关键词: C肽, 多拷贝基因, 大肠杆菌, 表达

Abstract:

Objective To obtain recombinant prokaryotic expression plasmid containing multi-copied human C-peptide in order to increase the expression of C-peptide in Escherichia coli(E.coli).Methods DNA sequence containing multiple copies of human C-peptide was obtained through introducing the restricted enzymatic site to ensure the multiple C-peptide encoding gene ligat head-to-tail.After being identified with DNA sequencing,the DNA sequence was cloned into the pET-30a expression vector.Then the recombinant expression plasmid was transformed into E.coli,induced with IPTG and purified with Ni-NTA resin to obtain the fusion C-peptide.Results A gene fragment encoding five copies of C-peptide was synthesized and cloned into the pET-30a vector.After being transformed into E.coli,induced with IPTG and purified with Ni-NTA resin,the fusion C-peptide tagged to six histidine was obtained.The fusion protein was expressed at high level as a soluble product in the cytoplasm.Ni-NTA affinity chromatography efficiently separated the expressed fusion protein from the supernatant,to obtain about 20～40mg/L of the fusion protein with 80% purity.Conclusion High-level expression of human C-peptide was obtained,which should lay an important basis for the study of the function of C-peptide.

Key words: C-peptide, multi-copied gene, escherichia coli, expression