggtc aatgtgactg gtgcgatacc actgacaaca 180acaccaacaa aatctcattt tgcaaatctc aaaggaacaa agaccagagg gaaactatgc 240ccaacctgtc tcaactgcac agatctggat gtggccttgg gcagaccaat gtgtgtgggg 300atcacacctt cggcaaaagc ttcaatactc cacgaagtca gacctgttac atccggatgc 360tttcctataa tgcacgacag aacaaaaatc agacagctac ccaatcttct cagaggatat 420gaaaaaatca gattatcaac ccaaaacgtt atcaacacag aaaaggcacc aggaggaccc 480tacagacttg gaacttcagg atcttgccct aacgctacca gtaaaagcgg atttttcgca 540acaatggctt gggctgtccc aagggacaac aacaaaacag caacgaatcc actaacagta 600gaagtaccac acatctgtac aaaagaagaa gaccaaatta ctgtttgggg gttccattct 660gataacaaaa cccaaatgaa aaacctctat ggagactcaa atcctcaaaa gttcacctca 720tctgctaatg ggataaccac acattatgtt tctcagattg gcggcttccc ggaccaaaca 780gaagacggag ggctaccaca aagcggcaga attgttgttg attacatggt gcaaaaacct 840gggaaaacag gaacaattgt ctatcaaaga gggattttgt tgcctcaaaa ggtgtggtgc 900gcgagtggca ggagcaaagt aataaaaggg tccttgcctt taattggtga agcagattgc 960cttcacgaaa aatacggtgg attaaacaaa agcaagcctt actacacagg agaacatgca 1020aaagccatag gaaattgccc aatatgggtg aaaacacctt tgaagcttgc caatggaacc 1080aaatatagac ctcctgcaaa actattaaag gaaaggggtt tcttcggagc tattgctggt 1140ttcttagaag gaggatggga aggaatgatt gcaggttggc acggatacac atctcacgga 1200gcacatggag tggcagtggc agcagacctt aagagtacgc aagaagccat aaacaagata 1260acaaaaaatc tcaattcttt gagtgagcta gaagtaaata accttcaaag actaagtggt 1320gccatggatg aactccataa cgaaatactc gagctggatg agaaagtgga tgatctcaga 1380gctgacacaa taagctcaca aatagaactt gcagtcttgc tttccaacga aggaataata 1440aacagtgaag atgagcatct attggcactt gagagaaaac taaagaaaat gctgggtccc 1500tctgctgtag acatagggaa tggatgcttc gaaaccaaac acaagtgcaa ccagacctgc 1560ttagacagga tagctgctgg cacctttaat gcagaagaat tttctcttcc cacttttgat 1620tcactgaaca ttactgctgc atctttaaat gatgatggat tggataacca tactatactg 1680ctctactact caactgctgc ttctagtttg gctgtaacat tgatgatagc tatttttatt 1740gtttatatga tctccagaga caatgtttct tgctccatct gtctataagg aaaattaagc 1800cctgtatttt cctttattgt ggtgcttgtt tgcttgttat cattacaaag aaacgttatt 1860gaaaaatgct cttgttacta ct 1882<210>23
<211>1844<212>DNA<213>流感B/Vienna/1/99/ca<400>23agcagaagca cagcattttc ttgtgaactt caagtaccag taaaagaact gaaaatcaaa 60atgtccaaca tggatattga cggtatcaac actgggacaa ttgacaaaac accggaagaa 120ataacttttg gaaccagtgg gacaaccaga ccaatcatca gaccagcaac ccttgcccca 180ccaagcaaca aacgaacccg taacccatcc ccggaaagag caaccacaag cagtgaagct 240gatgtcggga ggaaaaccca aaagaaacag accccgacag agataaagaa gagcgtctac 300aacatggtag tgaaactggg cgaattctac aaccagatga tggtcaaagc tggactcaac 360gatgacatgg agagaaacct aatccaaaat gcgcatgctg tggaaagaat tctattggct 420gccactgatg acaagaaaac tgaattccag aagaaaaaga ataccagaga tgtcaaagaa 480gggaaagaag aaatagatca caacaaaaca ggaggcacct tttacaagat ggtaagagat 540gataaaacca tctacttcag ccctataaga attacctttt taaaagaaga ggtgaaaaca 600atgtacaaaa ccaccatggg gagtgatggc ttcagtggac taaatcacat aatgattggg 660cattcacaga tgaatgatgt ctgtttccaa agatcaaagg cactaaaaag agttggactt 720gacccttcat taatcagtac ctttgcggga agcacaatcc ccagaagatc aggtgcaact 780ggtgttgcaa tcaaaggagg tggaacttta gtggctgaag ccattcgatt tataggaaga 840gcaatggcag acagagggct attgagagac atcaaagcca agactgccta tgaaaagatt 900cttctgaatc taaaaaacaa atgctctgcg ccccaacaaa aggctctagt tgatcaagtg 960atcggaagta gaaatccagg gattgcagac attgaagatc taaccctgct tgctcgtagt 1020atggtcgttg ttaggccctc tgtggcgagc aaagtggtgc ttcccataag catttacgcc 1080aaaatacctc aactagggtt caatgttgaa gagtactcta tggttgggta cgaagccatg 1140gctctttaca atatggcaac acctgtttcc atattaagaa tgggagatga tgcaaaagat 1200aagtcgcaat tattcttcat gtcttgcttc ggagctgcct atgaagacct gagagttttg 1260tctgcattaa caggcacaga attcaagcct agatcagcat taaaatgcaa gggtttccat 1320gttccagcaa aggaacaggt ggaaggaatg ggggcagctc tgatgtccat caagctccag 1380ttttgggctc caatgaccag atctgggggg aacgaagtag gtggagacgg agggtctggc 1440caaataagct gcagcccagt gtttgcagtg gaaagaccta ttgctctaag caagcaagct 1500gtaagaagaa tgctgtcaat gaatattgag ggacgtgatg cagatgtcaa aggaaatcta 1560ctcaagatga tgaatgactc aatggctaag aaaaccagtg gaaatgcttt cattgggaag 1620aaaatgtttc aaatatcaga caaaaacaaa accaatcccg ttgaaattcc aattaagcag 1680accatcccca atttcttctt tgggagggac acagcagagg attatgatga cctcgattat 1740taaagcaaca aaatagacac tatgactgtg attgtttcaa tacgtttgga atgtgggtgt 1800ttattcttat taaaataaat ataaaaaatg ctgttgtttc tact 1844<210>24<211>1557<212>DNA<213>流感B/Vienna/1/99/ca<400>24agcagaagca gagcatcttc tcaaaactga ggcaaatagg ccaaaaatga acaatgctac 60cttcaactat acaaacgtta accctattcc tcacatcagg gggagtgtta ttatcactat 120atgtgtcagc ttcactgtca tacttattat attcggatat attgctaaaa ttttcaccaa 180cagaaataac tgcaccaaca atgccattgg attgtgcaaa cgcatcaaat gttcaggctg 240tgaaccgttc tgcaacaaaa ggggtgacac ttcttctccc agaaccggag tggacatacc 300cgcgtttatc ttgcccgggc tcaacctttc agaaagcact cctaattagc cctcatagat 360
tcggagaaac caaaggaaac tcagctccct tgataataag ggaacctttt attgcttgtg 420gaccaaagga atgcaaacac tttgctctaa cccattatgc agcccaacca gggggatact 480acaatggaac aagagaagac agaaacaagc tgaggcatct aatttcagtc aaattgggca 540aaatcccaac agtagaaaac tccattttcc acatggcagc atggagcggg tccgcatgcc 600atgatggtaa agaatggaca tatatcggag ttgatggccc tgacagtaat gcattgctca 660aaataaaata tggagaagca tatactgaca cataccattc ctatgcaaac aacatcctaa 720gaacacaaga aagtgcctgc aattgcatcg ggggaaattg ttatcttatg ataactgatg 780gctcagcttc aggtattagt gaatgcagat ttcttaagat tcaagagggc cgaataataa 840aagaaatatt tccaacagga agagtagaac atactgaaga atgcacatgc ggatttgcca 900gcaataaaac catagaatgt gcctgtagag ataacagtta cacagcaaaa agaccctttg 960tcaaattaaa tgtggagact gatacagcag aaataagatt gatgtgcaca gagacttact 1020tggacacccc cagaccagat gatggaagca taacagggcc ttgtgaatct aatggggata 1080aagggagtgg aggcatcaag ggaggatttg ttcatcaaag aatggcatcc aagactggaa 1140ggtggtactc tcgaacaatg tctaaaacta aaaggatggg gatgggactg tatgtcaagt 1200atgatggaga cccatggact gacagtgatg cccttgctct tagtggagta atggtttcaa 1260tggaagaacc tggttggtac tcctttggct tcgaaataaa agataagaaa tgtgatgtcc 1320cctgtattgg gatagagatg gtacatgatg gtggaaagga gacttggcac tcagcagcaa 1380cagccattta ctgtttaatg ggctcaggac aactgctatg ggacactgtc acaggtgtta 1440atatggctct gtaatggagg aatggttgag tctgttctaa accctttgtt cctattttgt 1500ttgaacaatt gtccttactg aacttaattg tttctgaaaa atgctcttgt tactact1557<210>25<211>1190<212>DNA<213>流感B/Vienna/1/99/ca<400>25agcagaagca cgcactttct taagatgtcg ctgtttggag acacaattgc ctacctgctt 60tcattgacag aagatggaga aggcaaagca gaactagcag aaaaattaca ctgttggttc 120ggtgggaaag aatttgacct agactctgcc ttggaatgga taaaaaacaa aagatgctta 180actgatatac aaaaagcact aattggtgcc tctatctgct ttttaaaacc caaagaccag 240gaaagaaaaa gaagattcat cacagagccc ctatcaggaa tgggaacaac agcaacaaaa 300aagaaaggcc tgattctagc tgagagaaaa atgagaagat gtgtgagctt tcatgaagca 360tttgaaatag cagaaggcca tgaaagctca gcgctactat attgtctcat ggtcatgtac 420ctgaatcctg gaaattattc aatgcaagta aaactaggaa cgctctgtgc tttgtgcgag 480aaacaagcat cacattcaca cagggctcat agcagagcag cgagatcttc agtgcccgga 540gtgagacgag aaatgcagat ggtctcagct atgaacacag caaaaacaat gaatggaatg 600ggaaaaggag aagacgtcca aaaactggca gaagagctgc aaagcaacat tggagtactg 660agatctcttg gggcaagtca aaagaatggg gaaggaattg caaaggatgt aatggaagtg 720ctaaagcaga gctctatggg aaattcagct cttgtgaaga aatatctata atgctcgaac 780catttcagat tctttcaatt tgttctttta tcttatcagc tctccatttc gtggcttgga 840caatagggca tttgaatcaa ataaaaagag gagtaaacat gaaaatacga ataaaaagtc 900caaacaaaga gacaataaac agagaggtat caattttgag acacagttac caaaaagaaa 960tccaggccaa agaaacaatg aaggaagtac tctctgacaa catggaggta ttgggtgacc 1020acatagtaat tgaggggctt tctgccgaag agataataaa aatgggtgaa acagttttgg 1080agatagaaga attgcattaa attcaatttt tactgtattt cttactatgc atttaagcaa 1140attgtaatca atgtcagcaa ataaactgga aaaagtgcgt tgtttctact1190<210>26
<211>1097<212>DNA<213>流感B/Vienna/1/99/ca<400>26agcagaagca gagcatttgt ttagtcactg gcaaacagga aaaatggcga acaacataac 60cacaacacaa attgaggtgg gtccgggagc aaccaatgcc accataaact ttgaaacagg 120aattctggag tgctatgaaa ggctttcatg gcaaagagcc cttgactacc ctggtcaaga 180ccgcctaaac agactaaaga gaaaattaga gtcaagaata aagactcaca acaaaagtga 240gcctgaaagt aaaaggatgt ctcttgaaga gaggaaagca attggagtaa aaatgatgaa 300agtactccta tttatgaatc catctgctgg aattgaaggg tttgagccat actatatgaa 360aagttcctca aatagcaact gtccgaaata caattggacc gattaccctt caacaccagg 420gaggtgcctt gatgacatag aagaagaacc agaggatgtt gatggcccaa ctgaaatagt 480attaagggac atgaacaaca aagatgcaag gcaaaagata aaagaggaag taaacactca 540gaaagaaggg aagttccgtt tgacaataaa aagggatata cgtaatgtat tgtccttgag 600agtgttggta aacggaacat tcctcaaaca ccccaatgga tacaagtcct tatcaactct 660gcatagattg aatgcatatg accagagtgg aaggcttgtt gctaaacttg ttgctactga 720tgatcttaca gtggaggatg aagaagatgg ccatcggatc ctcaactcac tcttcgagcg 780tcttaatgaa ggacattcaa agccaattcg agcagctgaa actgcggtgg gagtcttatc 840ccaatttggt caagagcacc gattatcacc agaagaggga gacaattaaa ctggtcacag 900aagaacttta tcttttaagt aaaagaattg atgataacat attgttccac aaaacagtaa 960tagctaacag ctccataata gctgacatgg ttgtatcatt atcattatta gaaacattgt 1020atgaaatgaa ggatgtggtt gaagtgtaca gcaggcagtg cttgtgaatt taaaataaaa 1080atcctcttgt tactact1097<210>27<211>770<212>PRT<213>流感B/Vienna/1/99/ca<400>27Met Thr Leu Ala Lys Ile Glu Leu Leu Lys Gln Leu Leu Arg Asp Asn1 5 10 15Glu Ala Lys Thr Val Leu Lys Gln Thr Thr Val Asp Gln Tyr Asn Ile20 25 30Ile Arg Lys Phe Asn Thr Ser Arg Ile Glu Lys Asn Pro Ser Leu Arg35 40 45Met Lys Trp Ala Met Cys Ser Asn Phe Pro Leu Ala Leu Thr Lys Gly50 55 60Asp Met Ala Asn Arg Ile Pro Leu Glu Tyr Lys Gly Ile Gln Leu Lys65 70 75 80Thr Asn Ala Glu Asp Ile Gly Thr Lys Gly Gln Met Cys Ser Ile Ala85 90 95
Ala Val Thr Trp Trp Asn Thr Tyr Gly Pro Ile Gly Asp Thr Glu Gly100 105 110Phe Glu Lys Val Tyr Glu Ser Phe Phe Leu Arg Lys Met Arg Leu Asp115 120 125Asn Ala Thr Trp Gly Arg Ile Thr Phe Gly Pro Val Glu Arg Val Arg130 135 140Lys Arg Val Leu Leu Asn Pro Leu Thr Lys Glu Met Pro Pro Asp Glu145 150 155 160Ala Ser Asn Val Ile Met Glu Ile Leu Phe Pro Lys Glu Ala Gly Ile165 170 175Pro Arg Glu Ser Thr Trp Ile His Arg Glu Leu Ile Lys Glu Lys Arg180 185 190Glu Lys Leu Lys Gly Thr Met Ile Thr Pro Ile Val Leu Ala Tyr Met195 200 205Leu Glu Arg Glu Leu Val Ala Arg Arg Arg Phe Leu Pro Val Ala Gly210 215 220Ala Thr Ser Ala Glu Phe Ile Glu Met Leu His Cys Leu Gln Gly Glu225 230 235 240Asn Trp Arg Gln Ile Tyr His Pro Gly Gly Asn Lys Leu Thr Glu Ser245 250 255Arg Ser Gln Ser Met Ile Val Ala Cys Arg Lys Ile Ile Arg Arg Ser260 265 270Ile Val Ala Ser Asn Pro Leu Glu Leu Ala Val Glu Ile Ala Asn Lys275 280 285Thr Val Ile Asp Thr Glu Pro Leu Lys Ser Cys Leu Thr Ala Ile Asp290 295 300Gly Gly Asp Val Ala Cys Asp Ile Ile Arg Ala Ala Leu Gly Leu Lys305 310 315 320Ile Arg Gln Arg Gln Arg Phe Gly Arg Leu Glu Leu Lys Arg Ile Ser325 330 335Gly Arg Gly Phe Lys Asn Asp Glu Glu Ile Leu Ile Gly Asn Gly Thr340 345 350Ile Gln Lys Ile Gly Ile Trp Asp Gly Glu Glu Glu Phe His Val Arg
355 360 365Cys Gly Glu Cys Arg Gly Ile Leu Lys Lys Ser Lys Met Arg Met Glu370 375 380Lys Leu Leu Ile Asn Ser Ala Lys Lys Glu Asp Met Lys Asp Leu Ile385 390 395 400Ile Leu Cys Met Val Phe Ser Gln Asp Thr Arg Met Phe Gln Gly Val405 410 415Arg Gly Glu Ile Asn Phe Leu Asn Arg Ala Gly Gln Leu Leu Ser Pro420 425 430Met Tyr Gln Leu Gln Arg Tyr Phe Leu Asn Arg Ser Asn Asp Leu Phe435 440 445Asp Gln Trp Gly Tyr Glu Glu Ser Pro Lys Ala Ser Glu Leu His Gly450 455 460Ile Asn Glu Leu Met Asn Ala Ser Asp Tyr Thr Leu Lys Gly Val Val465 470 475 480Val Thr Lys Asn Val Ile Asp Asp Phe Ser Ser Thr Glu Thr Glu Lys485 490 495Val Ser Ile Thr Lys Asn Leu Ser Leu Ile Lys Arg Thr Gly Glu Val500 505 510Ile Met Gly Ala Asn Asp Val Ser Glu Leu Glu Ser Gln Ala Gln Leu515 520 525Met Ile Thr Tyr Asp Thr Pro Lys Met Trp Glu Met Gly Thr Thr Lys530 535 540Glu Leu Val Gln Asn Thr Tyr Gln Trp Val Leu Lys Asn Leu Val Thr545 550 555 560Leu Lys Ala Gln Phe Leu Leu Gly Lys Glu Asp Met Phe Gln Trp Asp565 570 575Ala Phe Glu Ala Phe Glu Ser Ile Ile Pro Gln Lys Met Ala Gly Gln580 585 590Tyr Ser Gly Phe Ala Arg Ala Val Leu Lys Gln Met Arg Asp Gln Glu595 600 605Val Met Lys Thr Asp Gln Phe Ile Lys Leu Leu Pro Phe Cys Phe Ser610 615 620
Pro Pro Lys Leu Arg Ser Asn Gly Glu Pro Tyr Gln Phe Leu Arg Leu625 630 635 640Val Leu Lys Gly Gly Gly Glu Asn Phe Ile Glu Val Arg Lys Gly Ser645 650 655Pro Leu Phe Ser Tyr Asn Pro Gln Thr Glu Val Leu Thr Ile Cys Gly660 665 670Arg Met Met Ser Leu Lys Gly Lys Ile Glu Asp Glu Glu Arg Asn Arg675 680 685Ser Met Gly Asn Ala Val Leu Ala Gly Phe Leu Val Ser Gly Lys Tyr690 695 700Asp Pro Asp Leu Gly Asp Phe Lys Thr Ile Glu Glu Leu Glu Lys Leu705 710 715 720Lys Pro Gly Glu Lys Ala Asn Ile Leu Leu Tyr Gln Gly Lys Pro Val725 730 735Lys Val Val Lys Arg Lys Arg Tyr Ser Ala Leu Ser Asn Asp Ila Ser740 745 750Gln Gly Ile Lys Arg Gln Arg Met Thr Val Glu Ser Met Gly Trp Ala755 760 765Leu Ser770<210>28<211>752<212>PRT<213>流感B/Vienna/1/99/ca<400>28Met Asn Ile Asn Pro Tyr Phe Leu Phe Ile Asp Val Pro Ile Gln Ala1 5 10 15Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Val Pro Pro Tyr Ser His20 25 30Gly Thr Gly Thr Gly His Thr Ile Asp Thr Val Ile Arg Thr His Glu35 40 45Tyr Ser Asn Lys Gly Lys Gln Tyr Val Ser Asp Ile Thr Gly Cys Thr50 55 60
Met Val Asp Pro Thr Asn Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser65 70 75 80Ala Tyr Ala Gln Leu Asp Cys Val Leu Glu Ala Leu Asp Arg Met Asp85 90 95Glu Glu His Pro Gly Leu Phe Gln Ala Ala Ser Gln Asn Ala Met Glu100 105 110Ala Leu Met Val Thr Thr Val Asp Lys Leu Thr Gln Gly Arg Gln Thr115 120 125Phe Asp Trp Thr Val Cys Arg Asn Gln Pro Ala Ala Thr Ala Leu Asn130 135 140Thr Thr Ile Thr Ser Phe Arg Leu Asn Asp Leu Asn Gly Ala Asp Lys145 150 155 160Gly Gly Leu Val Pro Phe Cys Gln Asp Ile Ile Asp Ser Leu Asp Lys165 170 175Pro Glu Met Thr Phe Phe Ser Val Lys Asn Ile Lys Lys Lys Phe Pro180 185 190Ala Lys Asn Arg Lys Gly Phe Leu Ile Lys Arg Ile Pro Met Lys Val195 200 205Lys Asp Arg Ile Ser Arg Val Glu Tyr Ile Lys Arg Ala Leu Ser Leu210 215 220Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala225 230 235 240Ile Ala Thr Ala Gly Ile Gln Ile Arg Gly Phe Val Leu Val Val Glu245 250 255Asn Leu Ala Lys Asn Ile Cys Glu Asn Leu Glu Gln Ser Gly Leu Pro260 265 270Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ser Asn Ala Val Ala Lys275 280 285Met Leu Ser Asn Cys Pro Pro Gly Gly Ile Ser Met Thr Val Thr Gly290 295 300Asp Asn Thr Lys Trp Asn Glu Cys Leu Asn Pro Arg Val Phe Leu Ala305 310 315 320
Met Thr Glu Arg Ile Thr Arg Asp Ser Pro Ile Trp Phe Arg Asp Phe325 330 335Cys Ser Ile Ala Pro Val Leu Phe Ser Asn Lys Ile Ala Arg Leu Gly340 345 350Lys Gly Phe Met Ile Thr Ser Lys Thr Lys Arg Leu Lys Ala Gln Ile355 360 365Pro Cys Pro Asp Leu Phe Ser Ile Pro Leu Glu Arg Tyr Asn Glu Glu370 375 380Thr Arg Ala Lys Leu Lys Lys Leu Lys Pro Phe Phe Asn Glu Glu Gly385 390 395 400Thr Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu405 410 415Ser Thr Val Leu Gly Val Ala Ala Leu Gly Ile Lys Asn Ile Gly Asn420 425 430Lys Glu Tyr Leu Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala Leu435 440 445Phe Val Asn Ala Lys Asp Glu Glu Thr Cys Met Glu Gly Ile Asn Asp450 455 460Phe Tyr Arg Thr Cys Lys Leu Leu Gly Ile Asn Met Ser Lys Lys Lys465 470 475 480Ser Tyr Cys Asn Glu Thr Gly Met Phe Glu Phe Thr Ser Met Phe Tyr485 490 495Arg Asp Gly Phe Val Ser Asn Phe Ala Met Glu Ile Pro Ser Phe Gly500 505 510Val Ala Gly Val Asn Glu Ser Ala Asp Met Ala Ile Gly Met Thr Ile515 520 525Ile Lys Asn Asn Met Ile Asn Asn Gly Met Gly Pro Ala Thr Ala Gln530 535 540Thr Ala Ile Gln Leu Phe Ile Ala Asp Tyr Arg Tyr Thr Tyr Lys Cys545 550 555 560His Arg Gly Asp Ser Lys Val Glu Gly Lys Arg Met Lys Ile Ile Lys565 570 575Glu Leu Trp Glu Asn Thr Lys Gly Arg Asp Gly Leu Leu Val Ala Asp
580 585 590Gly Gly Pro Asn Ile Tyr Asn Leu Arg Asn Leu His Ile Pro Glu Ile595 600 605Val Leu Lys Tyr Asn Leu Met Asp Pro Glu Tyr Lys Gly Arg Leu Leu610 615 620His Pro Gln Asn Pro Phe Val Gly His Leu Ser Ile Glu Gly Ile Lys625 630 635 640Glu Ala Asp Ile Thr Pro Ala His Gly Pro Val Lys Lys Met Asp Tyr645 650 655Asp Ala Val Ser Gly Thr His Ser Trp Arg Thr Lys Arg Asn Arg Ser660 665 670Ile Leu Asn Thr Asp Gln Arg Asn Met Ile Leu Glu Glu Gln Cys Tyr675 680 685Ala Lys Cys Cys Asn Leu Phe Glu Ala Cys Phe Asn Ser Ala Ser Tyr690 695 700Arg Lys Pro Val Gly Gln His Ser Met Leu Glu Ala Met Ala His Arg705 710 715 720Leu Arg Met Asp Ala Arg Leu Asp Tyr Glu Ser Gly Arg Met Ser Lys725 730 735Asp Asp Phe Glu Lys Ala Met Ala His Leu Gly Glu Ile Gly Tyr Thr740 745 750<210>29<211>726<212>PRT<213>流感B/Vienna/1/99/ca<400>29Met Asp Thr Phe Ile Thr Arg Asn Phe Gln Thr Thr Ile Ile Gln Lys1 5 10 15Ala Lys Asn Thr Met Ala Glu Phe Ser Glu Asp Pro Glu Leu Gln Pro20 25 30Ala Met Leu Phe Asn Ile Cys Val His Leu Glu Val Cys Tyr Val Ile
35 40 45Ser Asp Met Asn Phe Leu Asp Glu Glu Gly Lys Ala Tyr Thr Ala Leu50 55 60Glu Gly Gln Gly Lys Glu Gln Asn Leu Arg Pro Gln Tyr Glu Val Ile65 70 75 80Glu Gly Met Pro Arg Thr Ile Ala Trp Met Val Gln Arg Ser Leu Ala85 90 95Gln Glu His Gly Ile Glu Thr Pro Lys Tyr Leu Ala Asp Leu Phe Asp100 105 110Tyr Lys Thr Lys Arg Phe Ile Glu Val Gly Ile Thr Lys Gly Leu Ala115 120 125Asp Asp Tyr Phe Trp Lys Lys Lys Glu Lys Leu Gly Asn Ser Met Glu130 135 140Leu Met Ile Phe Ser Tyr Asn Gln Asp Tyr Ser Leu Ser Asn Glu Ser145 150 155 160Ser Leu Asp Glu Glu Gly Lys Gly Arg Val Leu Ser Arg Leu Thr Glu165 170 175Leu Gln Ala Glu Leu Ser Leu Lys Asn Leu Trp Gln Val Leu Ile Gly180 185 190Glu Glu Asp Val Glu Lys Gly Ile Asp Phe Lys Leu Gly Gln Thr Ile195 200 205Ser Arg Leu Arg Asp Ile Ser Val Pro Ala Gly Phe Ser Asn Phe Glu210 215 220Gly Met Arg Ser Tyr Ile Asp Asn Ile Asp Pro Lys Gly Ala Ile Glu225 230 235 240Arg Asn Leu Ala Arg Met Ser Pro Leu Val Ser Val Thr Pro Lys Lys245 250 255Leu Lys Trp Glu Asp Leu Arg Pro Ile Gly Pro His Ile Tyr Asn His260 265 270Glu Leu Pro Glu Val Pro Tyr Asn Ala Phe Leu Leu Met Ser Asp Glu275 280 285Leu Gly Leu Ala Asn Met Thr Glu Gly Lys Ser Lys Lys Pro Lys Thr290 295 300
Leu Ala Lys Glu Cys Leu Glu Lys Tyr Ser Thr Leu Arg Asp Gln Thr305 310 315 320Asp Pro Ile Leu Ile Met Lys Ser Glu Lys Ala Asn Glu Asn Phe Leu325 330 335Trp Lys Leu Trp Arg Asp Cys Val Asn Thr Ile Ser Asn Glu Glu Met340 345 350Ser Asn Glu Leu Gln Lys Thr Asn Tyr Ala Lys Trp Ala Thr Gly Asp355 360 365Gly Leu Thr Tyr Gln Lys Ile Met Lys Glu Val Ala Ile Asp Asp Glu370 375 380Thr Met Cys Gln Glu Glu Pro Lys Ile Pro Asn Lys Cys Arg Val Ala385 390 395 400Ala Trp Val Gln Thr Glu Met Asn Leu Leu Ser Thr Leu Thr Ser Lys405 410 415Lys Ala Leu Asp Leu Pro Glu Ile Gly Pro Asp Val Ala Pro Val Glu420 425 430His Val Gly Ser Glu Arg Arg Lys Tyr Phe Val Asn Glu Ile Asn Tyr435 440 445Cys Lys Ala Ser Thr Val Met Met Lys Tyr Val Leu Phe His Thr Ser450 455 460Leu Leu Asn Glu Ser Asn Ala Ser Met Gly Lys Tyr Lys Val Ile Pro465 470 475 480Ile Thr Asn Arg Val Val Asn Glu Lys Gly Glu Ser Phe Asp Met Leu485 490 495Tyr Gly Leu Ala Val Lys Gly Gln Ser His Leu Arg Gly Asp Thr Asp500 505 510Val Val Thr Val Val Thr Phe Glu Phe Ser Ser Thr Asp Pro Arg Val515 520 525Asp Ser Gly Lys Trp Pro Lys Tyr Thr Val Phe Arg Ile Gly Ser Leu530 535 540Phe Val Ser Gly Arg Glu Lys Ser Val Tyr Leu Tyr Cys Arg Val Asn545 550 555 560
Gly Thr Asn Lys Ile Gln Met Lys Trp Gly Met Glu Ala Arg Arg Cys565 570 575Leu Leu Gln Ser Met Gln Gln Met Glu Ala Ile Val Glu Gln Glu Ser580 585 590Ser Ile Gln Gly Tyr Asp Met Thr Lys Ala Cys Phe Lys Gly Asp Arg595 600 605Val Asn Ser Pro Lys Thr Phe Ser Ile Gly Thr Gln Glu Gly Lys Leu610 615 620Val Lys Gly Ser Phe Gly Lys Ala Leu Arg Val Ile Phe Thr Lys Cys625 630 635 640Leu Met His Tyr Val Phe Gly Asn Ala Gln Leu Glu Gly Phe Ser Ala645 650 655Glu Ser Arg Arg Leu Leu Leu Leu Ile Gln Ala Leu Lys Asp Arg Lys660 665 670Gly Pro Trp Val Phe Asp Leu Glu Gly Met Tyr Ser Gly Ile Glu Glu675 680 685Cys Ile Ser Asn Asn Pro Trp Val Ile Gln Ser Ala Tyr Trp Phe Asn690 695 700Glu Trp Leu Gly Phe Glu Lys Glu Gly Ser Lys Val Leu Glu Ser Val705 710 715 720Asp Glu Ile Met Asp Glu725<210>30<211>584<212>PRT<213>流感B/Vienna/1/99/ca<400>30Met Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn Ala Asp1 5 10 15Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val Lys20 25 30Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Ala Ile Pro Leu Thr35 40 45
Thr Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr Lys Thr50 55 60Arg Gly Lys Leu Cys Pro Thr Cys Leu Asn Cys Thr Asp Leu Asp Val65 70 75 80Ala Leu Gly Arg Pro Met Cys Val Gly Ile Thr Pro Ser Ala Lys Ala85 90 95Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro Ile100 105 110Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg Gly115 120 125Tyr Glu Lys Ile Arg Leu Ser Thr Gln Asn Val Ile Asn Thr Glu Lys130 135 140Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser Gly Ser Cys Pro Asn145 150 155 160Ala Thr Ser Lys Ser Gly Phe Phe Ala Thr Met Ala Trp Ala Val Pro165 170 175Arg Asp Asn Asn Lys Thr Ala Thr Asn Pro Leu Thr Val Glu Val Pro180 185 190His Ile Cys Thr Lys Glu Glu Asp Gln Ile Thr Val Trp Gly Phe His195 200 205Ser Asp Asn Lys Thr Gln Met Lys Asn Leu Tyr Gly Asp Ser Asn Pro210 215 220Gln Lys Phe Thr Ser Ser Ala Asn Gly Ile Thr Thr His Tyr Val Ser225 230 235 240Gln Ile Gly Gly Phe Pro Asp Gln Thr Glu Asp Gly Gly Leu Pro Gln245 250 255Ser Gly Arg Ile Val Val Asp Tyr Met Val Gln Lys Pro Gly Lys Thr260 265 270Gly Thr Ile Val Tyr Gln Arg Gly Ile Leu Leu Pro Gln Lys Val Trp275 280 285Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu Ile290 295 300Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys Ser
305 310 315 320Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys Pro325 330 335Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg340 345 350Pro Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala355 360 365Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly370 375 380Tyr Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu Lys385 390 395 400Ser Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser Leu405 410 415Ser Glu Leu Glu Val Asn Asn Leu Gln Arg Leu Ser Gly Ala Met Asp420 425 430Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp Leu435 440 445Arg Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu Ser450 455 460Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu465 470 475 480Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Asp Ile Gly Asn485 490 495Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp Arg500 505 510Ile Ala Ala Gly Thr Phe Asn Ala Glu Glu Phe Ser Leu Pro Thr Phe515 520525Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu Asp530 535 540Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu Ala545 550 555 560Val Thr Leu Met Ile Ala Ile Phe Ile Val Tyr Met Ile Ser Arg Asp565 570 575
Asn Val Ser Cys Ser Ile Cys Leu580<210>31<211>560<212>PRT<213>流感B/Vienna/1/99/ca<400>31Met Ser Asn Met Asp Ile Asp Gly Ile Asn Thr Gly Thr Ile Asp Lys1 5 10 15Thr Pro Glu Glu Ile Thr Phe Gly Thr Ser Gly Thr Thr Arg Pro Ile20 25 30Ile Arg Pro Ala Thr Leu Ala Pro Pro Ser Asn Lys Arg Thr Arg Asn35 40 45Pro Ser Pro Glu Arg Ala Thr Thr Ser Ser Glu Ala Asp Val Gly Arg50 55 60Lys Thr Gln Lys Lys Gln Thr Pro Thr Glu Ile Lys Lys Ser Val Tyr65 70 75 80Asn Met Val Val Lys Leu Gly Glu Phe Tyr Asn Gln Met Met Val Lys85 90 95Ala Gly Leu Asn Asp Asp Met Glu Arg Asn Leu Ile Gln Asn Ala His100 105 110Ala Val Glu Arg Ile Leu Leu Ala Ala Thr Asp Asp Lys Lys Thr Glu115 120 125Phe Gln Lys Lys Lys Asn Thr Arg Asp Val Lys Glu Gly Lys Glu Glu130 135 140Ile Asp His Asn Lys Thr Gly Gly Thr Phe Tyr Lys Met Val Arg Asp145 150 155 160Asp Lys Thr Ile Tyr Phe Ser Pro Ile Arg Ile Thr Phe Leu Lys Glu165 170 175Glu Val Lys Thr Met Tyr Lys Thr Thr Met Gly Ser Asp Gly Phe Ser180 185 190Gly Leu Asn His Ile Met Ile Gly His Ser Gln Met Asn Asp Val Cys195 200 205
Phe Gln Arg Ser Lys Ala Leu Lys Arg Val Gly Leu Asp Pro Ser Leu210 215 220Ile Ser Thr Phe Ala Gly Ser Thr Ile Pro Arg Arg Ser Gly Ala Thr225 230 235 240Gly Val Ala Ile Lys Gly Gly Gly Thr Leu Val Ala Glu Ala Ile Arg245 250 255Phe Ile Gly Arg Ala Met Ala Asp Arg Gly Leu Leu Arg Asp Ile Lys260 265 270Ala Lys Thr Ala Tyr Glu Lys Ile Leu Leu Asn Leu Lys Asn Lys Cys275 280 285Ser Ala Pro Gln Gln Lys Ala Leu Val Asp Gln Val Ile Gly Ser Arg290 295 300Asn Pro Gly Ile Ala Asp Ile Glu Asp Leu Thr Leu Leu Ala Arg Ser305 310 315 320Met Val Val Val Arg Pro Ser Val Ala Ser Lys Val Val Leu Pro Ile325 330 335Ser Ile Tyr Ala Lys Ile Pro Gln Leu Gly Phe Asn Val Glu Glu Tyr340 345 350Ser Met Val Gly Tyr Glu Ala Met Ala Leu Tyr Asn Met Ala Thr Pro355 360 365Val Ser Ile Leu Arg Met Gly Asp Asp Ala Lys Asp Lys Ser Gln Leu370 375 380Phe Phe Met Ser Cys Phe Gly Ala Ala Tyr Glu Asp Leu Arg Val Leu385 390 395 400Ser Ala Leu Thr Gly Thr Glu Phe Lys Pro Arg Ser Ala Leu Lys Cys405 410 415Lys Gly Phe His Val Pro Ala Lys Glu Gln Val Glu Gly Met Gly Ala420 425 430Ala Leu Met Ser Ile Lys Leu Gln Phe Trp Ala Pro Met Thr Arg Ser435 440 445Gly Gly Asn Glu Val Gly Gly Asp Gly Gly Ser Gly Gln Ile Ser Cys450 455 460
Ser Pro Val Phe Ala Val Glu Arg Pro Ile Ala Leu Ser Lys Gln Ala465 470 475 480Val Arg Arg Met Leu Ser Met Asn Ile Glu Gly Arg Asp Ala Asp Val485 490 495Lys Gly Asn Leu Leu Lys Met Met Asn Asp Ser Met Ala Lys Lys Thr500 505 510Ser Gly Asn Ala Phe Ile Gly Lys Lys Met Phe Gln Ile Ser Asp Lys515 520 525Asn Lys Thr Asn Pro Val Glu Ile Pro Ile Lys Gln Thr IIe Pro Asn530 535 540Phe Phe Phe Gly Arg Asp Thr Ala Glu Asp Tyr Asp Asp Leu Asp Tyr545 550 555 560<210>32<211>100<212>PRT<213>流感B/Vienna/1/99/ca<400>32Met Asn Asn Ala Thr Phe Asn Tyr Thr Asn Val Asn Pro Ile Pro His1 5 10 15Ile Arg Gly Ser Val Ile Ile Thr Ile Cys Val Ser Phe Thr Val Ile20 25 30Leu Ile Ile Phe Gly Tyr Ile Ala Lys Ile Phe Thr Asn Arg Asn Asn35 40 45Cys Thr Asn Asn Ala Ile Gly Leu Cys Lys Arg Ile Lys Cys Ser Gly50 55 60Cys Glu Pro Phe Cys Asn Lys Arg Gly Asp Thr Ser Ser Pro Arg Thr65 70 75 80Gly Val Asp Ile Pro Ala Phe Ile Leu Pro Gly Leu Asn Leu Ser Glu85 90 95Ser Thr Pro Asn100
<210>33<211>466<212>PRT<213>流感B/Vienna/1/99/ca<400>33Met Leu Pro Ser Thr Ile Gln Thr Leu Thr Leu Phe Leu Thr Ser Gly1 5 10 15Gly Val Leu Leu Ser Leu Tyr Val Ser Ala Ser Leu Ser Tyr Leu Leu20 25 30Tyr Ser Asp Ile Leu Leu Lys Phe Ser Pro Thr Glu Ile Thr Ala Pro35 40 45Thr Met Pro Leu Asp Cys Ala Asn Ala Ser Asn Val Gln Ala Val Asn50 55 60Arg Ser Ala Thr Lys Gly Val Thr Leu Leu Leu Pro Glu Pro Glu Trp65 70 75 80Thr Tyr Pro Arg Leu Ser Cys Pro Gly Ser Thr Phe Gln Lys Ala Leu85 90 95Leu Ile Ser Pro His Arg Phe Gly Glu Thr Lys Gly Asn Ser Ala Pro100 105 110Leu Ile Ile Arg Glu Pro Phe Ile Ala Cys Gly Pro Lys Glu Cys Lys115120 125His Phe Ala Leu Thr His Tyr Ala Ala Gln Pro Gly Gly Tyr Tyr Asn130 135 140Gly Thr Arg Glu Asp Arg Asn Lys Leu Arg His Leu Ile Ser Val Lys145 150 155 160Leu Gly Lys Ile Pro Thr Val Glu Asn Ser Ile Phe His Met Ala Ala165 170 175Trp Ser Gly Ser Ala Cys His Asp Gly Lys Glu Trp Thr Tyr Ile Gly180 185 190Val Asp Gly Pro Asp Ser Asn Ala Leu Leu Lys Ile Lys Tyr Gly Glu195 200 205Ala Tyr Thr Asp Thr Tyr His Ser Tyr Ala Asn Asn Ile Leu Arg Thr210 215 220
Gln Glu Ser Ala Cys Asn Cys Ile Gly Gly Asn Cys Tyr Leu Met Ile225 230 235 240Thr Asp Gly Ser Ala Ser Gly Ile Ser Glu Cys Arg Phe Leu Lys Ile245 250 255Gln Glu Gly Arg Ile Ile Lys Glu Ile Phe Pro Thr Gly Arg Val Glu260 265 270His Thr Glu Glu Cys Thr Cys Gly Phe Ala Ser Asn Lys Thr Ile Glu275 280 285Cys Ala Cys Arg Asp Asn Ser Tyr Thr Ala Lys Arg Pro Phe Val Lys290 295 300Leu Asn Val Glu Thr Asp Thr Ala Glu Ile Arg Leu Met Cys Thr Glu305 310 315 320Thr Tyr Leu Asp Thr Pro Arg Pro Asp Asp Gly Ser Ile Thr Gly Pro325 330 335Cys Glu Ser Asn Gly Asp Lys Gly Ser Gly Gly Ile Lys Gly Gly Phe340 345 350Val His Gln Arg Met Ala Ser Lys Thr Gly Arg Trp Tyr Ser Arg Thr355 360 365Met Ser Lys Thr Lys Arg Met Gly Met Gly Leu Tyr Val Lys Tyr Asp370 375 380Gly Asp Pro Trp Thr Asp Ser Asp Ala Leu Ala Leu Ser Gly Val Met385 390 395 400Val Ser Met Glu Glu Pro Gly Trp Tyr Ser Phe Gly Phe Glu Ile Lys405 410 415Asp Lys Lys Cys Asp Val Pro Cys Ile Gly Ile Glu Met Val His Asp420 425 430Gly Gly Lys Glu Thr Trp His Ser Ala Ala Thr Ala Ile Tyr Cys Leu435 440 445Met Gly Ser Gly Gln Leu Leu Trp Asp Thr Val Thr Gly Val Asn Met450 455 460Ala Leu465
<210>34<211>248<212>PRT<213>流感B/Vienna/1/99/ca<400>34Met Ser Leu Phe Gly Asp Thr Ile Ala Tyr Leu Leu Ser Leu Thr Glu1 5 10 15Asp Gly Glu Gly Lys Ala Glu Leu Ala Glu Lys Leu His Cys Trp Phe20 25 30Gly Gly Lys Glu Phe Asp Leu Asp Ser Ala Leu Glu Trp Ile Lys Asn35 40 45Lys Arg Cys Leu Thr Asp Ile Gln Lys Ala Leu Ile Gly Ala Ser Ile50 55 60Cys Phe Leu Lys Pro Lys Asp Gln Glu Arg Lys Arg Arg Phe Ile Thr65 70 75 80Glu Pro Leu Ser Gly Met Gly Thr Thr Ala Thr Lys Lys Lys Gly Leu85 90 95Ile Leu Ala Glu Arg Lys Met Arg Arg Cys Val Ser Phe His Glu Ala100 105 110Phe Glu Ile Ala Glu Gly His Glu Ser Ser Ala Leu Leu Tyr Cys Leu115 120 125Met Val Met Tyr Leu Asn Pro Gly Asn Tyr Ser Met Gln Val Lys Leu130 135 140Gly Thr Leu Cys Ala Leu Cys Glu Lys Gln Ala Ser His Ser His Arg145 150 155 160Ala His Ser Arg Ala Ala Arg Ser Ser Val Pro Gly Val Arg Arg Glu165 170 175Met Gln Met Val Ser Ala Met Asn Thr Ala Lys Thr Met Asn Gly Met180 185 190Gly Lys Gly Glu Asp Val Gln Lys Leu Ala Glu Glu Leu Gln Ser Asn195 200 205Ile Gly Val Leu Arg Ser Leu Gly Ala Ser Gln Lys Asn Gly Glu Gly210 215 220Ile Ala Lys Asp Val Met Glu Val Leu Lys Gln Ser Ser Met Gly Asn
225 230 235 240Ser Ala Leu Val Lys Lys Tyr Leu245<210>35 <211>109<212>PRT<213>流感B/Vienna/1/99/ca<400>35Met Leu Glu Pro Phe Gln Ile Leu Ser Ile Cys Ser Phe Ile Leu Ser1 5 10 15Ala Leu His Phe Val Ala Trp Thr Ile Gly His Leu Asn Gln Ile Lys20 25 30Arg Gly Val Asn Met Lys Ile Arg Ile Lys Ser Pro Asn Lys Glu Thr35 40 45Ile Asn Arg Glu Val Ser Ile Leu Arg His Ser Tyr Gln Lys Glu Ile50 55 60Gln Ala Lys Glu Thr Met Lys Glu Val Leu Ser Asp Asn Met Glu Val65 70 75 80Leu Gly Asp His Ile Val Ile Glu Gly Leu Ser Ala Glu Glu Ile Ile85 90 95Lys Met Gly Glu Thr Val Leu Glu Ile Glu Glu Leu His100 105<210>36<211>281<212>PRT<213>流感B/Vienna/1/99/ca<400>36Met Ala Asn Asn Ile Thr Thr Thr Gln Ile Glu Val Gly Pro Gly Ala1 5 10 15Thr Asn Ala Thr Ile Asn Phe Glu Thr Gly Ile Leu Glu Cys Tyr Glu20 25 30Arg Leu Ser Trp Gln Arg Ala Leu Asp Tyr Pro Gly Gln Asp Arg Leu35 40 45
Asn Arg Leu Lys Arg Lys Leu Glu Ser Arg Ile Lys Thr His Asn Lys50 55 60Ser Glu Pro Glu Ser Lys Arg Met Ser Leu Glu Glu Arg Lys Ala Ile65 70 75 80Gly Val Lys Met Met Lys Val Leu Leu Phe Met Asn Pro Ser Ala Gly85 90 95Ile Glu Gly Phe Glu Pro Tyr Tyr Met Lys Ser Ser Ser Asn Ser Asnl00 105 110Cys Pro Lys Tyr Asn Trp Thr Asp Tyr Pro Ser Thr Pro Gly Arg Cys115 120 125Leu Asp Asp Ile Glu Glu Glu Pro Glu Asp Val Asp Gly Pro Thr Glu130 135 140Ile Val Leu Arg Asp Met Asn Asn Lys Asp Ala Arg Gln Lys Ile Lys145150 155 160Glu Glu Val Asn Thr Gln Lys Glu Gly Lys Phe Arg Leu Thr Ile Lys165 170 175Arg Asp Ile Arg Asn Val Leu Ser Leu Arg Val Leu Val Asn Gly Thr180 185 190Phe Leu Lys His Pro Asn Gly Tyr Lys Ser Leu Ser Thr Leu His Arg195 200 205Leu Asn Ala Tyr Asp Gln Ser Gly Arg Leu Val Ala Lys Leu Val Ala210 215 220Thr Asp Asp Leu Thr Val Glu Asp Glu Glu Asp Gly His Arg Ile Leu225 230 235 240Asn Ser Leu Phe Glu Arg Leu Asn Glu Gly His Ser Lys Pro Ile Arg245 250 255Ala Ala Glu Thr Ala Val Gly Val Leu Ser Gln Phe Gly Gln Glu His260 265 270Arg Leu Ser Pro Glu Glu Gly Asp Asn275 280<210>37<211>122<212>PRT
<213>流感B/Vienna/1/99/ca<400>37Met Ala Asn Asn Ile Thr Thr Thr Gln Ile Glu Trp Arg Met Lys Lys1 5 10 15Met Ala Ile Gly Ser Ser Thr His Ser Ser Ser Val Leu Met Lys Asp20 25 30Ile Gln Ser Gln Phe Glu Gln Leu Lys Leu Arg Trp Glu Ser Tyr Pro35 40 45Asn Leu Val Lys Ser Thr Asp Tyr His Gln Lys Arg Glu Thr Ile Lys50 55 60Leu Val Thr Glu Glu Leu Tyr Leu Leu Ser Lys Arg Ile Asp Asp Asn65 70 75 80Ile Leu Phe His Lys Thr Val Ile Ala Asn Ser Ser Ile Ile Ala Asp85 90 95Met Val Val Ser Leu Ser Leu Leu Glu Thr Leu Tyr Glu Met Lys Asp100 105 110Val Val Glu Val Tyr Ser Arg Gln Cys Leu115 120
權利要求
1.一種生產完整病毒疫苗的方法，所述完整病毒疫苗最好是一種減毒活疫苗，所述方法包括下面步驟a)用一種所需病毒感染非洲綠猴腎(Vero)細胞，其中所述Vero細胞已經在無血清培養基中培養并從中分離，所述無血清培養基也不含非血清蛋白；b)將所述感染細胞與一種合適的無血清細胞培養基混合，所述無血清細胞培養基除蛋白酶和核酸酶外也不含非血清蛋白；然后c)在所述蛋白酶和所述核酸酶的存在下溫育所述細胞，使得生產傳染性病毒并同時消化釋放到細胞培養基中的核酸；d)通過收集離心所述細胞培養物獲得的包含病毒的上清液，收獲傳染性病毒；然后e)如下制備其疫苗用選自以下的至少一種加工步驟處理所述包含病毒的上清液過濾、濃縮、冰凍、凍干和加入穩定劑使之穩定。
2.依照權利要求1的方法，所述方法不涉及蛋白分離或純化的步驟。
3.依照權利要求1或2的方法，所述方法不涉及層析分離或純化的步驟，并且最好不包含除離心和/或過濾外的任何純化步驟。
4.依照權利要求1到3中任一項的方法，所述方法包括至少一步無菌過濾所述包含病毒的上清液。
5.依照權利要求1到4中任一項的方法，其中所述核酸酶具有DNA酶和/或RNA酶活性，并且最好是Benzonase。
6.依照權利要求1到5中任一項的方法，其中在溫育所述感染細胞開始前或開始時一次性在所述細胞培養基中加入所述蛋白酶和所述核酸酶。
7.依照權利要求1到6中任一項的方法，其中所述蛋白酶包括人重組來源或豬來源的胰蛋白酶和/或胰蛋白酶原，所述胰蛋白酶和/或胰蛋白酶原在所述細胞培養基中以初濃度每ml培養基0.5-10、最好2-5μg存在。
8.依照權利要求1到7中任一項的方法，其中所述細胞培養基以每ml培養基2到30、最好5到15U的初濃度包含核酸酶。
9.依照權利要求1到8中任一項的方法，其中步驟(a)中的溫育進行10到120分鐘，最好30到60分鐘。
10.依照權利要求1到9中任一項的方法，其中所述病毒選自野生型病毒、直接從感染個體獲得的原代分離物、重組病毒、減毒病毒、Vero適應性病毒、冷適應性病毒、溫度敏感性病毒以及重排列病毒。
11.依照權利要求1到10中任一項的方法，其中所述病毒是A型流感病毒或B型流感病毒，所述A型流感病毒最好是H3N2亞型或H1N1亞型。
12.依照權利要求1到11中任一項的方法，其中所述病毒具有誘導干擾素性表型和/或干擾素敏感性表型。
13.依照權利要求1到12中任一項的方法，其中所述病毒是選自以下的流感病毒A/Sing/1/57ca株、A/Sing/1/57ca/ΔNS87株、A/Sing/1/57ca/ΔNSPR8株、A/Sing/1/57ca/NS124PR8株、B/Vienna/1/99ca株、B/Vienna/99/ca37株以及來自這些病毒株中任一種的減毒變異體和重排列體。
14.一種完整病毒疫苗，所述完整病毒疫苗最好是一種減毒活疫苗，其特征在于它的現成使用形式包含基本未改變、任選過濾和/或濃縮的用于生產所述病毒的Vero細胞培養物的包含病毒的上清液，所述Vero細胞培養物中無血清并且無蛋白。
15.依照權利要求14的疫苗，所述疫苗的特征在于它選擇性凝集人紅細胞，而不凝集雞紅細胞。
16.依照權利要求14或15的疫苗，所述疫苗的特征在于它包含合適的穩定劑。
17.依照權利要求14到16中任一項的疫苗，所述疫苗的特征在于它為液體、冷凍或凍干制備物形式，任選適于經鼻傳遞。
18.依照權利要求14到17中任一項的疫苗，所述疫苗的特征在于它是一種活的減毒疫苗，最好包含完整流感病毒。
19.依照權利要求14到18中任一項的疫苗，所述疫苗的特征在于它包含至少一種流感病毒，所述流感病毒具有一個或多個選自以下特征的表型冷適應性、溫度敏感性、干擾素誘導性、干擾素敏感性。
20.依照權利要求18的疫苗，其中所述流感病毒選自A/Sing/1/57ca株、A/Sing/1/57ca/ΔNS87株、A/Sing/1/57ca/ΔNSPR8株、A/Sing/1/57ca/NS124PR8株、B/Vienna/1/99ca株以及來自這些病毒株中任一種的減毒變異體和重排列體。
21.依照權利要求14的疫苗，所述疫苗可以通過權利要求1到13中任一項定義的生產方法獲得。
22.一種完整病毒疫苗，所述完整病毒疫苗最好是一種減毒活疫苗，所述疫苗包括至少一種選自以下的流感病毒A/Sing/1/57ca株、A/Sing/1/57ca/ΔNS87株、A/Sing/1/57ca/ΔNSPR8株、A/Sing/1/57ca/NS124PR8株、B/Vienna/1/99ca株以及來自這些病毒株中任一種的減毒變異體和重排列體。
23.依照權利要求21的疫苗，所述疫苗的特征在于它選擇性凝集人紅細胞，而不凝集雞紅細胞。
24.依照權利要求22或23的疫苗，所述疫苗可以通過權利要求1到13任一項的生產方法獲得。
25.權利要求14到24中任一項定義的疫苗的應用，所述疫苗用于預防性或治療性給予對抗病毒感染。
26.至少一種選自以下的流感病毒的應用A/Sing/1/57ca株、A/Sing/1/57ca/ΔNS87株、A/Sing/1/57ca/ΔNSPR8株、A/Sing/1/57ca/NS124PR8株、B/Vienna/1/99ca株以及來自這些病毒病毒株中任一種的減毒變異體和重排列體，所述流感病毒用于生產疫苗，最好用于生產活的減毒流感疫苗。
全文摘要
本發明涉及一種從不同來源分離病毒以及在無血清Vero細胞培養物中生產活減毒流感疫苗的簡單有效的方法，其中所述生產在最小化或預防所述病毒的表面蛋白由于適應性選擇而發生變化的條件下進行。本方法不要求對從所述細胞培養物收獲的含病毒的上清液進行純化，也不要求對所述病毒進行溫育后處理以進行HA活化。本發明還涉及A型和B型流感原株候選物以及由其制造的疫苗。
文檔編號C12N7/04GK1582333SQ01819206
公開日2005年2月16日 申請日期2001年9月25日 優先權日2000年9月25日
發明者H·卡廷格爾, A·埃戈洛夫, B·費爾科, J·羅馬諾瓦, D·卡廷格爾 申請人:波利門科學生物免疫研究有限公司