Vero E6细胞中B23.1基因的扩增及其原核表达

摘要

应用RT-PCR技术,利用含有酶切位点的上、下游引物扩增得到Vero E6细胞中编码B23.1蛋白的基因片段,并进行序列测定及分析,结果表明,B23.1基因含有一个长901 bp的开放阅读框(ORF),未发现碱基的插入和缺失.将其进行BamH I和Xho Ⅰ双酶切,而后定向克隆到经相同双酶切的pGEX-6p-1载体中,构建的重组质粒命名为pGEX-6p-B23.1；将该质粒转化入大肠杆菌BL21( DE3)中,在IPTG诱导下进行表达；SDS- PAGE结果表明,表达出与预期大小相符的约68 ku的重组蛋白,重组蛋白主要以包涵体形式存在；Western blotting分析结果表明,表达的重组蛋白与B23蛋白单克隆抗体有很好的反应活性.%A fragment containing t323.1 protein gene of Veto E6 cell was amplified by reverse transcriptase-polymerase chain reaction(RT-PCR)using the upper/lower primers each including the recognition sequence for BamH I and Xho I re spectively.The results showed that the ORF of B23.1 gene was composed of 901 hp,deletion and insertion were not found.By digestion with restriction enzymes BamH I and Xho I,the B23.1 gene were cloned into pOEX-6p-1 vector to construct recombinant plasmid named pGEX-6p-B23.1.The recombinant plasmid was transformed into E.Coil BL21(DE3)and induced with IPTG.The results of SDS-PAGE assay showed that the protein was largely expressed,the GST-tagged protein was puri-fied.Western blotting analysis showed that the target protein had a good reactivity with B23 monoclonal antibody(F/louse).