H9N2亚型禽流感病毒M2e蛋白在原核系统中的表达

摘要

以含有全长H9N2亚型AIV M2基因的质粒为模板,经PCR扩增得到M2e基因;利用BglⅡ和BamHⅠ之间互为同尾酶关系,构建顺次连接的多拷贝体M2e,并连接入原核表达载体pGEX-6p-1,构成2M2e-pGEX、4M2e-pGEX、6M2e-pGEX、8M2e-pGEX和10M2e-pGEX重组质粒,并转化至表达宿主菌中;将测序正确的菌液经不同浓度IPTG进行诱导,SDS-PAGE电泳鉴定重组蛋白大小,对蛋白进行可溶性分析;利用Western blot分析5种重组蛋白的反应原性.结果显示,在37℃下,2M2e-pGEX、4M2e-pGEX、6M2e-pGEX、8M2e-pGEX和10M2e-pGEX重组蛋白分别经终浓度为0.25、0.25、0.5、0.25、0.75 mmol/L的IPTG诱导4 h时,表达量最高;2M2e-pGEX、4M2e-pGEX、6M2e-pGEX、8M2e-pGEX和10M2e-pGEX重组蛋白大小分别为31.7、37.2、42.7、48.2、53.9 ku;对重组蛋白进行可溶性分析显示,5种重组蛋白均以包涵体形式存在;Western blot分析显示,2M2e-pGEX、4M2e-pGEX、6M2e-pGEX、8M2e-pGEX和10M2e-pGEX重组蛋白与鼠抗GST标签的单克隆抗体具有良好的特异性反应,为后期进一步获得高免疫原性蛋白和筛选具有通用免疫原性的重组蛋白奠定基础.%The plasmid containing the full length M2 gene of H9N2 subtype AIV was used as template for M2e gene amplification by PCR method;the various numbers of copies of M2e gene were constructed using the isocaudamer enzyme ligation of BglⅡand BamHI,and the various numbers of copies of M2e gene were connected into the vector pGEX-6p-1 forming five recombinant plasmid 2M2e-pGEX,4M2e-pGEX,6M2e-pGEX,8M2e-pGEX and 10M2e-pGEX;five recombinant plasmids were transformed into expression host strain,respectively.The bacterial liquid containing correct recombinant plasmids were induced by different IPTG concentrations and the sizes of the proteins were identified using SDS-PAGE method;the analysis of soluble protein was carried;the immuneoreactivities of five proteins were analyzed using Western blot method.The result showed that the expression levels of 2M2e-pGEX,4M2e-pGEX,6M2e-pGEX,8M2e-pGEX and 10M2e-pGEX reached the highest yield respectively under 0.25,0.25,0.5,0.25 and 0.75 mmol/L final concentration of IPTG after being cultured for 4 h at 37℃;the sizes of the recombinant proteins of 2M2e-pGEX,4M2e-pGEX,6M2e-pGEX,8M2e-pGEX and 10M2e-pGEX were 31.7,37.2,42.7,48.2 and 53.9 ku,respectively;five proteins existed in a form of inclusion body;Western blot analysis showed that the 2M2e-pGEX,4M2e-pGEX,6M2e-pGEX,8M2e-pGEX and 10M2e-pGEX proteins had a good specific reaction with anti-mouse monoclonal antibody against GST.These results lay a foundation for the further obtaining high immunogenicity protein and screening the universal immunogen.