Construction Of Recombinant Lentiviral Multiple Expression Plasmid Of Human TGFb3, CTGF And TIMP1 Genes And Detection Of Activity

Objective:To construct the recombinant plasmid of lentivirus containing human transforming growth factorB3(TGFB3), connective tissue growth factor(CTGF) and tissue inhibitor of metalloproteinases(TIMP1), and then to observe the expression in transfected293cells and investigate its characteristics and patterns.Methods:we have used2A self-cleaving sequences to ligate the cDNAs of relative genes in a single open reading frame derived from plasmid of lentivirus. Then detect the expressions of relative mRNA and protein using RT-PCR and Western-blot techniques in different times after transfection.Results:the expressions of relative mRNA and protein of relative genes were detected, and the expression reached peak value48hours after transfection, and the protein level of downstream gene was about80%of upstream gene.Conclusion:The recombinant plasmid, pLVX-TGFB3-P2A-CTGF-T2A-TIMP1, was constructed successfully, providing a basic model for the packaging of virus and further study on therapy of intervertebral disc degeneration.