重组hPPAR-α受体表达质粒的构建

Constructing of Receptor Expression Plasmid Recombinant hPPAR-α

目的构建人PPAR-α受体的重组表达质粒。方法从基因库获取人PPAR-α基因蛋白编码区全长序列,依照引物设计原则及编码序列上已有酶切位点设计引物并添加酶切位点。从正常肝组织细胞提取总RNA,逆转录得cDNA,PCR扩增目的片段,克隆到pcDNA3.1真核表达载体,测序鉴定重组质粒目的基因蛋白编码区全长序列及开放阅读框的正确性,命名为pcDNA-hPPAR-α。结果核酸测序结果表明所得pcDNA-hPPAR-α的蛋白编码全长序列与基因库一致,正向插入载体pcDNA3.1多克隆位点NheⅠ和KpnⅠ之间,开放阅读框正确。结论成功构建重组质粒pcDNA-hPPAR-α。

OBJECTIVE To construct recombinant human PPAR-αreceptor expression vector.METHODS Coding domain sequences of hman gene PPAR-αfrom Genebank data was obtained , and PCR primer was designed according to the principle of primer design and the present restricted enzyme cutting site in the sequence.Total RNA was isolated from normal hepatic tissue ,then cDNA from total RNA by reverse transcription was amplified to get aim fragment by PCR mothod.The fragment was inserted into the multiple cloning sites of pcDNA 3.1 expression vec-tor.Product was sequenced to verify the coding sequences and open read frame , and termed pcDNA-hPPAR-α.RESULTS Sequencing results of pcDNA-hPPAR-αindicated that the sequences of inserted DNA fragment were coincident with the coding sequences of human PPAR-αgene in Genebank data and inserted in correct norientation between NheⅠand KpnⅠ multiple cloning sites,and the open read frame is correct.CONCLUSIONRecombinant plasmid of pcDNA-hPPAR-αare constructed successfully.