人源性MIF cDNA的克隆及其原核高效表达的研究

目的克隆人MIF cDNA并构建其高效原核表达载体,表达和纯化得到高纯度的可溶性hMIF蛋白.方法经RT-PCR从人乳腺癌细胞株MDA-MB453中扩增到hMIF cDNA,并克隆到原核表达载体pET11b中.测序验证后将其转入大肠杆菌BL21中表达,最后经离子交换和疏水层析法纯化hMIF蛋白.结果克隆到的hMIF cDNA与Genebank已发表的序列完全一致,纯化得到的产物经Western blotting鉴定证实为hMIF蛋白.结论应用基因重组技术成功构建了pET11b/hMIF重组质粒,在大肠杆菌中实现了高效表达;纯化到高纯度的可溶性hMIF蛋白,为治疗性抗hMIF抗体的研究创造了条件.

Cloning and expression of human macrophage migration inhibitory factor

Objective To clone the cDNA of human macrophage migration inhibitory factor (hMIF), construct its prokaryotic expression vector and purify hMIF protein. Methods RT-PCR was used to amplify the cDNA of hMIF from the total RNA of human breast cancer cell line MDA-MB453. The DNA fragment was cloned into vector pET11b. After its sequence was verified, recombinant plasmid was expressed in E.coli BL21 (DE3). The expressed product was purified through cation exchange and hydrophobic interaction chromatography and identified by Western blotting. Results A 353 bp-length DNA fragment was obtained by RT-PCR amplification and sequence analysis, indicating that the DNA was identical to the hMIF sequence reported in GenBank. The purified protein was proved to be soluble hMIF protein by Western blotting. Conclusion Full length hMIF cDNA was cloned from human breast cancer cell line MDA-MB453, and expressed in prokaryotic cells successfully. Purified soluble hMIF protein provides a good basis to prepare anti-hMIF antibody or humanized antibody.