小鼠FasL全长cDNA的克隆与FasL腺病毒载体的构建

目的: 克隆小鼠FasL全长cDNA, 构建FasL腺病毒载体。方法: 从经ConA(5mg/L)刺激 48h的BALB/c小鼠脾脏单个核细胞中克隆FasL全长cDNA, 构建以CMV启动子转录调控的含小鼠FasL全长cDNA的穿梭质粒mFasL pAdTrack。经PacI酶切后, 与腺病毒骨架质粒pAdEasy 1在大肠杆菌BJ5183中进行同源重组。挑选阳性重组子转染HEK- 293细胞, 构建CMV启动子转录调控的mFasL重组腺病毒载体。结果: 成功地克隆小鼠FasL全长cDNA, 经PCR、酶切及荧光检测等证实, 成功地构建了重组mFasL腺病毒载体。结论: 重组FasL腺病毒载体的构建为进一步研究FasL在肿瘤和自身免疫性疾病的作用奠定了基础。

Cloning of mouse FasL full-length cDNA and construction of its recombinant adenovirus vector

AIM: To clone mouse FasL full-length cDNA and construct recombinant adenovirus expression vector. METHODS: Mouse FasL full-length cDNA was cloned from BALB/c mouse's splenic mononuclear cells stimulated with ConA (5 mg/L) for 48 h, and then the adenovirus shuttle vector mFasL-pAdTrack containing mouse FasL full-length cDNA under the control of CMV promoter was constructed. The shuttle vector was linearized with Pme I and cotransformed into E.coli BJ5183 with pAdEasy-1, the adenovirus background plasmid vector. The positive recombinants were subsequently identified by restriction enzyme digestion, and transfected into HEK293 cells for preparing recombinant FasL adenoviruses. RESULTS: The mouse FasL full-length cDNA has been cloned and recombinant mFasL adenovirus has been constructed successfully, as shown by PCR, restriction endonuclease digestion analysis and fluorescence detection. CONCLUSION: The mouse FasL recombinant adenovirus prepared in this study can be used to study the role of FasL in tumor and autoimmune diseases.