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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 lentiviral vector (L-S157A) with a vasodilation-stimulated phosphoprotein (VASP) phosphorylation site 157 mutated to alanine and transfected into SGC-7901 cells. Methods: According to the primer of VASP157, the site of this site was mutated from serine to alanine by overlapping PCR. The lentiviral vector was recombined and the virus was infected and infected with SGC7901 cells. The mutation was verified by sequencing and western blot. Results: The phosphorylation site 157 of VASP was mutated to alanine and transfected into 293T and SGC-7901 cells. L-S157A significantly down-regulated the expression of p-VASPS157 in SGC-7901 cells. CONCLUSION: A mutant vector targeting serine 157 of VASP protein was successfully constructed and a SGC-7901 cell line stably expressing the mutant was obtained. Which lays the foundation for further study on the function of VASP and its phosphorylation in gastric cancer.