Construction Of Retrovirus Vector PLXPXSN-TCRα12-2-IRES-TCRVβ7.1 And Study Its Cytotoxicity To Hepatoma

Objective To construct a dicistronic retrovirus expression vector to coxpress both human TCRα12-2 and TCRVβ7.1genes and study the cytotoxicity to hepatoma cell. The use of Recombinant Retrovirus Vector pLXPXSN-TCRα12-2-IRES-Vβ7.1 could perhaps be potent tools for cancer gene therapy.Methods 1, construct the plasmid pLXPXSN-TCRα12-2-IRES-Vβ7.1. The TCRα12-2 and TCRVβ7.1 were amplified by PCR from plamid that we constructed before,which was cloned into vector pLXPXSN .2, package and identification of the recombinant retrovirus pLXPXSN-TCRα12-2-IRES-Vβ7.1. The plasmid pLXPXSN-TCRα12-2-IRES-Vβ7.1 was transfered into PA317 cells using Lipofectamine 2000 to generate recombinant retrovirus.A replication-defective retrovirus pLXPXSN-TCRα12-2-IRES-Vβ7.1 could be made by homologous recombinant,which can simultaneously express TCRα12-2 gene and TCRVβ7.1 gene.The recombinant retrovirus was analyzed by PCR to test target genes;The expression of pLXPXSN- TCRα12-2-IRES-Vβ7.1 in Hela cells was confirmed by reverse transcription polymerase chain reaction procedure(RT-PCR).The protein TCRVβ7.1 was detected by flow cytometry in PBMCs which were infected by pLXPXSN-TCRα12-2-IRES-Vβ7.1.The recombinant retrovirus were propagated on 3T3 cells,and the titer was measured. 3, the effect of pLXPXSN-TCRα12-2-IRES-Vβ7.1 on BEL-7402 cell and HepG2 cell was measured by MTT and flow cytometry. PBMC ,which was infected by pLXPXSN-TCRα12-2-IRES-Vβ7.1 and empty vector retrovirus,was added into the BEL-7402 cells and HepG2 cells . The killing effect on BEL-7402 cells and HepG2 cells was assayed by MTT and flow cytometry .Results The TCRα12-2 gene and TCRVβ7.1 gene were detected from genome of hela cells.Both the RT-PCR and flow cytometry had detected the expression of TCRα12-2 gene and TCRVβ7.1 gene. The killing effect on tumor cells in the group,in which PBMCs were infected by pLXPXSN-TCRα12-2-IRES-Vβ7.1 was obviously higher than the PBMC group and empty vector group.Conclusion Successfully construct the recombinant retrovirus pLXPXSN-TCRα12-2-IRES-Vβ7.1. By flow cytometry the TCRα12-2 and TCRVβ7.1 genes were expressed in PBMC transfected by retrovirus . PBMC infected by TCRα12-2 and TCRVβ7.1 genes had strong killing effect on BEL-7402 cells and HepG2 cells.