小鼠MIF基因的原核表达及纯化

Prokaryotic Expression and Purification of Mouse Macrophage Migration Inhibitory Factor Gene

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。

Objective To clone mouse macrophage migration inhibitor/ （MIF） gene, express in prokaryotie 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. eoli BL21 （DE3） for expression under induction of IPTG. The expressed product was identified by SDS-PAGE and Western blot, then purified by Ni^2＋-NTA agarose column chromatography. Results The length of amplified MIF gene tragment was consistent with that expected, and the sequence was completely identical to that reported in Gen- Bank. 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.