人PPARα受体cDNA全长序列的克隆及测序

Cloning and sequencing of the full-length of human PPARα cDNA

目的对人过氧化物酶体增殖物激活受体α(hPPARα)全长cDNA序列进行克隆并测序,为构建含hPPARα基因真核表达载体奠定基础。方法通过逆转录-聚合酶链式反应(RT-PCR)从HepG2细胞总RNA克隆hPPARα全长cDNA序列,胶回收目的条带后与pMD19-T载体连接并转化DH5α感受态菌,用BamH I、Sal I双酶切及基因测序筛选鉴定阳性克隆pMD19-hPPARα-T载体中hPPARα基因的完整性和忠实性。结果经酶切和DNA测序证实pMD19-hPPARα-T载体中插入的hPPARα基因序列与GeneBank中提交的序列(NM001001928)一致。结论成功克隆了hPPARα基因,构建了pMD19-hPPARα-T中间载体,为含hPPARα基因真核表达载体的构建奠定了基础。

Objective To clone and sequence the full-length of human PPARa cDNA in order to further construct the eukaryotic expression vector carrying hPPARa gene. Methods hPPARa cDNA was cloned from HepG2 cells total RNA by RT-PCR. The PCR product recovered from gel were ligated with pMD19-T vector and transformed into DH5α competent cell. The integrity and fidelity of hPPARα cDNA sequence inserted in T vector were verified by BamH Ⅰ and Sal Ⅰ double excising and DNA sequencing assays. Results The positive clone T vector plasmid containing correct sequence of hPPARa cDNA were verified by enzyme digestion as well as sequence analysis and was named as pMD19- hPPARα-T vector. The sequence of inserted hPPARa cDNA was in accordance with the corresponding sequence in C, eneBank database （NM001001928）. Conclusions Successfully clone hPPARa gene and construct the pMD19-hPPARa-T intermediate vector ,which provide a good basis for further ccnstruct the eukaryotic expression vector carrying hPPARa gene.