Construction of Recombinant Expression Plasmid plenti6N5-D-TOPO(R)- TβR Ⅱ DNglytk / 天津医药

ABSTRACT

Objective: To construct the lentiviral plenti6/V5-D-TOPO vector containing TβR Ⅱ Dnglytk and control vector by developing a lentivirus mediated gene transfer program incorporating a herpes simplex virus thymidine kinase(HSVtk) in the dominant negative TGF-β type Ⅱ receptor (TβR Ⅱ Dnglytk) expression vector. Methods: The PCR methods were used to amplify the genes TβR Ⅱ DN and HSV-tk from the respective plasmids. Then the genes were Linked by recombinant PCR technology to construct the fusion gene TβR Ⅱ Dnglytk and control vector TRANSglytk. According to the operation manual (from the Invitrogen company), ToPe cloning technology were used to construct the plasmids of plenti6/VS-D-TOPO(R)-TβRⅡ Dnglytk and plenti6/VS-D-TOPO -TRANSglytk. Both of the constructed plasmids were verified by sequencing.Results: The constructions of the plasmids of plenti6/V5-D-TOPO(R)-TβR Ⅱ Dnglytk and plenti6/V5-D-TOPO(R)-TRANSglytk were completed smoothly. The DNA sequencing results showed that both the plasmids were constructed correcdy and can be used in the production of infectious lentivirus vectors. Conclusion: Using TOPO cloning technology in the construction of the plasmids of plenti6/VS-D-TOPO -TβR Ⅱ Dnglytk and plenti6/VS-D-TOPO(R)-TRANSglytk and recombinant PCR in the fusion genes are feasible. The recombinant PCR combined with ToPe cloning technology can be the simple, highly efficient and rapid way to construct lentiviral vector and construction of plenti6/V5-D-TOPO(R)-TβR Ⅱ Dnglytk, which will lay a foundation for tumor immunotherapy.