Yang Jia-Hui, Shen Qian
Department of Clinical Diagnosis, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2005 Jan;21(1):6-8.
To clone mouse FasL full-length cDNA and construct recombinant adenovirus expression vector.
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.
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.
The mouse FasL recombinant adenovirus prepared in this study can be used to study the role of FasL in tumor and autoimmune diseases.
克隆小鼠FasL全长cDNA并构建重组腺病毒表达载体。
从用5mg/L刀豆蛋白A刺激48小时的BALB/c小鼠脾单核细胞中克隆小鼠FasL全长cDNA，然后构建在CMV启动子控制下含有小鼠FasL全长cDNA的腺病毒穿梭载体mFasL-pAdTrack。用Pme I将穿梭载体线性化，并与腺病毒背景质粒载体pAdEasy-1共转化到大肠杆菌BJ5183中。随后通过限制性内切酶消化鉴定阳性重组体，并转染到HEK293细胞中以制备重组FasL腺病毒。
通过PCR、限制性内切酶消化分析和荧光检测表明，已成功克隆小鼠FasL全长cDNA并构建了重组mFasL腺病毒。
本研究制备的小鼠FasL重组腺病毒可用于研究FasL在肿瘤和自身免疫性疾病中的作用。
