COPS3基因cDNA的克隆及表达

Cloning and expression of COPS3 cDNA

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。

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.