禽流感病毒H5N1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 H5N1 in E. coli

目的:克隆、表达和鉴定禽流感病毒H5N1血凝素基因(hemagglutinin,HA)和神经氨酸酶基因(neuram idinase,NA)序列,为制备抗体和基因工程疫苗打下基础。方法:在成功克隆禽流感病毒H5N1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(49～1587bp)、pET32a(+)/NA(121～1141bp)、pGEX4T-1/HA、pGEX4T-1/NA,转化大肠杆菌BL21/rosetta,IPTG诱导表达,利用Ni2+亲和层析柱和GSTraP4B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:重组蛋白在大肠杆菌中可以高效达,SDS-PAGE显示其相对分子质量与预计大小一致,蛋白纯度占总蛋白的90%以上。ELISA和Western blotting实验证实,重组蛋白具有良好的抗原性。结论:成功克隆和表达了禽流感病毒H5N1HA、NA基因序列,为禽流感病毒H5N1诊断试剂和疫苗的开发等进一步的研究奠定了基础。

Objective：To clone,express and characterize the HA（hemagglutinin） and NA（neuramidinase,NA） protein of avian influenza virus H5N1.Methods：On the basis of successful clone the full length HA and NA gene and sequence analysis of avian influenza virus H5N1,Ligated part of the gene into pET32a（＋）.full of the gene into pGEX4T-1.An expression vector pET32a（＋）/HA（49～1587bp）,pET32a（＋）/NA（121～1141bp）,pGEX4T-1/HA、pGEX4T-1/NA were constructed and expressed in E.coli BL21/rosetta induced by IPTG.recombinant protein was purified through Ni2＋ and GSTrap 4B 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 E.coli.SDS-PAGE showed that the gene could express product as same as I might expect.The purity of the protein is greater than 90%.ELISA and Western blotting showed that the recombinant protein has good antigenic.Conclusion：The HA and NA protein of avian influenza virus H5N1 has been successful cloned and expressed,which could be useful for developing diagnose reagents or vaccine of H5N1.