猪繁殖与呼吸综合征病毒SCQ株M蛋白基因截断片段原核表达载体构建及其表达的初步研究

为表达猪繁殖与呼吸综合征病毒(PRRSV)M蛋白主要抗原表位基因序列,参照GenBank中发表的PRRSV SCQ株M蛋白基因设计并合成一对特异性引物,通过PCR方法从重组质粒pMD18-T-M扩增得到缺失N端跨膜区的M蛋白基因片段dM(deleting M),将其与pMD19-T simple vector连接,经测序正确后克隆至高效原核表达载体pGEX-4T-1,得到重组表达载体pGEX-4T-1-dM,并将其转化大肠杆菌Rosetta(DE3),经IPTG于37℃诱导,PRRSV M基因获得表达。经SDS-PAGE分析,所表达的融合蛋白分子量约为35 kDa。以纯化的重组蛋白作为抗原,经Western Blot分析结果表明该重组蛋白可被PRRSV阳性血清所识别,可用于PRRSV的检测。

The Construction of Prokaryotic Expression Vector of M Protein Gene Truncated Fragment of Porcine Reproductive and Respiratory Syndrome Virus SCQ Strains and Preliminary Studies for its Expression

According to the PRRSV SCQ strain M gene nucleotide sequence reported in the GenBank,a pair of specific primers was designed and synthesized in order to successful expression of M protein of porcine reproductive and respiratory syndrome virus(PRRSV),the gene segment dM(deleting M)deleting N transmembrane domain sequence was successfully amplified from recombinant plasmids pMD18-T-M by PCR.Then,the dM gene was cloned into plasmid of PGEX-4T-1 and transformed into Rosetta(DE3).PRRSV M gene was expressed by IPTG induction at 37℃.SDS analysis showed that molecular weight of recombinant protein was about 35 kDa.The recombinant protein was analyzed by Western-blot,the result demonstrated that the recombinant protein was recognized by the PRRSV positive serum,so the M protein could be used for the detection of PRRSV.