Construction Of Recombinant Adenovirus Vector Ad. TCRα12-2-IRES-Vβ7.1 And Study Its Cytotoxicity To Hepatoma

Objective To construct the recombinant adenovirus Ad.TCRα12-2-IRES-Vβ7.1,and study its cytotoxicity to hepatoma cell. The use of Ad.TCRα12-2-IRES-Vβ7.1 could perhaps be potent tools for cancer gene therapy.Methods First, construct the shuttle plasmid pDC315-TCRα12-2-IRES-Vβ7.1. TCRα12-2 gene was obtained from peripheral blood mononuclear cell (PBMC) by RT-PCR,and TCRVβ7.1 gene was obtained from pCDNA3.1- Vβ7.1.Then,the two genes were connected by IRES and then cloned into the shuttle plasmid of pDC315.Second,package and identify the recombinant ahenovirus Ad.TCRα12-2-IRES- Vβ7.1. pDC315-TCRα12-2-IRES-Vβ7.1 and backbone plasmid of adenovierus were cotransfered into HEK293 cells using Lipofectamine2000 to generate recombinant adenovirus.A replication-defective adenovirus Ad.TCRα12-2-IRES- Vβ7.1 could be made by homologous recombinant,which can simultaneously express TCRα12-2 gene and TCRVβ7.1 gene.The recombinant adenovirus was analyzed by PCR to test target genes;The expression of Ad.TCRα12-2-IRES-Vβ- 7.1 in Hela cells was confirmed by reverse transcription polymerase chain reaction procedure(RT-PCR).The protein TCRVβ7.1 was detected by flow cytometry in PBMC which was infected by Ad.TCRα12-2-IRES-Vβ7.1 in 48hs.The recombinant adenovirus were propagated on HEK293 cells,and viruses were tittered useing TCID50 method.Third,the effect of Ad.TCRα12-2-IRES-Vβ7.1 on BEL-7402 cell and HepG2 cell was measured by MTT and flow cytometry. PBMC ,which was infected by Ad.TCRα12-2-IRES-Vβ7.1and Ad-GFP,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 after 48 hours.Results The TCRα12-2 gene and TCRVβ7.1 gene were detected from genome of the recombinant adenovirus.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 PBMC was infected by Ad.TCRα12-2-IRES-Vβ7.1 was obviously higher than the PBMC group and Ad.GFP group.Conclusion Successfully construct the recombinant adenovirus, Ad.TCRα12-2-IRES -Vβ7.1,which can simultaneously express TCRα12-2 and TCRVβ7.1 genes.PBMC infected by TCRα12-2 and TCRVβ7.1 genes had strong killing effect on BEL-7402 cells and HepG2 cells.