人胰岛细胞自身抗原69kD蛋白表达型质粒的构建

摘要

Objective By means of the gene engineering technique, it would be possible to construct an expressive plasmid for ICA cDNA for providing the diagnosis kits for type 1 diabetes mellitus. Methods Total RNA were extracted from the pancreatic cells of Caucasian and the insulinoma cells of Chinese and cDNA of islet cell autoantigen 69kD genes (ICA69) were synthesized by superscript reverse-transcriptase and polymerase chain reaction (PCR). After determining its nucleotide sequence by the dideoxy chain termination method, the encoding fragment of hICA69 gene was demonstrated to be composed of 1449bp nucleotides which would then be inserted into the pGEX-2T expression plasmids. Results It was confirmed that the nucleotide sequence was correct on the conjunction site between the recombinant DNA 5＇ terminal multi-cloning site and recombinant fragment after the analysis of the nucleotide sequence.Conclusion This investigation would be able to lay a foundation for hICA69 gene expression and clinical application of expression products.%目的为应用基因工程技术生产适用于1型糖尿病早期诊断的试剂盒打下基础。方法从白种人胰腺细胞和中国人胰岛细胞瘤组织总RNA中，经逆转录-聚合酶链反应(RT-PCR)克隆了胰岛细胞自身抗原69kD蛋白(ICA69)基因的cDNA，双脱氧末端终止法对其全部核苷酸序列予以确定后，将此编码483个氨基酸、全长1449bp的cDNA重组入表达型质粒中。结果核苷酸序列分析证实重组质粒接口处读码框架正确。结论此构建为ICA69重组基因的表达及表达产物在临床诊断中的应用奠定了基础。