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目的构建大鼠维生素D受体(VDR)蛋白表达载体,测定并分析遗传性高钙尿性结石(GHS)大鼠VDR cDNA序列。方法采用半定量逆转录-聚合酶链反应(RT-PCR)方法扩增含VDR蛋白编码基因序列,将扩增产物克隆至真核表达载体pcDNA3．1/Zero(+),对5只GHS大鼠和4只正常尿钙对照(NC)大鼠十二指肠VDR cDNA序列进行测序分析。结果琼脂糖凝胶电泳示PCR扩增产物碱基数目与目的片段大小一致。对重组质粒的分析表明,插入片段的序列与发表的VDR基因编码序列相同。5只GHS鼠和4只NC鼠肠VDR cDNA序列均有3个相同的位点不同于已公布的大鼠肠VDR cDNA序列：在256 bp位点：C取代G；569 bp位点：G代替A；1658位点：A替代G。另外,GHS鼠在1795 bp位点发生改变,G取代A,而NC鼠则无改变。结论大鼠维生素D受体蛋白编码序列被成功地克隆至表达载pcDNA3．1/Zero(+)上。GHS大鼠基因编码区的序列组成同NC大鼠相一致,但GHS鼠1795 bp位点的碱基改变未见于NC鼠。
Objective To construct the rat VDR protein expression vector and determine and analyze the VDR cDNA sequence of hereditary calculus calculi (GHS) rats. Methods The coding sequence of VDR protein was amplified by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The amplified product was cloned into eukaryotic expression vector pcDNA3.1 / Zero (+) and 5 GHS Mouse and four normal urinary calcium control (NC) rat duodenal VDR cDNA sequence analysis. Results The agarose gel electrophoresis showed that the base number of the PCR amplification product was consistent with the size of the target fragment. Analysis of the recombinant plasmids revealed that the insert has the same sequence as the published VDR gene coding sequence. Three identical sites in the 5 VG cDNA sequences of 5 GHS and 4 NC rats differ from the published rat VDR cDNA sequence: at 256 bp: C for G; 569 bp for G: A; position 1658: A instead of G. In addition, GHS rats changed at 1795 bp, G replaced A, while NC rats showed no change. Conclusion The rat vitamin D receptor protein coding sequence was successfully cloned into pcDNA3.1 / Zero (+). The sequence of the coding region of GHS rat was consistent with that of NC rats. However, the base change of 1795 bp in GHS rats was not found in NC rats.