PRRS病毒核衣壳蛋白基因的原核表达

EXPRESSION OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS NUCLEOPROTEIN GENE IN ESCHERICHIA COLI WITH pGEX-6p-1 VECTOR

根据猪繁殖与呼吸综合征病毒 ( PRRSV)核酸序列设计 1对引物 ,用 RT-PCR扩增出美洲型 PRRSV的核衣壳蛋白 ( N)基因 ,克隆到 p GEM-T easy载体中 ,再亚克隆到表达载体 p GEX-6p-1谷胱甘肽 S转移酶 ( GST)基因的下游。重组质粒转化大肠杆菌 BL2 1 ,用 IPTG于 3 7℃条件下诱导 ,经 SDS-PAGE和 Western blotting分析 ,GST-N融合蛋白的分子质量约为 3 9.5 ku,另 3种不同大小的 GST-N降解产物 ,分子质量分别为 3 3 .0、3 0 .5和 2 7.5 ku,其中 3 9.5和3 0 .5 ku的降解产物分别占菌体蛋白的 3 0 .9%和 1 5 .3 %。结果表明 :PRRSV核衣壳蛋白基因在大肠杆菌中以 GST融合蛋白的形式得到高效表达 ,且表达产物能与 PRRSV抗体阳性的猪血清发生特异性反应 ,可作为

Porcine reproductive and respiratory syndrome virus (PRRSV) nucleoprotein gene, amplified by reverse transcription PCR was cloned into pGEM T easy vector, and subcloned into expression plasmid pGEX 6p 1. The nucleoprotein (N) fused to glutathione S transfease (GST) protein was expressed in Escherichia coli BL21 induced by IPTG at 37 ℃. The resulting GST N recombinant fusion protein was identified to be consisted of 4 protein bands such as 39 5, 33.0, 30.5 and 27.5 ku by SDS PAGE and western blotting analysis, in which the 39.5 ku full size fusion protein and its 30.5 ku proteolytic fragment occupied 30.9% and 15.3% of total bacterial protein respectively. In this research, the N protein expressed in E.coli could react with pig serum containing antibody against PRRSV, it indicated that the recombinant fusion protein could be used as antigen of diagnostic assay for detecting antibodies.