【摘要】 应用带有His6尾的pET原核表达系统对禽流感病毒血凝素蛋白进行表达,将禽流感A/Turkey/Wisconsin/66(H9N2)的HA基因克隆至原核表达载体pET-30a中,经酶切、PCR扩增和测序分析确证其插入,并且阅读框正确,获得重组质粒pET30a-H9。将此重组质粒转化到宿主菌BL21中,用诱导剂异丙基硫代-β-D半乳糖苷(IPTG)进行诱导,产物处理后进行SDS-PAGE电泳、免疫印迹和薄层扫描分析。结果发现HA蛋白可被大量表达,主要以包涵体形式存在,表达产物分子质量约64 ku,与理论推测值一致;菌液OD600值为1.0、IPTG终浓度为0.3 mmol.L-1、诱导时间4 h,其蛋白表达量最大,约占菌体蛋白总量的35%左右;通过免疫印迹试验发现,表达的蛋白可被禽流感H9亚型阳性血清特异性识别。研究结果表明,可以用原核表达系统对禽流感病毒血凝素蛋白进行表达,其产物具有免疫原性,可以用作禽流感抗体检测的免疫诊断试剂。更多还原

【Abstract】 The HA protein of avian influenza virus(AIV) was expressed by pET expression systems in E.coli.The Hemagglutinin(HA)gene of A/Turkey/Wisconsin/66(H9N2)strain has been cloned into a prokaryotic expression plasmid pET30a with 6-His tag.That recombinant vector pET30a-H9 was transformed into E.coli.BL21,which was induction with IPTG,and product was collected.The expressed protein was identified with SDS-PAGE and Western Blotting.When the OD600 value of the bacteria containing pET30a-H9 was 1.0,the recombinant protein was highly expressed in E.coli BL21 at four hours later induced by 0.3 mmol·L-1 IPTG in the form of inclusion bodies.And the protein is about 64 Ku in size,the rate of the expressed protein in the induced bacteria protein was about 35%.Western-bolt analysis with AIV antibodies against H9 subtype showed the recombinant protein was distinguished clearly and specially.更多还原