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目的 克隆人ＬＩＧＨＴ基因，构建其重组腺病毒载体，以研究ＬＩＧＨＴ过表达与腺病毒感染对细胞生长的影响。方法用ＲＴ－ＰＣＲ法从人外周血单个核细胞（ＰＢＭＣ）中克隆人的ＬＩＧＨＴ全长基因。将ＬＩＧＨＴ ｃＤＮＡ克隆到穿棱载体ｐＡｄＴｒａｃｋ－ＣＭＶ－ＬＩＧＨＴ重组质粒中，经酶切线性化后，将重组质粒ｐＡｄＴｒａｃｋ－ＣＭＶ－ＬＩＧＨＴ和骨架质粒ｐＡｄＥａｓｙ－１，以电穿孔法共转染大肠杆菌ＢＪ５１８３，获得重组腺病毒质粒。最后，将线性化的重组腺病毒质粒转染２９３细胞包装获得重组腺病毒，用荧光显微镜、ＰＣＲ及Ｗｅｓｔｅｒｎ ｂｌｏｔ分析ＬＩＧＨＴ基因的表达。 结果 用ＲＴ－ＰＣＲ法从人的ＰＢＭＣ中，扩增出７２３ ｂｐ的ｃＤＮＡ，测序证实为人ＬＩＧＨＴ基因。荧光显微镜、ＰＣＲ及Ｗｅｓｔｅｒｎ ｂｌｏｔ证实，ＬＩＧＨＴ重组腺病毒可感染２９３细胞，并在２９３细胞内进行有效的复制。结论成功地克隆了人ＬＩＧＨＴ基因，并构建了其重组腺病毒载体，为进一步研究ＬＩＧＨＴ基因的功能提供了条件。
Objective To clone human LIGHT gene and construct its recombinant adenovirus vector to study the effect of LIGHT overexpression and adenovirus infection on cell growth. Methods Human LIGHT full-length gene was cloned from human peripheral blood mononuclear cells (PBMCs) by RT-PCR. The LIGHT cDNA was cloned into the vector pAdTrack-CMV-LIGHT. After linearized by restriction enzyme digestion, the recombinant plasmid pAdTrack-CMV-LIGHT and the backbone plasmid pAdEasy-1 were co-transfected into E. coli BJ5183 by electroporation, The recombinant adenovirus plasmid was obtained. Finally, the linearized recombinant adenovirus plasmid was transfected into 293 cells to obtain the recombinant adenovirus. The expression of LIGHT gene was analyzed by fluorescence microscope, PCR and Western blot. Results A 723 bp cDNA was amplified from human PBMC by RT-PCR and sequenced to be human LIGHT gene. Fluorescence microscopy, PCR and Western blot confirmed that LIGHT recombinant adenovirus infects 293 cells and efficiently replicates in 293 cells. Conclusion The human LIGHT gene was successfully cloned and its recombinant adenovirus vector was constructed, which provided conditions for the further study on the function of LIGHT gene.