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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: Polymerase chain reaction (PCR) was used to obtain the coding sequence of human islet antigen 69ku protein (hICA 69), which is necessary for gene probe and in vitro expression research. In order to investigate whether this gene is expressed in different lines and / or tissues Differences lay the foundation. Methods: The full - length cDNA encoding human pancreatic islets 69 ku protein (hICA 69) was reverse transcribed from normal human pancreatic cells and human islet cell tumor Poly (A ~ +) - RNA. As a template, the coding sequence of hICA 69 gene was amplified by polymerase chain reaction (PCR). Using gene recombination technology, the purified gene of interest was inserted into the multiple cloning site of pSPORT1 plasmid and initially identified by restriction endonucleases. Results: The hICA 69 gene was amplified successfully and cloned into pSPORT1 vector. The recombinant plasmid was identified by restriction enzyme digestion and positive cloned. The result was consistent with the expected value. Conclusion: The cloning of hICA 69 gene provides a basis for the production of a kit suitable for the diagnosis of type 1 diabetes using genetic engineering techniques to produce islet cells.