人CTLA4Ig腺病毒载体的构建及其生物学功能研究

Construction and biological activities of CTLA4Ig adenovirus vectors

目的:通过同源重组构建CTLA4Ig腺病毒载体,研究其生物学活性,并用于基因治疗诱导心脏移植耐受。方法:通过基因重组技术,将CTLA4Ig基因克隆至腺病毒穿梭质粒pCA13中。然后将该质粒与腺病毒辅助质粒同源重组,经过293细胞包装,构建CTLA4Ig腺病毒载体。用RT-PcR、SDS-PAGE及Western blot等技术,检测包装的病毒感染293细胞后CTLA4Ig蛋白的表达及分泌,通过体外实验,观察病毒感染的293细胞上清对混合淋巴细胞反应(MLR)的抑制作用。通过大鼠体内实验,检测用CTLA4Ig腺病毒载体基因治疗后,CTLA4Ig在体内的表达。结果:CTLA4Ig腺病毒载体构建成功。体外实验证实,CTLA4Ig腺病毒感染的293细胞上清液,能够抑制异基因脾细胞的单向MLR。体内实验表明,经过静脉途径给予受体大鼠CTLA4Ig腺病毒载体,能够诱导移植耐受,延长心脏移植物的存活。结论:构建的CTLA4Ig腺病毒载体,体外感染293细胞能够分泌CTLA4Ig蛋白。该蛋白可抑制T细胞的活化,CTLA4Ig腺病毒载体可用于体内基因治疗诱导移植耐受。

AIM: To construct CTLA41g adenovirus vectors (AdCTLA4lg) by homologous recombination and study their activity, and to employ the vectors to induce cardiac transplantation tolerance by gene therapy. METHODS; CTLA4lg gene was cloned to pCA13 adenovirus shuttle plasmid by recombination strategy. Construction of CTLA4lg adenovirus vectors was performed by homologous recombination of pCA13 plasmid containing CTLA4lg gene with adenovirus helper plasmid, followed by packaged with 293 cells. Expression and secretion of CTLA4lg was confirmed by RT-PCR, SOS-PAGE and Western blot. The inhibitory effect of supernate of 293 cells infected with AdCTLA4lg on MLR in vitro was observed. A biological activity of CTLA4lg adenovirus vectors was determined by AdCTLA4lg gene therapy in rats in vivo. RESULTS; The Construction of CTLA4lg adenovirus vector was successful. It was confirmed that the supernatant of 293 cells infected with AdCTLA4lg could inhibit MLR in vitro. It was also showed that CTLA4lg adenovirus vectors could induce transplantation tolerance and prolong allograft survival when they were administrated in rats in vivo. CONCLUSION; CTLA4lg adenovirus vectors successfully constructed can infect 293 package cells and secrete CTLA4lg. The CTLA4lg protein can inhibit T cell activation. The CTLA4lg adenovirus vectors can be employed to gene therapy in vivo, and induce transplantation tolerance.