人PPARγ1受体cDNA全长序列的克隆及测序

Cloning and sequencing of the full-length of human PPARγ1 cDNA

目的对人过氧化物酶体增殖物激活受体γ1(hPPARγ1)全长cDNA序列进行克隆并测序,为构建含hPPARγ1基因真核表达载体奠定基础。方法通过逆转录-聚合酶链反应(RT—PCR)从HepG2细胞总RNA克隆hPPARγ1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XhoI、SmaI双酶切及基因测序筛选鉴定阳性克隆pMD19-hPPARγ1-T载体中hPPARγ1基因的完整性和忠实性。结果经酶切和测序证实,pMD19-hPPARγ1-T载体中插入的hPPARγ1基因序列与GeneBank中提交的序列一致。结论成功克隆了hPPARγ1基因,构建了pMD19-hPPARγ1-T中间载体。

[Objective] To clone and sequence the full-length of human PPARγ1 eDNA in order to further construct the eukaryotic expression vector carrying hPPARγ1 gene, [Methods] hPPARγ1 cDNA was cloned from HepG2 cells total RNA by RT-PCR, The PCR product recovered from gel was ligated with pMD19-T vector and transformed into DH5α competent cell, The integrity and fidelity of hPPARγ1 eDNA sequence inserted in T vector were verified by Xho Ⅰ,Sma Ⅰdouble excising and DNA sequencing assays. [Results] The positive clone T vector plasmid containing correct sequence of hPPARγ1 eDNA were verified by enzyme digestion as well as se quence analysis and was named as pMD19- hPPARγ1-T vector. The sequence of inserted hPPARγ1 cDNA was in accordance with the corresponding sequence in GeneBank database. [Conclusion] Successfully clone hPPARγ1 gene and construct the pMD 19-hPPARγ1-T intermediate vector.