人胰岛细胞自身抗原69ku蛋白基因的分离及重组质粒鉴定

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（hＩＣＡ６９）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（hＩＣＡ６９）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hＩＣＡ６９基因

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

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.