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  

Construction of SHP-2 WT/mutant eukaryotic expression vector and identification of protein phosphatase activity

作　　者：胡中倩[1] 汪心怡[2] 李菲菲[1] 储著朗[1] 邢小翠[1] 李娟[1] 查欣[1] 瞿成奎[1,3] 汪思应[1] 

机构地区：[1]安徽医科大学病理生理学教研室,合肥230032 [2]安徽医科大学临床学院,合肥230032 [3]Case Western Reserve University,Cleveland OH 44106

出　　处：《安徽医科大学学报》2012年第7期767-771,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:30873046、30973424、81072663);安徽省留学回国人员科研项目(编号:2009-2011);安徽省学科学术带头人基金;安徽省博士点基金

摘　　要：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pcDNA3.1SHP-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pcDNA3.1SHP-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。Objective To establish eukaryotic expression vector of pcDNA3.1 SHP-2 wild-type （WT） and mutant SHP-2D61G（MT） successfully,and to provide a basis of the effects of SHP-2 tyrosine phosphatase on malignant bi- ological 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 PTPNll 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 identifica- tion; The NIH3T3 cells were transfected with vector of pcDNA3.1SHP-2WT and pcDNA3.1SHP-2D61G. The pro- tein 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 Ptpnl 1 cDNA was mutated at nucleotides 181 （ 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.

关 键 词：SHP-2 蛋白酪氨酸磷酸酶 真核表达 突变 

分 类 号：R363[医药卫生—病理学]