von Hippel-Lindau基因两种异构体在真核细胞中高效表达和对细胞凋亡信号Bax/Bcl-2的影响

Overexpression and impact on cell apoptosis signaling of two isoforms of von Hippel-Lindau gene in 293 cells

目的构建von Hippel-Lindau(VHL)基因的两种异构体的重组表达载体并研究异构体与细胞凋亡信号之间的关系。方法将VHL基因的两种异构体VHL19、VHL30分别克隆入真核表达载体pcDNA3.1中,测序鉴定。转染293细胞,通过蛋白印记分析细胞内两种异构体高效表达与凋亡信号Bax、Bcl-2之间的关系。结果重组质粒经酶切获得与目的基因大小相当的条带(pVHL30约660 bp,pVHL19约500 bp),测序结果显示与目的基因完全相同。蛋白印记分析结果显示VHL19和VHL30在293细胞中获得表达,Bax和Bcl-2在空白对照、载体对照和pcDNA3.1-VHL19、pcDNA3.1-VHL30各组细胞之间的表达均没有观察到明显的差异。结论成功构建VHL两种异构体VHL19、VHL30的高效表达质粒,并获得高效表达。当细胞内VHL19、VHL30高效表达时凋亡信号Bax、Bcl-2表达并没有明显变化。

Objective To investigate expression profile and impact on cell apoptosis pathway of two isoforms of yon Hippel-Lindau gene in human embryonic kidney cells （ 293 cells ）. Methods Two isoforms of VHL（ VHL19 and VHL30） were cloned into eukaryotic expression plasmid pcDNA3. 1 and transfect 293 cells after sequencing. Western blot assay was performed to analyze overexpression of the two isoforms and their impacts on cell apoptosis signaling pathway （ Bax and Bcl-2 ）. Results Recombinant plasmids were indicated sizable strips and identical genes with targeted genes after enzyme restriction and sequencing. Overexpression of the two VHL isoforms （ pcDNA3.1- VHL19 and pcDNA3.1-VHL30 ） were detected in 293 cells whereas Bax and Bcl-2 expression were observed no significant differences within controls and the two VHL isoforms. Conclusion Overexpression of the two isoforms of VHL （ VHL19 and VHL30 ） were observed and didnt cause significant changes on cell apoptosis signal Bax and Bcl-2 respectively.