H9亚型禽流感病毒血凝素蛋白在原核系统中的表达

Expression of HA protein of H9 subtype avian influenza virus in prokaryotic system

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。

The HA protein of avian influenza virus（AIV） was expressed by pET expression systems in E.colL The Hemagglutinin（HA）gene of NTurkey/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.