SHP-2酪氨酸磷酸酶野生、突变型真核表达载体构建及其表达产物磷酸酶活性检测

摘要

Objective To establish eukaryotic expression vector of pcDNA3. 1 SHP-2 wild-type ( WT ) and mutant SHP-2D61 G( MT ) successfully, and to provide a basis of the effects of SHP-2 tyrosine phosphatase on malignant biological behaviors of tumor cells. Methods The SHP2 cDNA was obtained by the technology of RT-PCR from cells of mouse embryonic fibroblast ( MEF ), cDNAs encoding mutant SHP2 proteins were made from PTPN11 cDNA by site-directed mutagenesis and were cloned into pcDNA3. 1 vector. The recombinant plasmid pcDNA3. 1SHP-2-WT and pcDNA3. 1SHP-2D61G were constructed successfully, followed by sequencing after enzyme digestion identification; The NIH3T3 cells were transfected with vector of pcDNA3. 1 SHP-2 WT and pcDNA3.1SHP-2D61G. The protein expression levels of SHP-2 were tested by Western blot; pNPP and co-immunoprecipitation( IP )assay were used to study the protein phosphatase activity and protein expression of SHP-2 in stable transfected NIH3T3 cell lines. Results Sequencing and enzyme digestion identification showed that eukaryotic expression vector of pcDNA3. 1 SHP-2-WT and pcDNA3.1SHP-2D61G had been successfully established. The WT Ptpn11 cDNA was mutated at nucleotides 18l(G>T)by Sequencing. IP analysis revealed that the SHP-2 proteins was expressed in NIH3T3 cells when compared 48 h after transfection. Moreover, compared with WT group, protein phosphatase activity of the mutant SHP-2 was more significantly 2 times in NIH3T3 cells( P < 0. 01 ). Conclusion Eukaryotic expression vector pcDNA3. 1 SHP-2-WT and pcDNA3. 1SHP-2D61G are successfully established. The mutant SHP-2D61G can rise its phosphatase activity.%目的 构建pcDNA3.1 SHP-2野生型(WT)及SHP-2D61G突变型(MT)真核表达载体,为研究SHP-2激活突变对肿瘤恶性生物学行为的影响提供基础.方法 用RT-PCR 及定点突变法从小鼠胚胎成纤维细胞(MEF)中扩增出目的片段,双酶切后定向连入 pcDNA3.1载体,构建pcDNA3.1 SHP-2野生型及SHP-2D61G/+真核表达质粒,经酶切、测序检测其构建的准确性;Western blot法检测转染 NIH3T3细胞中SHP-2蛋白的表达水平;免疫共沉淀法获得表达在NIH3T3细胞中的SHP-2蛋白,用 pNPP法检测其磷酸酶活性.结果 构建的pcDNA3.1 SHP-2野生型及SHP-2D61G突变质粒经酶切初步检测后,测序检测发现MT SHP-2在181位核苷酸由WT的G突变为T,转染NIH3T3细胞株能够表达SHP-2蛋白;且转染MT的NIH3T3细胞株内SHP-2磷酸酶活性较WT组升高2倍(P<0.01).结论 成功构建了pcDNA3.1 SHP-2野生型及SHP-2D61G突变型真核表达载体,SHP-2D61G突变的蛋白磷酸酶活性升高.