| 庞晓斌1 刘永真2 谢欣梅1 李晓婷1 赵清辉1 杜冠华3△.人源蛋白酪氨酸磷酸酶SHP-2 基因克隆与原核表达[J].现代生物医学进展英文版,2014,14(5):846-849. |
| 人源蛋白酪氨酸磷酸酶SHP-2 基因克隆与原核表达 |
| Cloning and Prokaryotic Expression of Human ProteinTyrosine Phosphatase SHP-2* |
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| DOI： |
| 中文关键词: SHP-2 基因克隆 蛋白表达 |
| 英文关键词: SHP-2 Gene cloning Protein expression |
| 基金项目:国家自然科学基金项目（81273652） |
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| 中文摘要: |
| 摘要目的：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。 |
| 英文摘要: |
| ABSTRACT Objective:This study is aimed to constructe prokaryotic expression vector of protein tyrosine phosphatase SHP-2 and
express the soluble protein in .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. Conclusion:this study constructed the SHP-2 prokaryotic
expression vector successfully, recombinant expression soluble protein of SHP-2 has the higher phosphatase activity. |
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