大鼠维生素D受体蛋白表达载体的构建及遗传性高钙尿性结石大鼠十二指肠维生素D受体cDNA序列分析

Construction of rat VDR protein expression plasmid and analysis of GHS rats duodenum VDR cDNA

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 expression plasmid of rat VDR protein and analyze the genetic hypercalciuria stone-forming （GHS） rats duodenum VDR eDNA sequence. Methods The coding sequence of rat VDR protein gene was amplified by RT-PCR. The PCR product was cloned into the eu- karyotic expression vector pcDNA3.1/Zero（ ＋ ）. VDR eDNA sequences of 5 GHS rats and 4 normocalciuric control （NC） rats were analyzed. Results A specific band of 2 039 bp from PCR amplification was seen in gel electrophoresis. The sequence of the insert in the plasmid was identical to the published codingregion sequence of VDR gene. Differences in basepairs were discovered at three sites when the sequences of VDR eDNA from both normocalciuric and GHS rats were compared with the published rat intestinal VDR eDNA sequence：at bp 256 was C→G; at bp 569 G→A; and at bp 1 658,A→G. Alteration at bp 1 795,G→A,was also found in GHS rats but not in NC rats. Conclusion The coding sequence of rat VDR protein gene was successfully cloned into the eukaryotic expression vector peDNA3.1/Zero（ ＋ ）. Coding-region sequences of GHS rats and NC rats intestinal VDR gene are identical, but alteration of bp 1795 in GHS rats has not been found in NC rats.