高致病性禽流感病毒血凝素基因定点突变及其原核表达

目的改构高致病性禽流感病毒血凝素基因,建立有效的原核表达体系。方法利用位点特异PCR突变技术同义突变HA1基因中的稀有密码子,将改构的HA1基因克隆到pGEX-4T-1载体中建立4T-1-HA1-m 重组表达质粒,然后转入E·coliBL21DE3中并用IPTG诱导表达HA1-GST融合蛋白,SDS-PAGE电泳检测目的蛋白,Western印迹分析表达产物的免疫活性。结果改构的HA1基因能够在大肠埃希菌中有效表达,表达产物能与H5N1亚型高致病性禽流感病毒免疫鼠血清特异性结合。结论提示高致病性禽流感病毒血凝素基因经位点特异PCR突变技术改构后,可以在大肠埃希菌中表达出有免疫活性的血凝素蛋白。

Site-directed Mutations and Prokaryotic Expression of Hemagglutinin Gene of High Pathogenic Avian Influenza Virus

Objective To modify the HA1 gene of high pathogenic avian influenza virus (HPAIV) and to build an efficient prokaryotic expression system of it.Methods Site-directed synonymous mutation, by using PCR, was used to substitute the low-usage codons in the HA1 gene, and the modified HA1 gene was cloned into pGEX-4T-1 to build 4T-1-HA1-m~ recombinant plasmid. Then the recombinant 4T-1-HA1-m~ was transferred into E. coli BL21 DE3 and was induced by IPTG to express HA1-GST, which was detected by SDS-PAGE and analyzed by Western-blotting to show whether it had immune function.Results The modified HA1 gene could be well expressed, and the expressed HA1-GST had specific reaction to the blood serum of the mouse immunized by the H5N1 subtype HPAIV.Conclusion The HA1 gene of HPAIV could be well expressed in E. coli to generate HA1-GST fusional protein with immune function after being modified by site-directed synonymous mutation by using PCR.