SRG基因的克隆及原核表达

目的克隆SRG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SRG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SRG基因亚克隆到pET-28(a )表达载体中,构建SRG表达载体,IPTG诱导表达2h~6h,做SDS-PAGE分析,鉴定SRG蛋白的表达。结果DNA测序证明,获得了SRG基因,其序列与GenBank中报道序列完全一致。SDS-PAGE分析表明,SRG蛋白获得高效表达,其相对分子质量为22kDa,表达量约占菌体总蛋白的25%。结论SRG基因的克隆和表达均获得了成功,为进一步探讨SRG基因在骨肉瘤诊治中应用奠定了基础。

Cloning and prokaryotic expression of SRG

Objective: To clone, express and identify human SRG gene. Methods:Total RNA was extracted from human osteosarcoma cells and the full length cDNA of SRG was obtained by RT-PCR. The SRG gene was cloned into pGEM-T-Easy vector and sequenced. Then the gene was inserted to BamHI and Sal I site of pET-28(a ) expression vector to construct the expression vector which was transformed into E.coil BL21.After the transformed bacteria were induced at IPTG for 2-6h the expressed protein was analyzed by SDS-PAGE. Results:DNA sequencing results showed that SRG gene was exactly consistent with the sequence reported in GenBank. SDS-PAGE analysis demonstrated that SRG protein was expressed in E.coli and the relative molecular mass of it was 22 kDa. The protein band amounted to 25% of total bacteria total protein. Conclusions:The results show that SRG gene has been successfully cloned and expressed. It lays a foundation for further studies of the applications of SRG in the osteosarcoma diagnosis and therapy.