人胰岛素样生长因子1前体基因的重组、表达及纯化  

Construction,expression and purification of recombinant human insulin-like growth factor-1 precursor

作　　者：姜苏蓉[1] 刘莉[1] 陈晨[1] 程蕴琳[1] 

机构地区：[1]南京医科大学第一附属医院老年医学科,210029

出　　处：《江苏医药》2006年第2期136-138,共3页Jiangsu Medical Journal

基　　金：江苏省卫生厅"135"重点医学工程基金(项目编号135-14)

摘　　要：目的构建含有人胰岛素样生长因子1(hIGF-1)前体编码基因的重组载体,在E.coliBL21中表达,并探讨其抗原性和应用价值。方法用PCR法,从现有重组质粒pBakPAK8/hIGF-1中,扩增编码hIGF-1前体的基因片段,经双酶切、纯化、连接后得到pET29a(+)/hIGF-1重组体,再将其转化大肠杆菌BL21-CodonPlus并诱导表达。表达产物经镍柱亲和层析纯化后,用Western blot法检测重组蛋白的抗原活性。结果重组持粒pET29a(+)/hIGF-1经双酶切鉴定和测序分析证实构建成功。在大肠杆菌BL21-CodonPlus中经IPTG诱导表达,有一相对分子质量15000的重组蛋白hIGF-1前体,该蛋白经镍柱亲和层析获得了良好的纯化效果,具有特异的抗原活性。结论成功地克隆并表达相对分子质量为15000的hIGF-1前体编码基因,为进一步制备抗hIGF-1抗体和后续hIGF-1生物学功能研究打下了良好基础。Objective To construct a recombinant vector containing gene encoding human insulin-like growth factor-1 （hIGF-1） precursor, express in E. coli BL21, and investigate its antigenicity and application. Methods By PCR technique,the target gene was amplified from pBakPAK 8/hIGF-1 and then inserted into the expression vector pET29a（＋）. The recombinant vector was expressed in E. coli BL21-CodonPlus after induced by IPTG. By Ni^2＋ affinity chromatography, purified recombinant protein was obtained and analyzed by Western blot. Results Enzyme digestion analysis and sequencing showed that the target gene had been inserted into the recombinant vector. After induced by IPTG,SDS-PAGE showed that relative molecule mass of expressed product was 15000 and after Ni^2＋ affinity chromatography, the highly purified protein with specific antigenicity was obtained. Conclusion The gene coding for hIGF-1 precursor is cloned and expressed successfully. The results lay a foundation for further research on its antibody and biological function.

关 键 词：人胰岛素样生长因子1前体 基因 抗原性 人胰岛素样生长因子1 亲和层析纯化 前体基因 重组载体 相对分子质量 WESTERN 编码基因 

分 类 号：R394.8[医药卫生—医学遗传学]