水牛MD-2 cDNA基因的克隆与原核表达

Cloning and Prokaryotic Expession of MD-2 from Buffalo

为探讨水牛抵抗革兰阴性菌感染的免疫机制,克隆、表达了水牛髓样分化蛋白-2(bMD-2)基因,并用Western blot进行鉴定。采用RT-PCR方法从水牛外周血白细胞中扩增bMD-2基因,构建pET28a-bMD-2表达载体,转化至大肠埃希菌BL21(DE3)中,IPTG诱导表达,进行SDS-PAGE和Western blot分析。结果显示,bMD-2基因含有一个483 bp的开放阅读框,编码161个氨基酸;37℃条件下,经1 mmol/LIPTG诱导表达6 h后,His-bMD-2在E.coliBL21中表达量最高,并以包涵体的形式存在,融合蛋白的分子质量约为25 ku。结果表明水牛MD-2原核表达载体成功构建并表达,为继续深入对水牛MD-2基因功能的研究提供参考。

This study was designed to clone and express buffalo myeloid differentiation-2,and then carry out Western blot for it.RT-PCR was used to clone CDS area of buffalo MD-2,and the PCR products of bMD-2 were cloned into pET-28a,and transformed into E.coli BL21.The bioinformatics analyses were performed.The protein expression was induced by IPTG and analyzed by SDS-PAGE and Western blot.The results showed that the bMD-2 CDS contain a 483 bp ORF,which encodes 161 amino acids,and His-bMD-2 fusion protein was expressed in E.coli BL21（DE3） with molecular weight of 25 ku induced at 37 ℃,6 h by 1 mmol/L IPTG,which indicated that prokaryotic expression vector of buffalo MD-2 was constructed and expressed successfully.