人胰岛素样生长因子前体基因的重组、表达及其蛋白纯化

目的：构建含有人胰岛素样生长因子（human insulin-like growth factor-1，hIGF-1）前体编码全长cDNA的重组载体，使其在E-coli BL21（DE3）中表达，并对此表达菌株所表达的重组蛋白进行纯化，同时探讨其抗原性及应用价值。方法：采用PCR法，扩增编码hIGF-1前体cDNA的片段，通过双酶切、胶回收纯化、连接后得到pET32a（＋）／hIGF-1重组载体，然后将其转化入BL21（DE3）中，经IPrG诱导后表达的重组蛋白经镍柱亲和层析纯化，并用Western blot法检测重组蛋白的抗原活性。结果：重组质粒pET32a（＋）／hIGF-1经双酶切鉴定和测序分析后证实构建成功。BL21（DE3）表达的重组蛋白经SDS-PAGE电泳后显示．其表达量占细菌总蛋白量的25％左右．该重组蛋白经镍柱亲和层析纯化后其纯度高达90％以上。Western blot结果显示非常清晰的免疫印迹条带，表明此重组蛋白具有免疫学特异性。结论：pET32a（＋）／hIGF-1重组载体构建成功，并能够高效表达。

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

Objective:To construct a recombinant vector containing the cDNA which encodes human insulin-like growth factor-1 precursor and to transfect it in E.coli BL21(DE3),then to purify recombinant protein,and to investigate the antigenicity and application of the recombinant protein.Methods:By using PCR technique,the target fragment which coded human insulin-like growth factor-1 precursor cDNA was amplified,and then inserted into the expression vector pET32a( ).Subsequently,the recombinant vector was transformed into BL21(DE3).Induced by IPTG,the recombinant protein was purified with Ni affinity chromatography.Its antigenic activity was assessed with western blot.Results:Enzyme digestion analysis and sequencing showed that the target fragment had been successfully inserted into the vector pET32a( ).SDS-PAGE showed that BL21(DE3)expression accounted for 25% of all protein the bacteria expressed.The purity of recombinant protein obtained by Ni affinity chromatography exceeded 90%.Western blot analysis showed it had immunology specificity.Conclusion:The recombinant vector pET32a( )/hIGF-1 has been constructed successfully,and it could express with high performance.