重组人干扰素α2a和GFE-1多肽融合基因的克隆、表达及活性测定  

Recombinant IFN-α2a-GFE-1 fusion protein gene cloning,expression and biological activity

作　　者：谷仲平[1] 朱以芳[1] 张涛[1] 周勇安[1] 杨晔[1] 

机构地区：[1]第四军医大学唐都医院胸外科,陕西西安710038

出　　处：《现代肿瘤医学》2007年第11期1550-1554,共5页Journal of Modern Oncology

摘　　要：目的:构建IFN-α2a-GFE-1融合蛋白基因表达载体,获得高质量生物产品。方法:应用聚合酶链式反应技术(PCR)对干扰素(IFN)α2a3'端酶切位点进行改造,人工合成编码能特异性高效结合肺组织的GFE-1寡核苷酸片段,二者连接后克隆入pGEM3Zf质粒,进行序列分析,将融合蛋白基因克隆入pBV220表达载体,在大肠杆菌中表达,对IFN-α2a-GFE-1表达产物纯化、鉴定及体外活性检测。结果:利用PCR的方法成功地改造了IFN-α2a3'端酶切位点,成功的将GFE-1多肽与IFN-α2a3'端连接,构建了IFN-α2a-GFE-1融合蛋白的基因克隆载体pGEM3Zf-GFE-1,DNA测序完全正确。构建了IFN-α2a-GFE-1融合蛋白基因原核表达载体pBV220-IFN-α2a-GFE-1,经温度诱导在大肠杆菌中有效表达。纯化了IFN-α2a-GFE-1融合蛋白,SDS-PAGE凝胶电泳检测纯度大95%,比活性达5.8×109U/mg。结论:IFN-α2a-GFE-1融合蛋白基因的成功克隆、表达、纯化和活性测定,为研制一种具有导向性治疗肺癌、肺纤维化等疾病的有效药品奠定基础。Construct IFN - α2a - GFE - 1 fusion gene expression vector and obtain high quality products. Methods ： Amplified IFN - α2a gene and changed its enzyme digestion sites of C - terminus by PCR. Got a 13 - amino acid peptide GFE - 1 from phage - display library which can bind lung tissue specifically and effectively. Synthesized the nucleotide fragment encoding GFE- 1 and linked it with IFN -α2a, the fusion gene was cloned into pGEM -3zf and identified by DNA sequencing. Cleaved out the fusion gene from pGEM -3zf and cloned it into FBV220 expression vector. After expressed in E. coli, a purification procedure to obtain high quality fusion protein was set. Results： The enzyme digestion sites of C -terminus of IFN -α2a gene changed successfully and linked with GFE -1 gene. The fusion gene was cloned into pGEM -3zf cloning vector and identify positive clones with DNA sequencing, pBV220 - IFN - α2a - GFE - 1 expression vector was constructed and its expression induced in E. coli by temperature change. Purify the expressed fusion protein, SDS - PAGE showed that target protein purity was over 95 % and activity assay showed the activity was 5.8×10^9/ mg. Conclusion： IFN - α2a - GFE - 1 fusion protein was successfully cloned, expressed and purified, its activity was assayed. The research showed that it may be useful andeffective in targeted - drug therapy of diseases such as lung cancer and pulmonary fibrosis.

关 键 词：干扰素Α2A GFE-1多肽 融合蛋白基因 克隆 表达 

分 类 号：R730[医药卫生—肿瘤]