COPS3基因cDNA的克隆及表达

目的:克隆COPS3基因cDNA,构建其原核融合蛋白表达载体并表达和鉴定。方法:从培养的人骨肉瘤细胞系SOSP-9607细胞中提取总RNA,经RT-PCR获得COPS3基因。将该基因克隆到pGEM-T-Easy载体中,酶切及测序鉴定。将COPS3基因插入pET28 a融合蛋白表达载体中,IPTG诱导表达,进行SDS-PAGE分析。免疫印迹法鉴定COPS3蛋白的表达。结果:cDNA测序证明,获得了COPS3基因cDNA,其序列与Genebank中报道序列完全一致。酶切分析表明,成功构建了含COPS3基因的pET28 a融合蛋白表达载体。SDS-PAGE以及免疫印迹法鉴定分析表明,COPS3蛋白获得高效表达,分子质量为51Ku,表达量约占菌体总蛋白的30%。结论:成功克隆和表达了COPS3 cDNA。

Cloning and expression of COPS3 cDNA

Objective: To clone,express and identify human COPS3 gene.Methods: Total RNA was extracted from human osteosarcoma cell line SOSP-9607,and the whole length gene of COPS3 was obtained by RT-PCR.The COPS3 gene was cloned into pGEM-T-Easy vector and sequenced.Then the gene was inserted to NdeI and BamHI site of pET28a expression vector.After the recombinant bacteria was induced with IPTG,the express protein was analysed by SDS-PAGE and western blot.Resuits: DNA sequencing result showed that COPS3 gene was exactly consistent with the sequence reported in genebank.SDS-PAGE and Western blot analysis demonstrated that COPS3 protein was expressed in E.coli,and the molecular mass of it is 51Ku.The protein band amounted to 30%of total bacteria protein.Conclusion: The results showed that COPS3 gene was successfully cloned and expressed.