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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。
Objective: To construct prokaryotic expression vector of protein tyrosine phosphatase SHP-2 and express in Escherichia coli. Methods: Human brain tissue mRNA was used as a template to amplify the target cDNA by RT-PCR and to construct the recombinant protein tyrosine phosphatase SHP-2-pEASY-E1. The recombinant plasmids were transformed into E. coli TOP10 competent cells, and the positive clones were screened and verified by colony PCR and sequencing. The correct plasmids were transformed into E. coli Transetta competent cells and subjected to SDS-PAGE and western-blot Protein Detection and Validation, Enzymatic Kinetics Analysis of SHP-2 Soluble Protein Activity. Results: The SHP-2 domain was successfully cloned, and the prokaryotic expression vector SHP-2-pEASY-E1 was constructed to complete the expression of soluble protein. The kinetic kinetic analysis showed that the Michaelis constant Km was 0.97mmol / L, Vmax was 13.57mmol / L / s. Conclusion: The prokaryotic expression vector of SHP-2 was successfully constructed in this study. The SHP-2 protein expressed recombinant has high phosphatase activity.