【作者】 李龙江； 龚浩； 温玉明； 王昌美； 李声伟； 陈俊杰； 王若菡；

【Author】 Li Longjiang Gong Hao Chen Junjie Wang Ruohan Wen Yuming Wang Changmei Li shenwei Department of Oral Maxillofacial Surgery of West China College of Stomatology, Sichuan University Mollecullar Laboratory of West China College of Basic and Forensic Medicine, Sichuan University

【机构】 四川大学华西口腔医学院口腔颌面外科； 四川大学华西基础医学院分子生物学开放实验室；

【摘要】 <正>目的:人类和小鼠恶性肿瘤细胞的MHC Ⅰ表达降低,导致恶性肿瘤弱抗原性和免疫逃逸。拟克隆615小鼠MHC Ⅰ K基因座上的H-2Kk基因cDNA,以便进行恶性肿瘤的MHC Ⅰ转基因治疗研究。材料和方法:根据小鼠MHC Ⅰ基因K基因座的特征,设计合成引物,提取615小鼠肝组织总RNA, 逆转录,PCR扩增,分离回收1．4kb条带,EcoRI和BamHI双酶消化1．4kb目的基因cDNA平和更多还原

【Abstract】 Object: The expressing of MHC of malignant tumors has been depressed. This makes the tumor cell sneak through the immuno-surveillance. We attempted to clone the cDNA in the K locus of MHC I(H-2) of 615 mice so as to be employed to the MHC I transgenic research on malignant tumors in 615 mice. Materials and Methods: The H-2KkcDNA was amplified from the liver total RNA of a 615 mouse by RT-PCR with the K locus-specific primers. The cDNA was inserted into PGEM3Zf(+) vectors , and the competent E.coli JM109 were transformed by the ligated product. The recombinant plasmids were obtained by restricted enzyme analysis of the transfectants. The complete sequences of 615 mouse H-2KkcDNA were determined by Sanger’s method, and the sequence analysis was performed by the online Blastn query. Result: A 1.4kb cDNA band was acquired by RT-PCR. After the ligation and transformation, a recombinant with a 1.4kb insert , which had a same restricted map with the known H-2KkcDNA. was picked out . The online query of 615 mouse H-2KkcDNA sequences indicated: The sequences have 99% similarity with the known H-2KkcDNA whose accession number is U47330. Conclusion: The sequences of 615 mouse H-2KkcDNA which we cloned are credible and can be applied to MHC I transgenic research of malignant tumors. Because the 615 mouse H-2KkcDNA has same amino-residues in antigen recognizing sites with the known H-2KkcDNA, it can not be regarded as a new member of H-2 . The cDNA cloning of MHC by RT-PCR is sure and recommendable to the studies on MHC.更多还原