禽流感病毒H5N1亚型血凝素基因在昆虫细胞中的表达  

Expression of Hemagglutinin Gene of Avian Influenza Virus Subtype H5N1 in Insect Cells

作　　者：王桂军[1] 邵红霞[1] 金文杰[1] 秦爱建[1] 

机构地区：[1]江苏省动物预防医学重点实验室扬州大学,江苏扬州225009

出　　处：《安徽农业科学》2008年第32期13988-13990,共3页Journal of Anhui Agricultural Sciences

基　　金：国家十五攻关项目(2004BA519A05);江苏省十五攻关项目(BE2002346);江苏高校高新技术产业发展项目(JH03-028)

摘　　要：[目的]研究禽流感病毒H5N1亚型血凝素基因在昆虫细胞中的表达。[方法]将禽流感病毒H5N1亚型HA基因以BamHI和NotI双酶切插入pFastBacI载体构建pFast-H5HA转移载体质粒,然后将pFast-H5HA转入含有穿梭质粒的感受态DH10Bac中发生转座作用,在含有X-gal、IPTG、庆大霉素、卡那霉素和四环素的琼脂平板上筛选白色菌落,通过连续传代4次后获得稳定的重组质粒reBac-mid-H5HA。[结果]将重组质粒reBacmid-H5HA转染Sf9昆虫细胞,获得含有禽流感病毒H5N1亚型HA基因的重组杆状病毒,经间接免疫荧光和Western-blot证实HA基因在昆虫细胞中获得表达。重组杆状病毒表达产物能够凝集鸡红细胞,并能被抗H5N1 HA单抗所抑制。[结论]重组血凝素蛋白在杆状病毒中能获得正确表达,为研制AIVH5亚型检测抗原和亚单位疫苗奠定基础。[Objective ] The study aimed to study the expression of Hemagglutinin （HA） gene of avian influenza virus subtype HSN1 in insect cells. [ Method] The HA gene of avian influenza virus subtype H5N1 was cleaved with BamHI and NotI from plasmid pcHSHA and inserted into baculovirus transfer vector pFastBacl for constructing the recombinant transfer vector pFast-HSHA. The pFast-H5HA was transfonnated into E. coli DH10Bac cells with shuttle plasmid by transposition. The white colonies were screened on the agar plate with X-gal, IPTG, genta- micin, kanamycin and tetracycline and then the stable recombinant plasmid was constructed as reBacmid-HSHA through continuous passage for 4 times. [ Result] The recombinant baclovirus with HA gene of avian influenza virus subtype HSN1 was obtained by transfecting sf9 cells with the recombinant plasmid reBacmid-H5HA. The results of IFA and Western-blot assay demonstrated that HA protein was expressed in the sf9 in- sect cells infected with recombinant baculovirus and could agglutinate chicken red blood cells, which could be inhibited by the monoclonal an- tibody against H5NI HA. [ Conclusion] The recombinant HA protein could get the right expression in baculovirus, which give a good base for replacements of AIV in developing detection antigen and subunit vaccine

关 键 词：禽流感病毒 H5N 血凝素基因 重组杆状病毒 

分 类 号：S858.2[农业科学—临床兽医学]