HCCR-2基因的克隆及原核表达

目的:克隆HCCR-2基因、原核表达并鉴定。方法:从培养的人骨肉瘤细胞SOSP-9607中提取总RNA,经RT-PCR获得HCCR-2基因。将该基因克隆到pGEM-T-Easy克隆载体中,测序、鉴定。将测序正确的HCCR-2基因亚克隆到pET-28a( )表达载体中,构建HCCR-2表达载体,IPTG诱导表达1~5h,做SDS-PAGE分析,鉴定HCCR-2蛋白的表达。结果:DNA测序证明,获得了HCCR-2基因,其序列与GenBank中报道序列完全一致。SDS-PAGE分析表明,HCCR-2蛋白获得高效表达,其相对分子质量为39kDa,表达量约占菌体总蛋白的25%。结论:HCCR-2基因的克隆和表达均获得了成功,为进一步探讨HCCR基因在骨肉瘤诊治中应用奠定了基础。

Cloning and prokaryotic expression of HCCR-2

Objective:To clone, express and identify HCCR-2.Methods:Total RNA was extracted from human osteosarcoma cells and the full-length cDNA of HCCR-2 was obtained by RT-PCR. The HCCR-2 was cloned into pGEM-T-Easy vector and sequenced. Then the gene was inserted into BamHI and Sal I site of pET-28a( ) expression vector to construct the expression vector which was transformed into E.coil BL21. After the transformed bacteria were induced at IPTG for 1-5 h, the expressed protein was analyzed by SDS-PAGE.Results:DNA sequencing results showed that HCCR-2 was exactly consistent with the sequence reported in GenBank. SDS-PAGE analysis demonstrated that HCCR-2 protein was expressed in E.coli and its relative molecular mass was 39 kDa. The protein band amounted to 25% of total bacteria total protein.Conclusions:HCCR-2 has been successfully cloned and expressed. It lays a foundation for further studies of the applications of HCCR-2 in osteosarcoma diagnosis and therapy.