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-2K^k Gene cDNA of 615 Mice

目的 :克隆 6 15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 6 15小鼠肝组织总RNA中获得 1 4kb的H_2Kk 基因cDNA ,将其定向连接至PGEM3Zf(+)载体 ,转化E coliJM10 9,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MHCⅠ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 6 15小鼠MHCⅠ分子抗原识别基因 。

Objective:The purposes of this study were to clone and sequence the major histocompatibility complex type Ⅰ (MHCⅠ) molecular antigen recognizing gene (H-2K+k) of 615 mice, and to provide the functional gene for transgenic therapy. Methods: The 1.4 kb full-length fragment of H-2K+k gene complementary DNA (cDNA) was amplified from the total RNA of 615 mouse liver by using reverse transcription polymerase chain reaction (RT-PCR). The cDNA was inserted into PGEM3Zf(+) vector directionally, and the competent E.coli JM109 was transformed with the ligated product. The recombinant PGEM3Zf(+)-H-2K+k cDNA plasmid was obtained using restricted enzyme analysis of the transfectants. The complete sequence of 615 mouse H-2K+k cDNA was determined by using Sanger′s method.Results: The sequences of 615 mouse H-2K+k cDNA were 99% similar with those of H-2K+k cDNA which were reported by other researchers, and the sequences encoding antigen recognizing regions (ARS) were identical with each other.Conclusion: The authors cloned the MHC Ⅰ moleculur antigen recognizing gene (H-2K+k ) of 615 mice successfully and got the functional gene of MHC Ⅰ.