小鼠MIF基因的原核表达及纯化

目的克隆小鼠巨噬细胞移动抑制因子(MIF)基因,在原核系统中表达并进行初步纯化,为研究MIF的功能奠定基础。方法提取小鼠肺组织总RNA,采用RT-PCR技术扩增MIF基因,克隆入pMD18-T载体,酶切鉴定及测序后,再定向插入原核表达载体pET-28a(+)中,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE及Western blot鉴定后,进行纯化。结果克隆得到的目的基因片段大小与预期相符,测序结果与GenBank中报道的序列完全一致。重组MIF蛋白相对分子质量约为15000,以包涵体形式存在,表达量约占菌体总蛋白的40%,且具有良好的反应原性。经初步纯化后,纯度达95%以上。结论已成功克隆了小鼠MIF基因,并在原核系统中表达了重组蛋白,纯化的蛋白纯度较高,可用于后续试验。

Prokaryotic Expression and Purification of Mouse Macrophage Migration In-hibitory Factor Gene

Objective To clone mouse macrophage migration inhibitory(MIF)gene,express in prokaryotic cells and purify the expressed product.Methods The total RNA of lung tissue of mice were extracted for amplification of MIF gene by RT-PCR.The amplified MIF gene was cloned into vector pMD18-T,and the constructed recombinant plasmid pMD18-M was identified by restriction analysis and sequencing.The target gene was recovered and cloned into expression vector pET-28a(+),and the constructed recombinant plasmid pET-28a-M was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot,then purified by Ni2+-NTA agarose column chromatography.Results The length of amplified MIF gene fragment was consistent with that expected,and the sequence was completely identical to that reported in GenBank.The expressed recombinant MIF,with a relative molecular mass of about 15 000,existed in a form of inclusion body and contained about 40% of total somatic protein.It showed good reactogenicity and reached a purity of more than 95% after purification.Conclusion Mouse MIF gene was successfully cloned and expressed in prokaryotic cells,and the expressed product reached a high purity,which laid a foundation of study on the function of MIF.