Gene cloning and prokaryotic expression of human CC-chemokine receptor 5

Aim:To construct a prokaryotic expression vector of human CC-chemokine receptor 5(CCR5) protein and to express it in E.coli.Methods:The total RNA was extracted from normal human PBMC and used as a template for RT-PCR.A 1 059 bp fragment containing the whole length of gene coding region of protein CCR5 was obtained and cloned into pGEM-T vector.The recombinant CCR5 which had analyzed in restriction endonucleases and PCR was sequenced to confirm its identity.The fragment of CCR5 form the right recombinant pGEM-T-CCR5 was ligated with vector pQE80L and transformed into E.coli DH5α,From which expressed the recombinant protein with identication.Results:After being induced by IPTG at 30 ℃ for 4 h,a recombinant protein CCR5 with an apparent MW of 41 000 was obtained in 120 g/L SDS-PAGE gel and accounted for 25% of the total cellular protein.The Western Blot showed that there was the specific binding of CCR5 mcAb with aim protein.Conclusion:Human CCR5 recombinant protein was obtained successfully.