PIG11基因逆转录病毒载体构建及其高表达对HepG2细胞凋亡影响.pdfVIP

0.40% ，且差异有显著性（P0.01 ）。 结论：成功构建了逆转录病毒载体pLXSN-PIG11 ，获得稳定高表达PIG11 蛋白的HepG2 细胞株；PIG11 蛋白高表达能明显促进HepG2 细胞凋亡。 关键词：PIG11 ，逆转录病毒载体，HepG2 ，凋亡 5 Recombinant PIG11 Gene Retroviral Vector Construction and the Induced Apoptosis Effect of Its High Expression in HepG2 Cells ABSTRACT Objective: To construct the human PIG11 gene retroviral vector and obtain a HepG2 cell line high expressing PIG11gene by infection, which can be used for the further research on the function of PIG11 gene in liver cancer cell and the role of which to promote apoptosis. Explore the effect of high expression PIG11 gene can induce HepG2 cells apoptosis. Methods: Amplified PIG11 gene fragment by PCR from expression vector pcDNA3.1/NT-GFP-PIG11, after enzyme digestion and T4 DNA ligase to link, put this fragment inserted into retroviral vector pLXSN. Then this new vector pLXSN-PIG11 was identified by PCR and DNA sequencing analysis. Transfecting this vector into packaging cell PA317 mediate by lipofectamineTM2000, which can induce retrovirus release. The recombinant retroviral virus was collected and virus titer detected by infect NIH3T3 cell. Then we used this retrovirus infected HepG2 cell and achieved PIG11 gene high expression HepG2 cell line after G418 screening. The expression of PIG11 cells was detected by RT-PCR and PIG11 protein was analyzed by western blot. Using HE stain, DNA ladder and flow cytometry to observe HepG2 cell apoptosis when PIG11 gene high expression. Results: Obtained recombinant retroviral vector pLXSN-PIG11, which the same as PIG11 cDNA in GenBank report. The viral, released from packaging cell PA317