流感病毒H3N2血凝素基因和神经氨酸酶基因在真核酵母菌中的表达  

Expression of the Hemagglutinin and Neuramidinase Gene of Influenza A Virus H3N2 in P. methanolica

作　　者：李梓[1] 张烨[1] 唐启慧[2] 陈禹宝 于在江[1] 辛丽[1] 陈永坤[1] 陈清轩[2] 舒跃龙[1] 

机构地区：[1]中国疾病预防控制中心病毒病所国家流感中心,北京100052 [2]北京标凯科技有限公司,北京100094 [3]北京中亚国瑞生物经济研究所,北京102206

出　　处：《实验动物科学》2010年第4期6-10,共5页Laboratory Animal Science

基　　金：国家科技支撑计划项目(项目编号:2006BAD06A15)

摘　　要：目的克隆、表达和鉴定流感病毒H3N2血凝素基因(hemagglutinin,HA)和神经氨酸酶基因(neuramidinase,NA)序列,为制备抗体和基因工程疫苗打下基础。方法在成功克隆流感病毒H3N2全长HA、NA基因并测序的基础上,将部分基因序列克隆到表达载体pMET A上,构建了重组表达质粒pMET A/HA(52 bp～1 549 bp)、pMET A/NA(121 bp～1 260 bp),电转化真核酵母菌pMAD16,甲醇诱导表达,利用Ni2+亲和层析柱对重组蛋白进行纯化,并用Western Blotting和ELISA方法检测其抗原性。结果重组蛋白在酵母菌中可以高效表达,SDS-PAGE显示蛋白表达后形成了二聚体,蛋白纯度占总蛋白的95%以上,ELISA和Western Blotting实验证实,重组蛋白具有良好的抗原性。结论本研究成功克隆和表达了流感病毒H3N2 HA、NA基因序列,为流感病毒H3N2诊断试剂和疫苗开发等进一步研究奠定了基础。Objective To clone,express and characterize the HA（hemagglutinin）and NA（neuramidinase,NA） protein of influenza virus H3N2.Methods On the basis of successful clone the full length HA and NA gene and sequence analysis of influenza virus H1N1,and the genes were ligated into pMET A.An expression vector pMET A / HA（52 bp-1 549 bp）,pMET A /NA（121 bp-1 260 bp）were constructed and expressed in pMAD16 induced by methanol.Recombinant protein was purified through Ni2 ＋ affinity chromatography column.Western Blotting and ELISA were used to determine the antigenic of the recombinant protein.Results The recombinant capsid gene can be overexpressed in P.methanolica.SDS-PAGE showed that the product is a dimer.The purity of the protein is greater than 95%.ELISA and Western Blotting showed that the recombinant protein has good antigenic.Conclusion the HA and NA Protein of influenza virus H3N2 has been successful cloned and expressed,which could be useful for developing diagnose reagents or vaccine of H3N2.

关 键 词：流感病毒H3N2 血凝素 神经氨酸酶 酵母菌 表达 

分 类 号：S852.65[农业科学—基础兽医学]