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

作　　者：石锐[1] 包红梅[2] 姜永萍[2] 乔传玲[2] 陈化兰[2] 王秀荣[2] 陈维多[3] 

机构地区：[1]黑龙江民族职业学院,哈尔滨150081 [2]中国农业科学院哈尔滨兽医研究所农业部动物流感重点开放实验室及兽医生物技术国家重点实验室,哈尔滨150001 [3]东北农业大学生命科学学院,哈尔滨150030

出　　处：《东北农业大学学报》2011年第12期86-90,共5页Journal of Northeast Agricultural University

基　　金：国家科技攻关项目(2004BA519A23)

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。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.

关 键 词：禽流感 H9 原核表达 

分 类 号：S855.3[农业科学—临床兽医学] S852.65[农业科学—兽医学]