前列腺癌免疫治疗中质粒pLenti6／V5-D-TOPO-TβRⅡDNglytk构建的意义及相应慢病毒的产生

目的构建含有TβRⅡDNglytk的慢病毒质粒载体，生产出相应的慢病毒，用于对TGF—β失敏感的CTL的制备。方法先以原始质粒为模板，PCR扩增TβRⅡDN和HSV—tk基因，用重组PCR方法获得TβRⅡDNglytk融合基因，用PCR扩增出TRANSglytk基因；再用TOPO克隆技术将其连接到慢病毒表达质粒，经测序验证；最后用Invitrogen公司提供的ViraPowerTMLentiviralSystem和293细胞合成慢病毒，并用293细胞完成其滴度测定。结果完成慢病毒质粒TβRⅡDNglytk的构建，经测序验证序列正确；生产的慢病毒具有感染能力，其滴度达到实验要求，叮用于对TGF—B不敏感的肿瘤特异性CTL的制备。结论运用重组PCR技术合成TβRⅡDNglytk及TRANSglytk两个融合基因，并用TOPO克隆技术连接到pLenti6／V5-D-TOPO载体，成功构建了慢病毒表达载体，说明此方法用于病毒载体的构建是快捷可行的；用ViraPowerTMLentiviralSystem和293FT细胞成功制备了慢病毒，为生产TGF—B不敏感的人肿瘤特异性CTL提供了基础。

Construction of recombinant expression plasmid pLenti6/VS-D-TOPO- TβRⅡ DNglytk and production of the lentivirus in the immunotherapy for prostate cancer

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 thy- midine kinase（HSV-tk） in our dominant negative TGF-βtype Ⅱ receptor （TβRⅡDNglytk） expression vector. So we first need to construct the lentiviral pLenti6/V5-D-TOPO vector containing TβRⅡ DNglytk and produce the recombinant lentivirus as the transfection vector. Methods PCR were used to amplify the genes TβRⅡ DN and HSV-tk from the respective plasmids. Then the genes were linked by recombinant PCR technology to construct the fusion gene TβRⅡ DNglytk 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-TβRⅡ DNglytk and pLenti6/V5-D-TOPO-TRANSglytk. Both of the constructed plasmids were verified by sequencing. ViraPow- erTM Lentiviral System and 293 FT cells provided by Invitrogen were used to produce the recombinant lentivirus vector, the tilters of the lentivirus were determined by 293 cells. Results The construction of the plasmids of pLenti6/V5-D-TOPO-TβRⅡ DNglytk and pLenti6/V5-D-TOPO-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-TβRⅡ DNglytk and pLenti6/V5-D-TOPO- TRANSg- lytk and recombinant PCR in the fusion genes is feasible. Recombinant PCR combine with TOPO cloning technolo- gy can be a simple, highly efficient and rapid way to construct lentiviral vector. The production of infectious lenti- virus with appropriate tilters using 293FT is suitable and feasible. The construction of pLe