人胰岛细胞自身抗原69ku蛋白基因的分离及重组质粒鉴定

Separation of Full-Length cDNA Encoding Human Islet Cell Autoantigen 69ku Protein and Identification of Recombinant Plasmid

目的:应用聚合酶链技术获取作为基因探针和体外表达研究所必需的人胰岛细胞自身抗原69ku蛋白(hICA 69)编码基因,为探讨该基因在不同种系间和(或)组织来源间的差异打下基础。方法:分别从正常白人胰腺细胞和国人胰岛细胞瘤Poly(A^+)-RNA中逆转录合成编码人胰岛细胞自身抗原69ku蛋白(hICA 69)的全长cDNA,以双链cDNA(ds cDNA)为模板,经聚合酶链反应(PCR)特异性扩增出hICA 69基因的编码序列。利用基因重组技术,将纯化的目的基因插入pSPORT1质粒的多克隆位点,经限制性内切酶加以初步鉴定。结果:成功地扩增了hICA 69基因,并正确地克隆进pSPORT1载体,酶切鉴定筛选出正向插入重组子,其结果与预期值一致。结论:hICA 69基因的克隆为应用基因工程技术生产胰岛细胞自身抗原、生产适用于1型糖尿病诊断的试剂盒打下一定的基础。

Objective: As an essential gene probe and the important expression research in vitro, the encoding genes of hICA69 were obtained by using the polymerase chain reaction. This investigation would be able to lay a foundation for exploring the deviation of their nucleotide sequences between the races and/or the original tissues. Methods:By means of reverse transcrip-tion, it has been successful in synthesizing the encoding sequences of islet cell autoantigen 69 ku gene (ICA 69) from the pancreatic cells of Caucasian (the white race) and the insulinoma cells of Chinese (the Mongolian race). With double-strand cDNA (ds cDNA) as template,cDNA encoding human ICA 69 was amplified by PCR. After recovery from gel and purifica-tion cDNA fragment were inserted into the multiple cloning sites of the pSPORT 1 plasmids by the gene recombinant tech-niques, and the recombinant plasmids were identified using the restriction endonuleases. Results:The complete fragments encoding hICA 69 gene were amplified,and cDNA for hICA 69 gene was inserted into the pSPORT 1 vectors via ligation. The positive recombinants were selected by means of enzyme cleavage identification,and the results were coincident with the expectation. Conclusion:The cloning of hICA 69 gene would be possible to lay a foundation for obtaining islet cell autoantigen with gene engineering technique and producing diagnosis kits for type 1 diabetes mellitus.