【摘要】 利用PCR技术扩增编码水疱性口炎病毒印第安纳型(VSV-Ind)糖蛋白基因的主要抗原表位区,通过引入其两端的EcoRⅠ和XhoⅠ酶切位点,定向插入到pET30c质粒T7启动子下游的多克隆区,构建重组质粒pET30c-vsvG,并转化至宿主菌BL21(DE3)株中。用1 mmol/L IPTG诱导后,SDS-PAGE电泳表明,重组蛋白在大肠埃希氏菌中得到了高效表达,经过4 h表达即可达到高峰;融合蛋白的相对分子质量为57 ku。重组蛋白含有六聚组氨酸尾,主要以包涵体形式存在,Ni柱纯化后的蛋白经Western-blotting鉴定,具有良好的抗原性和特异性。更多还原

【Abstract】 The glycoprotein gene of vesicular stomatitis virus Indiana（VSV-Ind） without signal and transmembrane region was amplified by PCR.The DNA sequenced EcoRⅠ and XhoⅠ restricted core fragment was cloned in a frame downstream of T7 promoter in the pET30c vector,then the recombinant plasmid pET30c-vsvG was transformed into E.coli BL21(DE₃).The recombinant fusion protein was expressed highly in E.coli BL21 four hours later induced by 1 mmol/L of IPTG and the molecular weight of the（fusion） protein was about 57ku.The recombinant protein containing a six-histidine tag aggregated into the inclusion body was purified by Ni-column,subsequently examined and analysed by SDS-PAGE and（Western-blotting） tests.The results showed the purified protein was of good antigenicity and specificity.更多还原