Cloning of HCRTRI Gene by Overlap-PCR from Qinchuan Cattle and Its Expression in Escherichia coli

A 1 278 bp coding sequences of hypocretin receptor 1(HCRTR1)gene(GenBank accession No.:DQ874350)in Qinchuan cattle(Bos tams)was obtained by the Overlap-PCR methods and cloned into pMD18-T vector,and the positive clone plasmid(pMD18T-HCRTR1)was sequenced.Results showed that the cloned fragments had 98% homology with the sequence of GenBank after being blasted.After digested the plasmid with BamHⅠand HindⅢ,the fragment was ligated with the expression pET32 a(+)vector.The positive recombined plasmid(pET32 a(+)-HCRTR1)was transformed into Escherichia coli BI21(DE3)and induced with IPTG.SDS-PAGE result showed that the bovine HCRTR1 gene cannot be expressed in E.coli.And by bioinformatics analysis results indicated that the HCRTR1 protein was a member of G-protein-coupled receptor family which possessed seven transmembrane helices.The transmembrane structure of the HCRTRI protein was too complex to express in the E.coli.