多拷贝人C肽基因的构建及其原核表达研究

目的构建多拷贝的人C肽基因,选用合适的表达载体,以多拷贝基因的形式在大肠杆菌中高效表达人C肽。方法采用PCR、克隆、DNA序列分析、IPTG诱导表达和亲和层析等方法构建多拷贝的人C肽基因。结果构建成含5拷贝C肽基因的重组pET-30 a表达载体,经转化及诱导,可在大肠杆菌中高效稳定表达可溶性的带6个组氨酸标签的C肽融合蛋白。结论在大肠杆菌中获得了人C肽的高表达,为下一步C肽的功能研究创造了重要条件。

Construction and Expression of Multi-copied Human C-peptide Gene in Escherichia Coli

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.