Construction of recombinant expression plasmid pLenti6/V5-D-TOPO(R)-TβRⅡDNglytk and production of the lentivirus in the immunotherapy for prostate cancer / 中华内分泌外科杂志

ABSTRACT

Objective Our hypothesis was if we rendered host' s cytotoxic tumor lymphocytes insensitive to TGF-β,these immune cells could be able to overcome the TGF-β mediated immunosuppression and reject the tumor.We aimed to develop a lentivirus mediated gene transfer program incorporating a herpes simplex virus thymidine kinase(HSV-tk)in our dominant negative TGF-β type Ⅱ receptor(TβRⅡDNglytk)expression vector.So we first need to construct the lentiviral pLenti6/V5-D-TOPO vector containing TβRⅡDNglytk and produce the recombinant lentivirus as the transfection vector.Methods PCR were used to amplify the genes TβRⅡDN and HSV-tk from the respective plasmids.Then the genes were linked by recombinant PCR technology to construct the fusion gene TβRⅡDNglytk and control vector TRANSglytk.According to the operation manual from the Invitrogen company,TOPO cloning technology was used to construct the plasmids of pLenti6/V5-D-TOPO(R)-TβRⅡDNglytk and pLenti6/V5-D-TOPO(R)-TRANSglytk.Both of the constructed plasmids were verified by sequencing.ViraPowerTM Lentiviral System and 293 FT cells provided by Invitrogen were used to produce the recombinant lentivirus vector,the tillers of the lentivirus were determined by 293 cells.Results The construction of the plasmids of pLenti6/V5-D-TOPO(R)-TβRⅡDNglytk and pLenti6/V5-D-TOPO(R)-TRANSglytk were completed succefully.The DNA sequencing results showed that both the plasmids were constructed correctly.Using them we successfully produced infectious lentivirus vectors with appropriate tilters.Conclusions Using TOPO cloning technology in the construction of the plasmids of pLenti6/V5-D-TOPO(R)-TβRⅡDNglytk and pLenti6/V5-D-TOPO(R)-TRANSglytk and recombinant PCR in the fusion genes is feasible.Recombinant PCR combine with TOPO cloning technology can be a simple,highly efficient and rapid way to construct lentiviral vector.The production of infectious lentivirus with appropriate tilters using 293FT is suitable and feasible.The construction of pLenti6/V5-D-TOPO(R)-TβRⅡDNglytk and production of the infectious lentivirus will lay a foundation for immunotherapy of prostate cancer.