活化型及失活型n-Src真核表达载体的构建及活性鉴定

Construction of the eukaryotic expression vectors of active and inactive n-Src and identification of their kinase activity

目的 构建神经型酪氨酸蛋白激酶n-Src的活化型（aSrc,SrcY535F）及失活型（iSrc,SrcK303R）真核表达载体并鉴定其在HEK293T细胞中的表达及活性.方法 以野生型pRc/CMV-n-Src真核表达载体为模板,采用PCR定点突变技术分别扩增活化型及失活型pRc/CMV-n-Src全长序列,并转染入HEK293T细胞,通过免疫印迹检测n-Src的蛋白表达及其自身磷酸化水平.结果 aSrc及iSrc目的 基因成功扩增,且测序结果与预期结果一致.免疫印迹分析显示,aSrc及iSrc均能在HEK293T细胞中表达,aSrc自身磷酸化水平显著升高.结论 成功构建活化型及失活型n-Src真核表达载体,并能于HEK293T细胞中高效表达.

Objective To construct active type （aSre, SreY535F） and inactive type （iSrc, SrcK303R） eukaryotic expression vector of neural type tyrosine kinase n - Src, and to identify their protein expression and kinase activity in HEK293T cells. Methods PCR site - directed mutagenesis was used to amplify the active and inactive full - length pRc/CMV - n - Src using wild type pRc/CMV - n - Src as template. The recombinants were then transfected into HEK293T cells, and protein expression and autophosphorylation level were detected by Western blot. Results Target genes of aSrc and iSrc were successfully amplified and sequencing results were consistent with expectations. Western blot analysis showed that aSrc and iSrc vectors were expressed in HEK293T cells and aSrc presented higher autophosphoryla- tion level. Conclusion Activated and inactivated n - Src eukaryotic expression vectors were successfully constructed, and were highly expressed in HEK293T cells.