口疮病毒ORF020基因的原核表达与纯化

Prokaryotic expression of ORF020 gene from orf virus and purification of the expressed ORF020 protein

根据Gen Bank中口疮病毒ORF020基因序列,利用DNAMAN软件设计引物,以口疮病毒基因组为模板,采用PCR扩增出552bp的目的基因片段。凝胶回收纯化目的片段,将其连接入p MD20-T载体,转化E.coli DH5α并测序。测序正确后再其再连接入p ET-28a(+)表达载体,构建重组质粒p ET28a(+)-ORF020,转化入E.coli BL21(DE3),经IPTG诱导表达,并用SDS-PAGE和Western-blot鉴定诱导表达的目的蛋白,镍亲和层析纯化目的蛋白。结果表明,成功构建了原核表达载体p ET28a(+)-ORF020,并在E.coli BL21中表达了ORF020基因,诱导得到的融合蛋白与目的蛋白大小一致,证明目的基因被成功表达并得到纯化蛋白。上述研究结果为开展ORF020的功能研究奠定了一定基础。

The specific primers were designed by DNAMAN software according to ORF020 gene sequence of orf virus in GenBank,and then the ORF020 gene, which was 522 bp in size, was amplified by PCR from the orfV genome. After being purified,the ORF020 gene was inserted into pMD20-T vector to construct recombinant plasmid pMD2OT-ORFO20. The pMD2OT-ORF020 was transformed into E coil DH5- and identified by sequencing,then, the ORF020 was ligated into pET-28a （＋）. The recombinant plasmid pET28a （＋）-ORF020 was transformed into E. coil BL21（DE3） for expression under induction with IPTG. Lastly,the recombinant protein His-ORF020 was identified by SDS-PAGE and Western-blot. The expression product was purified by nickel affinity column. The results showed that the prokaryotic expression vector was successfully constructed, the ORF020 gene was expressed in E .coli BL21, and the recombinant 0RF020 protein was purified. The abovementioned results laid the foundation for study on the functions of the protein.