胰腺十二指肠同源框基因PDX-1的体外扩增

Clone of pancreatic duodenal homeobox-1

目的提取大鼠胰岛细胞瘤细胞株的RNA,以其中的mRNA为模板,扩增胰腺十二指肠同源框-1(PancreaticDuodenalHomeobox-1)编码基因,并测定核苷酸序列,为进一步克隆及表达PDX-1cDNA奠定基础。方法采用TIZOL方法进行RNA提取;通过RT-PCR方法扩增大鼠PDX-1cRNA;琼脂糖凝胶电泳检测后进行序列测定。结果从大鼠胰岛细胞瘤细胞中提取了RNA,以其中的mRNA为模板扩增出大鼠PDX-1基因,经琼脂糖凝胶电泳鉴定为目的片断,经序列测定与Genebank公布的序列一致。结论在国内首次成功的克隆了PDX-1基因。扩增时在5'端以限制性酶切位点进行限制,对进一步进行其克隆、表达研究以及对其生物学功能进行研究奠定了基础。

[Objective] RNA was extracted form insulinoma cell fine of rat, PDX-1 cDNA was amplified using mRNA as template and then nuclotide suquence was determined. All of this work is the basis of further study of cloning and expression of PDX-1 cDNA. [Methods] RNA was extracted by Trixol method; PDX-1 cDNA was amplified by RT-PCR technology, then nucleoside sequence was identified. [Results] RNA was extracted from the instdinoma cell line of rat, mRNA of RNA served as template. The segment was identified as aimed DNA by agarose electrophoresis, and necleotide wequence accorded to actual sequence. [Conclusions] Rat PDX-1 cDNA was amphfied by RT-PCR technology using mRNA from insuhnoma cell hne as template in china for the first time and cDNA 5＇ end was modified with cutting-site of restriction endonuclease, with is convenient for the cloning and expression of PDX-1 cDNA.