Overlap-PCR克隆秦川牛HCRTR1基因及其在大肠杆菌中的表达分析

通过Overlap-PCR方法克隆了秦川牛的HCRTR1基因编码区全长，获得1278 bp编码区全长序列，将其克隆至pMD-18T载体上，经测序表明获得的cDNA序列与GenBank公布的序列同源性均为98%。提取阳性克隆的质粒经BamHI和HindⅢ双酶切后，回收到1278 bp的目的片段，定向克隆到pET32a（+）表达载体上，连接产物转化Bl21(DE3)感受态大肠杆菌，对长出的菌落进行菌落PCR和质粒酶切鉴定正确后，用IPTG诱导目的蛋白的表达。SDS-PAGE表明秦川牛HCRTR1基因在大肠杆菌中不表达，生物信息学分析表明，HCRTR1基因的表达产物为具有7个跨膜螺旋的G蛋白偶联受体，可能是此类蛋白在原核系统的表达将会影响细菌本身存活，因此原核表达系统也成为研究此类蛋白的瓶颈。

The CDS Cloning of HCRTR1 Gene with Over-lap PCR from Qinchuan Cattle and its Expression in E.coli

With the Overlap-PCR methods, the 1 278 bp cds sequences of HCRTR1 gene in Qinchuan cattle was got. The fragments were cloned into the pMD18-T vector and the positive clone plasmids was sequenced. Results showed that the cloned fragments had 98% homology with the sequence of GenBank after being blasted. Digested the plasmid with BamHⅠand HindⅢ and the 1 278 bp was obtained. The obtained fragment was ligated with the longer fragment digested with the same restriction enzyme from the expression vector, pET32 a(+). The positive recombinated plasmid was transformed Bl21(DE3) and induced with IPTG. But the HCRTR1 gene cannot be expressed by E.coli. And the tranmembrane structure may explain why.