摘要
本试验旨在克隆并表达中国荷斯坦奶牛髓样分化蛋白-2(myeloid differentiation protein-2,MD-2)基因,并用West-ern blotting进行鉴定。采用RT-PCR技术从中国荷斯坦奶牛外周血白细胞中扩增MD-2cDNA,并将扩增产物与pMD20-T载体连接,重组质粒经测序鉴定后,构建pET28a-MD-2重组质粒,转化大肠杆菌BL21(DE3),经IPTG诱导表达后进行SDS-PAGE和Western blotting分析。结果表明,目的基因含有1个483bp的开放阅读框,编码161个氨基酸;与黄牛、黑猩猩和小鼠MD-2的cDNA序列同源性分别为99.38%、77.02%、68.12%,氨基酸同源性分别为98.75%、65.00%和58.13%。融合蛋白以包涵体的形式存在,分子质量约为25ku。说明本研究成功克隆并表达了中国荷斯坦奶牛MD-2cDNA,为其进一步的功能研究提供了理论依据。
In order to clone,express and identify myeloid differentiation-2 of Holstein dairy cow,total RNA was isolated from Chinese Holstein dairy cow,RT-PCR was used to amplify MD-2 cDNA.The PCR products were and cloned into pMD20-T vector and the recombinant plasmid was confirmed by PCR,endonuclease digestion.MD-2 was cloned into pET28a vector to construct the pET28a-MD-2,protein expression was induced by IPTG and analyzed by SDS-PAGE and Western blotting.The results showed that the MD-2 gene contain a 483 bp ORF encoding 161 amino acids;His-MD-2 fusion protein was expressed in E.coli BL21（DE3） with molecular weight of 25 ku after induced by IPTG.
出处
《中国畜牧兽医》
CAS
北大核心
2012年第2期20-24,共5页
China Animal Husbandry & Veterinary Medicine
基金
国家转基因生物新品种培育重大专项(2009ZX08007-009B)
