ALK5激活型突变体在肾癌细胞中的表达及功能

The expression and functional vertification of constitutively activated ALK5 in kindney cancer cells

目的:构建ALK5激活型真核表达质粒Flag-ALK5 T204D,证实融合蛋白在肾癌细胞内表达,并验证其对TGF-β信号通路的激活作用。方法:以野生型ALK5质粒为模板,采用重叠延伸PCR方法进行定点突变;将构建的重组质粒测序并转染到肾癌细胞ACHN中,提取细胞蛋白进行Western blot检测;利用双荧光素酶报告基因分析,检测该突变体对TGF-β信号通路典型靶基因启动子的激活作用。结果:对ALK5的表达序列进行定点突变,构建了ALK5激活型真核表达质粒Flag-ALK5 T204D;Western blot检测到融合蛋白的表达,分子量约为58k Da;Flag-ALK5 T204D能够上调TGF-β信号通路典型靶基因启动子活性。结论:成功构建了Flag-ALK5 T204D并验证了其对TGF-β信号通路的激活作用,为进一步研究ALK5的生物学特性及其功能奠定了基础。

Objective： To construct site-directed mutation plasmid of Flag-ALK5 T204 D,and identify the expression and activity of this recombinant protein. Methods： Mutation was constructed by overlap-extension PCR method. The plasmid pRK-Flag-ALK5 was used as template. After the target region was sequenced,the plasmid was transfected into ACHN cell line. The expression of the recombinant plasmid was proved by Western blot. Identify the activity of TGF-β target gene promotor by the mutated ALK5 with Dual-Luciferase reporter assay. Results： By site-directed mutation,the Flag-ALK5 T204 D plasmid was constructed. The expression of Flag-ALK5 T204 D fusion protein was detected by Western blot with a molecular weight 58 k Da. Flag-ALK5 T204 D up-regulated the activity of TGF-β target gene promotor. Conclusion： The construction of ALK5 T204 D and establishment its activity on TGF-β signal pathway,provide a foundation for further studies on ALK5.