关键词: 克隆, 分子, 原核表达, 蛋白质纯化

Abstract: Abstract:Objective To clone the human TNF-like molecular 1A (TL1A) gene, and construct prokaryotic plasmid of TL1A and express it in E.coli, further more, to obtain high pure HCA661 protein.Methods The gene encoding TL1A was amplified using the total RNA of human umbilical vein epithelial cells (HUVECs) as template by RT-PCR, and inserted into pTA2 vector, then identified by restriction enzyme and sequencing.The recombinant expression plasmid PQE-TL1A was constructed and transferred into E.coli M15.The recombinant protein was expressed under the induction of IPTG, identified by Western blotting, purified by Ni-NTA affinity chromatography column.Results The identification of target gene by restriction enzyme was the same as the expectation.The sequence of target gene was identical with that registered in GenBank.With induction of IPTG, a new fusion protein with relative molecular mass of 22 000 was expressed and mainly located in inclusion bodies; the expressed 6×his-rhTL1A fusion proteins were identified by Western blotting with anti-His monoclonal antibody.Conclusion The　 recombinant prokaryotic expression plasmid pQE-Tl1A is constructed successfully, and recombinant TL1A protein with high purity coefficient is gained.

Key words: cloning, molecular, prokaryon expression, protein purification