Recombinant PIG11 Gene Retroviral Vector Construction And The Induced Apoptosis Effect Of Its High Expression In HepG2 Cells

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 after transfection, titer reached 1×105cfu/ml. Using RT-PCR and western blot detected infected HepG2 cells, which indentified PIG11 mRNA and PIG11 protein both up-regulated. Continue experiments indicated that the high expression PIG11 HepG2 cells can see much more apoptotic cells in cytomorphology and find clearing DNA ladder. The result of flow cytometry also expressed the transfected cell type apoptotic rate was significantly step up to 41.13%±2.29% , compared with the untransfected cells 3.33%±0.81 %and transfected zero load cells 3.53%±0.40% (P<0.01).Conclusion: The retroviral vector pLXSN-PIG11 was successfully constructed and infected HepG2 cells, obtain a new HepG2 cell line which highly express PIG11 protein and PIG11 gene can promote cell apoptosis.