人源蛋白酪氨酸磷酸酶SHP-2基因克隆与原核表达

目的:构建蛋白酪氨酸磷酸酶SHP-2的原核表达载体并在大肠杆菌中表达。方法:以人脑组织mRNA为模板,通过RT-PCR扩增出目标cDNA,构建蛋白酪氨酸磷酸酶SHP-2-pEASY-E1重组质粒。将重组质粒转化进E.coli TOP10感受态细胞中,通过菌落PCR和测序进行阳性克隆的筛选和验证,将正确的质粒转化E.coli Transetta感受态细胞中,通过SDS-PAGE和western-blot进行蛋白检测和验证,酶促动力学分析SHP-2可溶性蛋白的活性。结果:成功克隆SHP-2功能域,构建SHP-2-pEASY-E1原核表达载体,完成可溶性蛋白的表达;酶促动力学分析结果为:米氏常数Km=0.97mmol/L,Vmax为13.57mmol/L/s。结论:本研究成功构建SHP-2的原核表达载体,重组表达的SHP-2蛋白具有较高的磷酸酶活性。

Cloning and Prokaryotic Expression of Human Protein Tyrosine Phosphatase SHP-2*

Objective： This study is aimed to constructe prokaryotic expression vector of protein tyrosine phosphatase SHP-2 and express the soluble protein in E.coli. Methods： With mRNA from human brain tissue as a template, cloned SHP-2 gene with RT-PCR, constructed recombinant plasmid of the protein tyrosine phosphatase SHP-2-pEASY-E1. Recombinant plasmid was transformed into competence cell top 10, positive clones were screened and verificated by colony PCR and DNA sequencing, the right recombinant plasmid was transformed into Transetta competence cell, the expression protein was detected and validated with SDA-PAGE and western blot, the activity of soluble protein SHP-2 was analysed with enzymatic dynamics analysis. Results： cloned SHP-2 functional domain successfully, constructed the SHP-2-pEASY-E1 prokaryotic expression vector, obtained the soluble protein; Enzymatic kinetics analysis indicated： its Michaelis constant （Km） was 0.97 mmol/L, Vmax was 13.57mmol/L/s. Conclusions： this study constructed the SHP-2 prok- aryotic expression vector successfully, recombinant expression soluble protein of SI-IP-2 has the higher phosphatase activity.