流感病毒H3N2血凝素基因和神经氨酸酶基因在大肠杆菌中的表达  

Expression of the Hemagglutinin and Neuramidinase Gene of Influenza A Virus H3N2 in E. coli

作　　者：张烨[1] 于在江[1] 黄捷勤 唐启慧[2] 陈禹保 辛丽[1] 陈永坤[1] 舒跃龙[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,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：《生物技术通报》2011年第1期213-218,共6页Biotechnology Bulletin

基　　金：国家科技支撑计划资助项目(2006BAD06A15)

摘　　要：克隆、表达和鉴定流感病毒H3N2 HA,NA基因序列,为制备抗体和基因工程疫苗打下基础。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pET32a(+)上,全基因序列克隆到表达载体pGEX4T-1上,构建了重组表达质粒pET32a(+)/HA(截短),pET32a(+)/NA(截短),pGEX4T-1/HA,转化大肠杆菌BL21/Rosetta,IPTG诱导表达,利用Ni2+亲和层析柱和GSTrap 4B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。结果显示,重组蛋白在大肠杆菌中可以高效表达,SDS-PAGE显示其相对分子质量与预计大小一致。ELISA和Western blotting试验证实,重组蛋白具有良好的抗原性。成功克隆和表达了流感病毒H3N2 HA、NA基因序列,可为流感病毒H3N2诊断试剂和疫苗的开发等进一步的研究提供参考。It was to clone,express and characterize the HA and NA protein of influenza virus H3N2.On the basis of successful cloning the full length HA and NA gene and sequence analysis of influenza virus H3N2,part of the genes were ligated into pET32a（＋）,and full length gene into pGEX4T-1.An expression vector pET32a（＋）/HA（cut）,pET32a（＋）/NA（cut）,pGEX4T-1/HA were constructed and expressed in E.coli BL21/Rosetta induced by IPTG.Recombinant protein was purified through affinity chromatography column.Western blotting and ELISA were used to determine the antigenic of the recombinant protein.Results showed that the recombinant capsid gene was expressed in E.coli.SDS-PAGE showed that the gene expression product was as same as expected.ELISA and Western blotting showed that the recombinant protein has satisfactory antigenic.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 克隆表达 

分 类 号：R346[医药卫生—基础医学] Q789[生物学—分子生物学]