人用H5N1禽流感疫苗毒种检定及毒种库建立
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朱智勇，丁晓航，朱函坪，李岩金，陈学奎，沈吉友，张涛，何培江，姚苹苹，徐芳，翁景
禽流感疫苗;毒种;检定;毒种库,,禽流感疫苗;毒种;检定;毒种库,1材料与方法,2结果,3讨论,参考文献
参见附件(17kb)。
摘要： 目的 为了研制人用H5N1禽流感疫苗,按照《中国药典》三部 “生物制品生产和检定用菌毒种管理规程” 中的要求,建立用于生产禽流感疫苗的毒种库。方法 从国外引进2株H5N1禽流感疫苗的毒种 (R1194和R1203), 进行全面检定,建立三级种子库，即原代、主代和工作代，用于疫苗生产。结果 引进的R1194和R1203 2个毒株能在胚蛋中大量增殖并稳定传代；与普通流感A1，A3，B作交叉血凝抑制试验和交叉单向免疫扩散试验表明,符合H5N1禽流感病毒的特征；经对血凝素序列测定，证实了引进的2毒株为H5N1禽流感病毒；测得的序列与原始株序列比较可知,2毒株均已去除有毒基因,属弱毒株；确定2毒株的P2为原代，P4为主代，P5为工作代。结论 引进的R1194和R1203毒种为H5N1禽流感减毒株,可用于禽流感疫苗的生产。
关键词： 禽流感疫苗;毒种;检定;毒种库
Study on avian influenza (H5N1) vaccine used in human body and establishment of virus seed bank
ZHU Zhiyong,DING Xiaohang,ZHU Hanping,et al.
Key Lab of Vaccine against Hemorrhagic Fever with Renal Syndrome, Center for Disease Control and Prevention, Zhejiang Province(Hangzhou 310009,China)
Abstract： Objective To develop avian influenza (H5N1) vaccines used in human body.To establish the virus seed bank used to produce avian influenza vaccines according to “Requirements for Bacterial and Viral StrainsSeeds Used for Production and Quality Control of Biologics” from the book called PHARMACOPOEIA OF THE PEOPLE’S REPUBLIC OF CHINA Volume III.Methods To importe from abroad and checked up entirely the twovirus seeds of R1194 and R1203 for the avian influenza vaccine production.To establish three virus seed lot systems (seed bank) i.e.the primary seed lot, master seed lot and working seed lot.Results The two seeds of avian influenza vaccines can be multiplied greatly and passaged steadily in egg embryos; The cross hemagglutinininhibiting test and immunodiffusion test with the antigen and antibody of human influenza A1, A3 and B and avian influenza H5N1 indicated that the above two seeds had the character of avian influenza H5N1. The complete HA genes, that were sequenced, approved that the seeds were avian influenza H5N1 viruses.The virulence genes were deleted and the two seeds belonged to feebleness virulence strain by sequence compared with wild types. The two seeds, which were passed 2，4 and 5 times in egg embryos respectively, were confirmed as the primary seed lot, master seed lot and working seed lot.Conclusion The twovirus strain R1194 and R1203 imported from abroad can be used to produce the avian influenza vaccines.
Key words： avian influenza vaccine;virus seed;check up;virus seed bank
自2003年12月～2006年10月16日，世界各地共发生由禽传人的H5N1禽流感病例256人，死亡151人，病死率高达59 %〔1〕。由于流感病毒极易变异，一旦获得在人间传播的能力，数亿人的生命将受到威胁，其潜在危害性远超过严重急性呼吸道综合征(SARS)，已引起WHO和世界各国政府的重视〔2〕，并把人用禽流感疫苗的研究提到重要位置〔3〕。2005年我们从国外引进2株H5N1亚型禽流感病毒，用于疫苗的研究，由于内容较多，现把研究结果分3部分：(1)疫苗毒种的检定及毒种库的建立；(2)疫苗检定评价；(3)疫苗免疫动物的效果观察。本文报告第一部分。
1 材料与方法
11 材料 (1)毒种: NIBRG-14株(简称R1194)(英国国家生物制品检定所)。该毒株是1194株(A/Viet Nam/1194/2004 H5N1)和PR8株(H1N1)2病毒,通过反相遗传的方法改造而成；VNH5N1-PR8/CDC-RG株(简称R1203)(美国疾病控制中心)，该毒株是用1203株(A/VietNam/1203/2004H5N1)和PR8株(H1N1)病毒,通过反相遗传的方法改造而成。2种病毒均在胚蛋中传二代(P2)。(2)血清:禽流感病毒H5血凝素及抗血清(英国国家生物制品检定所);A1、A3、B型人流感病毒血凝素及抗血清(英国国家生物制品检定所);R1203抗血清(本实验室用R1203疫苗免疫家兔获得)。(3)无致病菌(SPF)鸡蛋(北京实验动物中心SPF鸡群)。
12 方法 (1)血凝抑制试验和单向免疫扩散试验:按常规法。(2)病毒滴度测定:按常规鸡胚感染法，每稀释度接种4只胚蛋 ......
摘要： 目的 为了研制人用H5N1禽流感疫苗,按照《中国药典》三部 “生物制品生产和检定用菌毒种管理规程” 中的要求,建立用于生产禽流感疫苗的毒种库。方法 从国外引进2株H5N1禽流感疫苗的毒种 (R1194和R1203), 进行全面检定,建立三级种子库，即原代、主代和工作代，用于疫苗生产。结果 引进的R1194和R1203 2个毒株能在胚蛋中大量增殖并稳定传代；与普通流感A1，A3，B作交叉血凝抑制试验和交叉单向免疫扩散试验表明,符合H5N1禽流感病毒的特征；经对血凝素序列测定，证实了引进的2毒株为H5N1禽流感病毒；测得的序列与原始株序列比较可知,2毒株均已去除有毒基因,属弱毒株；确定2毒株的P2为原代，P4为主代，P5为工作代。结论 引进的R1194和R1203毒种为H5N1禽流感减毒株,可用于禽流感疫苗的生产。
关键词： 禽流感疫苗;毒种;检定;毒种库
Study on avian influenza (H5N1) vaccine used in human body and establishment of virus seed bank
ZHU Zhiyong,DING Xiaohang,ZHU Hanping,et al.
Key Lab of Vaccine against Hemorrhagic Fever with Renal Syndrome, Center for Disease Control and Prevention, Zhejiang Province(Hangzhou 310009,China)
Abstract： Objective To develop avian influenza (H5N1) vaccines used in human body.To establish the virus seed bank used to produce avian influenza vaccines according to “Requirements for Bacterial and Viral StrainsSeeds Used for Production and Quality Control of Biologics” from the book called PHARMACOPOEIA OF THE PEOPLE’S REPUBLIC OF CHINA Volume III.Methods To importe from abroad and checked up entirely the twovirus seeds of R1194 and R1203 for the avian influenza vaccine production.To establish three virus seed lot systems (seed bank) i.e.the primary seed lot, master seed lot and working seed lot.Results The two seeds of avian influenza vaccines can be multiplied greatly and passaged steadily in egg embryos; The cross hemagglutinininhibiting test and immunodiffusion test with the antigen and antibody of human influenza A1, A3 and B and avian influenza H5N1 indicated that the above two seeds had the character of avian influenza H5N1. The complete HA genes, that were sequenced, approved that the seeds were avian influenza H5N1 viruses.The virulence genes were deleted and the two seeds belonged to feebleness virulence strain by sequence compared with wild types. The two seeds, which were passed 2，4 and 5 times in egg embryos respectively, were confirmed as the primary seed lot, master seed lot and working seed lot.Conclusion The twovirus strain R1194 and R1203 imported from abroad can be used to produce the avian influenza vaccines.
Key words： avian influenza vaccine;virus seed;check up;virus seed bank
自2003年12月～2006年10月16日，世界各地共发生由禽传人的H5N1禽流感病例256人，死亡151人，病死率高达59 %〔1〕。由于流感病毒极易变异，一旦获得在人间传播的能力，数亿人的生命将受到威胁，其潜在危害性远超过严重急性呼吸道综合征(SARS)，已引起WHO和世界各国政府的重视〔2〕，并把人用禽流感疫苗的研究提到重要位置〔3〕。2005年我们从国外引进2株H5N1亚型禽流感病毒，用于疫苗的研究，由于内容较多，现把研究结果分3部分：(1)疫苗毒种的检定及毒种库的建立；(2)疫苗检定评价；(3)疫苗免疫动物的效果观察。本文报告第一部分。
1 材料与方法
11 材料 (1)毒种: NIBRG-14株(简称R1194)(英国国家生物制品检定所)。该毒株是1194株(A/Viet Nam/1194/2004 H5N1)和PR8株(H1N1)2病毒,通过反相遗传的方法改造而成；VNH5N1-PR8/CDC-RG株(简称R1203)(美国疾病控制中心)，该毒株是用1203株(A/VietNam/1203/2004H5N1)和PR8株(H1N1)病毒,通过反相遗传的方法改造而成。2种病毒均在胚蛋中传二代(P2)。(2)血清:禽流感病毒H5血凝素及抗血清(英国国家生物制品检定所);A1、A3、B型人流感病毒血凝素及抗血清(英国国家生物制品检定所);R1203抗血清(本实验室用R1203疫苗免疫家兔获得)。(3)无致病菌(SPF)鸡蛋(北京实验动物中心SPF鸡群)。
12 方法 (1)血凝抑制试验和单向免疫扩散试验:按常规法。(2)病毒滴度测定:按常规鸡胚感染法，每稀释度接种4只胚蛋 ......
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