615小鼠H-2K~k基因cDNA的克隆及序列测定和分析
The Cloning and Sequencing of H-2KkGene cDNA of 615 Mice
作者：李龙江,龚浩,陈俊杰,王若菡,温玉明,王昌美
Author：Li Longjiang, Gong Hao, Wen Yuming, et al
收稿日期：2002-10-25 年卷（期）页码：2002,20(05):313-
期刊名称：华西口腔医学杂志
Journal Name：West China Journal of Stomatology
关键字：主要组织相容性复合体,基因,克隆,小鼠,
Key words：H-2K+k gene,major histocompatibility complex typeⅠ,sequence clone mice,
基金项目：国家自然科学基金资助项目(编号39700162)
中文摘要
目的:克隆615小鼠主要组织相容性复合体(MHC)Ⅰ分子抗原识别基因H-2Kk并测序分析,为转基因治疗提供目的基因。方法:采用RT-PCR法从615小鼠肝组织总RNA中获得114 kb的H-2Kk基因cDNA,将其定向连接至 PGEM3Zf(+)载体,转化E.coliJM109,限制性内切酶筛选出阳性克隆PGEM3Zf(+)-H-2KkcDNA,末端终止法完成 H-2KkcDNA的全序列测定。结果:测得的H-2KkcDNA序列与文献报道序列同源性高达99%,编码MHC I分子抗原识别部位的氨基酸残基的核苷酸序列完全相同。结论:成功克隆了615小鼠MHCⅠ分子抗原识别基因,获得了表达MHC Ⅰ 功能的目的基因。
英文摘要
Objective:The purposes of this study were to clone and sequence the major histocompatibility complex typeÑ(MHCÑ) molecular antigen recognizing gene (H-2Kk) of 615 mice, and to provide the functional gene for transgenic therapy.Methods: The 1.4 kb full-length fragment of H-2Kkgene complementary DNA (cDNA) was amplified from the total RNAof 615 mouse liver by using reverse transcription polymerase chain reaction (RT-PCR). The cDNAwas inserted into PGEM3Zf(+) vector direction- ally, and the competentE.coliJM109 was transformed with the ligated product. The recombinant PGEM3Zf(+)-H-2KkcDNA plasmid was obtained using restricted enzyme analysis of the transfectants. The complete sequence of 615 mouse H-2KkcDNAwas determined by using Sangercs method.Results:The sequences of 615 mouse H-2KkcDNAwere 99% similarwith those of H-2Kk cDNAwhich were reported by other researchers, and the sequences encoding antigen recognizing regions (ARS) were identical with each other.Conclusion:The authors cloned the MHCÑmoleculur antigen recognizing gene (H-2Kk) of 615 mice success- fully and got the functional gene of MHCÑ.
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