人源性MIFcDNA的克隆及其原核高效表达的研究  

Cloning and expression of human macrophage migration inhibitory factor

作　　者：刘进军[1] 李鹏[1] 郭鹰[1] 潘自铁[1] 胡川闽[1] 

机构地区：[1]第三军医大学医学检验系临床生物化学教研室,重庆400038

出　　处：《第三军医大学学报》2005年第10期938-941,共4页Journal of Third Military Medical University

基　　金：重庆市科委自然科学基金资助重点项目 ( 2 0 0 4BA50 17) ;创伤烧伤复合伤国家重点实验室开放基金资助项目 ( 2 0 0 317)~~

摘　　要：目的　克隆人MIFcDNA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 MB45 3中扩增到hMIFcDNA ,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BL2 1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hMIFcDNA与Genebank已发表的序列完全一致,纯化得到的产物经Westernblotting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。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.

关 键 词：hMIF 乳腺癌 原核表达 蛋白纯化 

分 类 号：R392.11[医药卫生—免疫学] R394-33[医药卫生—基础医学]