血管扩张刺激磷蛋白磷酸化157位点突变的慢病毒载体的构建与鉴定

Construction and Identification of Lentiviral Vector Expressing VASP Phosphorylation Site Alanine157 Mutant

目的:构建含血管扩张刺激磷蛋白(VASP)磷酸化位点157突变成丙氨酸的慢病毒载体(L-S157A)并转染SGC-7901细胞。方法:针对VASP157位点的引物,利用重叠PCR的方法定点突变该位点从丝氨酸至丙氨酸,重组入慢病毒载体,包装病毒后感染SGC7901细胞,并利用测序和westernblot方法对突变进行验证。结果:VASP磷酸化157位点突变成丙氨酸,并且转导入293T和SGC-7901细胞,L-S157A明显下调SGC-7901细胞中p-VASPS157的表达。结论:成功地构建了针对VASP蛋白丝氨酸157位点的突变的表达载体,并获得了稳定表达该突变体的SGC-7901细胞株。为进一步研究VASP及其磷酸化在胃癌中的功能奠定了基础。

Objective：To construct a recombinant lentiviral vector carrying the mutagenesis of the VASP phosphorylation site alanine157（L-S157A）,and to transfect L-S157A into SGC-7901 cells.Methods：Design primers directed to VASP phosphorylation site 157,and overlapped PCR was applied to mutate the serine 157 to alanine,then the mutant was recombinated into lentivirus vector,packed lentivirus was transfected into SGC-7901 cells,gene sequence and western blot assay were applied to verify the mutant.Results：The VASP phosphorylation site 157 mutated to alanine,the L-S157A was transfected into 293T and SGC-7901 cells,L-S157A obviously decreased the p-VASP S157 expression in SGC-7901.Conclusion：We successfully constructed S157A expressing lentiviral vector and transfected it into SGC-7901 cells to obtain the genetically modified SGC-7901 cells.This study provided the basis for further investigating VASP and its phosphorylation in gastric cancer.