H9N2亚型禽流感病毒M1和NA基因在昆虫细胞中的表达  

Expression of M1 and NA Genes of H9N2 Subtype Avian Influenza Virus in Insect Cells

作　　者：王粲 张民秀 谢芝勋 李孟 罗思思 李丹 阮志华 谢丽基 谢志勤 Wang Can;Zhang Minxiu;Xie Zhixun;Li Meng;Luo Sisi;Li Dan;Ruan Zhihua;Xie Liji;Xie Zhiqin(Key Laboratory of China(Guangxi)-ASEAN Cross-border Animal Disease Prevention and Control,Ministry ofAgriculture and Rural Affairs,Guangxi Key Laboratory of Veterinary Biotechnology,Guangxi Veterinary ResearchInstitute,Nanning 530001,Guangxi,China)

机构地区：[1]广西壮族自治区兽医研究所,广西兽医生物技术重点实验室,农业农村部中国(广西)-东盟跨境动物疫病防控重点实验室,广西南宁530001

出　　处：《中国动物检疫》2024年第4期103-110,共8页China Animal Health Inspection

基　　金：广西重点研发计划项目(桂科AB21076004);“广西八桂学者”项目(2019A50);广西重大专项项目(桂科AA23062050);广西科技先锋队项目(桂农科盟202409-01)。

摘　　要：为通过昆虫细胞表达出H9N2亚型禽流感病毒M1和NA蛋白,并鉴定其免疫活性,利用PCR技术扩增H9N2亚型禽流感病毒M1和NA基因,以pFastBacDual为转移载体构建重组转移载体pFastBacDual-M1和pFastBacDual-NA;将阳性重组转移载体分别转化至DH10Bac感受态细胞,得到重组杆粒rBacmid-M1和rBacmid-NA;将重组杆粒分别转染Sf9昆虫细胞,获得含M1和NA基因的重组杆状病毒rBV-M1和rBV-NA;运用IFA和Western-blot鉴定M1和NA蛋白的表达情况,同时以NA蛋白为包被抗原,运用间接ELISA方法鉴定NA蛋白的反应活性。结果显示,IFA鉴定均出现特异性绿色荧光,Western-blot检测M1和NA蛋白大小分别约为28和52 ku,ELISA检测NA蛋白对抗H9N2阳性血清有很高的反应值。结果表明,M1和NA蛋白可在昆虫细胞中特异性表达且具有反应原性。本试验为进一步研发H9N2亚型禽流感诊断技术和疫苗奠定了基础。In order to express M1 and NA proteins of H9N2 subtype avian influenza virus(AIV)through insect cells and to identify their immune activity,M1 and NA genes were amplified by PCR,recombinant transfer vectors of pFastBacDual-M1 and pFastBacDual-NA were constructed based on pFastBacDual acting as a recombinant transfer vector;the positive recombinant transfer vectors were respectively transformed into DH10Bac competent cells to obtain recombinant bacmids of rBacmid-M1 and rBacmid-NA;Sf9 insect cells were transfected by the recombinant bacmids to obtain recombinant baculovirus containing M1 and NA genes,that were,rBV-M1 and rBV-NA;the expression of M1 and NA proteins was identified by IFA and Western-blot,and NA protein was used as a coated antigen to identify its reactivity by the indirect ELISA.The results showed that specific green fluorescence was observed by IFA identification,M1 and NA proteins were about 28 and 52 ku,respectively,as tested by Western-blot,and the expressed NA protein could specifically react with H9N2 positive serum.In conclusion,M1 and NA proteins with reactivity could be specifically expressed in insect cells.A foundation was laid for further development of diagnostic techniques and vaccine for H9N2 subtype AIV.

关 键 词：禽流感病毒 H9N2亚型 M1蛋白 NA蛋白 Bac-to-Bac杆状病毒表达系统 

分 类 号：S852.65[农业科学—基础兽医学]