人胰岛素样生长因子-1的融合表达及纯化  

Fusion expression and purification of human insulin-like growth factor-1

作　　者：曹玉锋[1] 李霄培[1] 陈子杨[1] 王健 常东英[1] 贾媛[1] 张健锋[1] 吴菲[1] 王伟善[1] 时成波[1] 

机构地区：[1]长春生物制品研究所有限责任公司生物技术室,吉林长春130062 [2]长春祈健生物制品有限公司,长春130000

出　　处：《中国生物制品学杂志》2013年第12期1725-1728,1733,共5页Chinese Journal of Biologicals

摘　　要：目的利用原核表达系统融合表达人胰岛素样生长因子-1(human insulin-like growth factor-1,hIGF-1),并进行纯化及鉴定。方法根据大肠埃希菌密码子偏好性对hIGF-1天然基因序列进行同义突变,并人工合成,PCR扩增后,克隆至pET48b(+)载体,构建重组原核表达质粒,转化大肠埃希菌BL21(DE3),IPTG诱导表达。表达的融合蛋白Trx A-hIGF-1经Ni2+亲和层析进行纯化,纯化产物经肠激酶酶切后,进行Western blot鉴定。结果重组表达质粒pET48b-Trx A-hIGF-1经菌落PCR及测序证实构建正确;表达融合蛋白相对分子质量约为26 000,表达量约为菌体总蛋白的40%,主要以可溶形式存在于菌体裂解上清中,可溶性蛋白占总目的蛋白的比例不低于90%,纯化后纯度不低于90%,蛋白浓度为0.5 mg/ml;纯化的Trx A-hIGF-1融合蛋白可被肠激酶切割为相对分子质量约为8 000的hIGF-1蛋白和18 000的Trx A蛋白,hIGF-1蛋白可与兔抗hIGF-1多克隆抗体特异性结合。结论成功表达了hIGF-1融合蛋白,为其生物学活性的研究及规模化生产奠定了基础。Objective To express fusion human insulin-like growth factor-1 （hIGF-1） in prokaryotic cells, and purify and identify the expressed product. Methods The sequence of natural hIGF-1 gene was subjected to samesense mutation according to the E. coli preferred codons, synthesized amplified by PCR and cloned into vector pET48b（＋）. The construct recombinant plasmid pET48b-Trx A-hIGF-1 was transformed to E. coli BL21 （DE3） for expression under induction of IPTG. The expressed fusion protein Trx A-hIGF-1 was purified by nickel ion affinity chromatography, digested with enterokinase and identified by SDS-PAGE. Results Recombinant plasmid pET48b-Trx A-hlGF-1 was constructed correctly as proved by colony PCR and sequencing. The expressed fusion protein, with a relative molecular mass of about 26 000, contained about 40% of total somatic protein and mainly existed in a soluble form, of which the proportion of soluble protein to total target protein was not less than 90%. The fusion protein reached a purity of not less than 900/＊ and a concentration of 0. 5 mg/ml, which was digested with enterokinase into hlGF-1 with a relative molecular mass of about 8 000 and Trx A with a relative molecular mass of about 18 000. The hIGF-1 showed specific binding to rabbit anti-hIGF-1 polyclonal antibody. Conclusion The hIGF-1 fusion protein was successfully expressed, which laid a foundation of study on biological activity and large-scale production of hIGF-1.

关 键 词：胰岛素样生长因子-1 融合蛋白 原核细胞 基因表达 硫氧还蛋白A 

分 类 号：R587.1[医药卫生—内分泌] Q786[医药卫生—内科学]