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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 inhibitory factor (MIF) gene, express it in prokaryotic system and preliminarily purify it, which lays the foundation for studying the function of MIF. Methods The total RNA was extracted from the lungs of mice. The MIF gene was amplified by RT-PCR and cloned into pMD18-T vector. The recombinant plasmid was identified by restriction enzyme digestion and sequencing. The MIF gene was inserted into prokaryotic expression vector pET-28a (+ BL21 (DE3), IPTG induced expression. The expressed product was identified by SDS-PAGE and Western blot, then purified. Results The size of the target gene cloned was in line with the expectation. The sequencing result was exactly the same as that reported in GenBank. The relative molecular mass of recombinant MIF protein is about 15000, which exists in the form of inclusion bodies. The expression level of recombinant MIF protein accounts for about 40% of the total bacterial protein, and has good reactionogenicity. After the initial purification, the purity of more than 95%. Conclusion MIF gene has been successfully cloned and expressed in prokaryotic system. The purity of the purified protein is high and can be used in subsequent experiments.