水泡性口炎病毒糖蛋白主要抗原决定区片段基因的原核表达和纯化

Expression and purification of protective antigen partial genes of vesicular stomatitis virus G protein

参照GenBank中水泡性口炎病毒(Vesicular stomatitis virus,VSV)G蛋白基因的核苷酸序列,设计合成1对特异性引物,采用RT-PCR方法扩增约为810bp的G基因片段,并将其克隆到pGEX-4T-1表达载体上,将经PCR、酶切鉴定以及测序分析正确的阳性重组质粒命名为PGEX-G810,之后将其转化至表达菌株E.coli BL21(DE3),经IPTG进行诱导表达;表达产物经SDS-PAGE、Western blotting等方法进行检测。SDS-PAGE结果表明,重组蛋白主要以包涵体形式存在,相对分子质量约为60 000;包涵体经变性、复性、Glutathione Sepharose 4B亲和层析纯化后,分离得到复性的融合蛋白。纯化的融合蛋白能与VSV阳性血清发生特异性免疫反应,证明表达的目的蛋白具有良好的抗原性。

According to the published sequence of vesicular stomatitis virus（VSV） G gene in GenBank,a pair of primers was designed and synthesized.The sequence of G gene about 810 bp was amplified by RT-PCR.The amplicon was inserted into pGEX-4T-1 vector,and the recombinant plasmid was identified by PCR amplification and digestion with restriction endonucleases,and then sequenced.The positive recombinant pGEX-G810 was transformed into E.coli BL21（DE3）.The expressed recombinant protein was identified by SDS-PAGE and Western blotting.The results showed that,the recombinant protein with a molecular weight of 60ku was soluble in the form of inclusion bodies.The inclusion bodies were denatured,refoloded and purified by glutathione sepharose 4B affinity chromatography.The purified protein could reacted to positive sera against VSV,indicating that recombinant protein had good antigenicity.