H5N1亚型禽流感病毒HA蛋白的原核表达

Prokaryotic Expression of HA Protein of Avian Influenza Virus H5N1

血凝素(hemagglutinin,HA)是禽流感病毒的主要抗原之一,原核表达HA是该抗原特性研究和疫苗制备的关键。利用分子生物学方法构建H5N1亚型的禽流感病毒HA基因原核表达载体,转化至大肠埃希菌Rosetta菌株,用不同IPTG浓度和时间诱导HA表达,检测蛋白表达量,分析不同诱导条件对HA表达效率的影响,以确定最佳诱导条件。重组表达载体pET32a-HA经BamHⅠ和EcoRⅠ双酶切鉴定,获得预期大小的DNA片段,说明HA基因成功插入质粒pET32a。Western blot检测结果表明,IPTG诱导重组菌株成功表达HA蛋白,当诱导浓度为0.1mmol/L,诱导时间为6h时,HA蛋白表达量最高。研究结果为HA抗原制备及禽流感疫苗的研究奠定基础。

Hemagglutinin(HA)is one of vital antigens of avian influenza virus.Expression of HA in prokaryotic cells plays an important role in exploration of HA characteristics and preparation of vaccine.In this study,HA of H5N1 was cloned into the prokaryotic expression vector,and transformed into Escherichia coli Rosetta.In order to optimize induction conditions,recombinant bacteria were induced with different IPTG concentrations and induction time,and then ratio of target protein/total proteins was measured.The result of Bam HⅠand Eco RⅠdigestion of pET32a-HA demonstrated that the expected length of HA fragment was inserted into pET32a.Western blot result illustrated that HA protein was successfully expressed in the recombinant strain via IPTG induction.With optimal conditon of 0.1 mmol/L IPTG and 6 h induction time,HA expression level reached highest.This study provided the foundation for the preparation of HA antigen and avian influenza vaccine research.