H9N2亚型禽流感病毒HA基因的克隆及其DNA疫苗的动物免疫试验  

Cloning of HA Gene of H9N2 Subtype Avian Influenza Virus and Immune Protection Test of Its DNA Vaccine

作　　者：何宏轩[1,2] 秦曦明[1] 张强哲[1] 汤义[1] 刘丽艳[1,2] 郑昌学[1] 段明星[1] 

机构地区：[1]清华大学生物科学与技术系,生物膜与膜生物工程国家重点实验室,北京100084 [2]河南职业技术师范学院动物科学系,新乡453003

出　　处：《生物化学与生物物理进展》2004年第2期163-166,共4页Progress In Biochemistry and Biophysics

摘　　要：血凝素 (HA)是决定禽流感病毒的毒力强弱和免疫原性的主要蛋白质 .根据已发表的H9亚型AIV的HA基因序列 ,设计合成了 1对H9HA特异引物 ,以AIVA/Chicken/Henan/ 1 / 1 999/ (H9N2 )核酸为模板 ,通过RT PCR扩增出 1条 1 6kbcDNA片段 .将HA基因插入pVAX1中 ,构建了真核表达质粒pVAX H9.采用活体电击法免疫 3周龄SPF鸡 1 0只 ,剂量为 5 0 μg/只 ,3周后加强免疫一次 ,5周后以 1 0 0倍鸡胚感染剂量 (EID)的HA基因同源病毒对所有鸡进行攻毒 .其间每周检测抗体水平变化 ,6周后以棉拭子进行泄殖腔病毒分离 .结果为攻毒后免疫组鸡HI效价为 9log2～ 1 0log2 ,对照组为 2log2～ 4log2 ;免疫组病毒分离数为 0 / 1 0 ,对照组为 1 0 / 1 0 .Avian influenza (AI) was caused by A avian influenza virus in chicken all over the world. One 1 6 kb DNA fragment was amplified by RT PCR from the RNA of AIV A/Chicken/Henan/1/1999(H9N2) and proved to be HA gene by sequencing. The HA gene expression plasmid pVAX H9 was constructed by inserting the fragment into the pVAX1 vector. 3 week old SPF chickens were inoculated with 50 μg DNA of plasmid pVAX H9 by electroporation, 5 weeks later, all chickens were challenged with 100×EID50 of AIV A/Chicken/Henan/1/1999(H9N2), 1 week post challenge all birds were sampled by cloacal swabbing to isolate virus. The virus isolation was negative in all vaccinated birds and positive in all control birds. The HI titers reached to 10lg2 in vaccinated group, in contrast to 2lg2～4lg2 in control group post challenge respectively. These results showed that the plasmid pVAX H9 designed as DNA vaccine would be able to elicit a firm protective immune response against AIV infection.

关 键 词：禽流感病毒 血凝素基因 RT-PCR 克隆 DNA疫苗 

分 类 号：S852.659.5[农业科学—基础兽医学] S858.3[农业科学—兽医学]