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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 construct a recombinant expression vector of two isoforms of von Hippel-Lindau (VHL) gene and study the relationship between isoforms and apoptosis signals. Methods The two isoforms VHL19 and VHL30 of VHL gene were cloned into the eukaryotic expression vector pcDNA3.1 respectively and sequenced. The 293 cells were transfected, and the relationship between the expression of Bax and Bcl-2 was analyzed by Western blotting. Results The recombinant plasmid was digested with restriction endonucleases (about 660 bp in pVHL30 and about 500 bp in pVHL19). The sequencing result showed that the target gene was exactly the same as the target gene. Western blot analysis showed that VHL19 and VHL30 were expressed in 293 cells. The expression of Bax and Bcl-2 in blank control, vector control and pcDNA3.1-VHL19, pcDNA3.1-VHL30 cells were not observed The difference. Conclusion High efficient expression plasmids of VHL19 and VHL30, two isoforms of VHL, were successfully constructed and highly expressed. When the intracellular VHL19, VHL30 expression of apoptotic signals Bax, Bcl-2 expression did not change significantly.