人真核表达慢病毒质粒载体plenti6/V5-D-TOPO~-TβR Ⅱ DNglytk的构建及意义

Construction of Recombinant Expression Plasmid plenti6/V5-D-TOPO~-TβR Ⅱ DNglytk

目的:构建含有TβRⅡ DNglytk的慢病毒质粒载体plenti6/V5-D-TOPO-TβRⅡ DNglytk,用含有TβRⅡ DNglytk质粒的慢病毒感染T淋巴细胞使其对转化生长因子-β(TGF-β)失敏感,恢复其对肿瘤的杀伤活性。方法:以原始质粒为模板,PCR扩增TβRⅡ DN和HSV-tk基因,运用重组PCR方法将2个基因连接起来,获得TβRⅡDNglytk融合基因,再以此基因为模板,PCR扩增获得其对照基因TRANSglytk。应用Invitrogen公司提供的系统,通过TOPO技术将2个融合基因分别连接到慢病毒表达质粒,并经过测序验证。结果:完成慢病毒表达质粒载体TβRⅡ DNglytk的构建,经测序验证序列正确,可以用于相应慢病毒的生产。结论:运用重组PCR技术和TOPO技术成功构建了plenti6/V5-D-TOPO-TβRⅡ DNglytk载体,为产生对TGF-β不敏感的人肿瘤特异性淋巴细胞,并用于肿瘤的免疫治疗提供了基础。

Objective：To construct the lentiviral plenti6/V5-D-TOPO vector containing TβRⅡ DNglytk and control vector by developing a lentivirus mediated gene transfer program incorporating a herpes simplex virus thymidine kinase（HSV- tk） 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）, TOPO cloning technology were used to construct the plasmids of plenti6/V5-D-TOPO-TβRⅡDNglytk and plenti6/V5-D-TOPO-TRANSglytk. Both of the constructed plasmids were verified by sequencing. Results： The constructions of the plasmids of plenti6/V5-D-TOPO-TβRⅡDNglytk and plenti6/V5-D-TOPO-TRADNSglytk were completed smoothly. The DNA sequencing results showed that both the plasmids were constructed correctly and can be used in the production of infectious lentivirus vectors. Conclusion： Using TOPO cloning technology in the construction of the plasmids of plenti6/V5-D-TOPO-TβRⅡDNglytk and plenti6/V5-D-TOPO-TRADNSglytk and recombinant PCR in the fusion genes are feasible. The recombinant PCR combined with TOPO cloning technology can be the simple, highly efficient and rapid way to construct lentiviral vector and construction of plenti6/V5-D-TOPO-TβRⅡDNglytk, which will lay a foundation for tumor immunotherapy.