利用重组酵母菌生产人类胰岛素--作业向 上传【荐】.docVIP

利用重组酵母菌生产人类胰岛素 某某人 114204XXXX 摘要：利用PCR技术合成重组人胰岛素基因，并在A链和B链之间加入Kex2酶切位点。将合成的重组人胰岛素基因以正确的阅读框插入到组成型分泌表达载体pGAPZα-A的α-factor信号肽序列的下游，构建成pGAPZα-A/Insulin重组分泌表达载体。表达载体用Bln I线性化处理后电击转化毕赤酵母GS115感受态细胞，构建成分泌型表达Insulin的工程菌。SDS2PAGE和Native2PAGE电泳说明A链跟B链是否正确地进行了切割并形成有高级结构的重组人胰岛素。WesternBlot结果表明：重组蛋白是否能够与鼠抗人Insulin抗体发生特异性反应而具有与天然Insulin相同的免疫原性。通过单因素分析和正交实验法对巴斯德毕赤酵母的高密度发酵培养基进行了筛选和优化；对高效表达人生长激素的培养条件进行了优化；根据优化的摇瓶发酵结果进行了补料分批发酵；对表达产物进行了初步鉴定。 关键词：重组人胰岛素，毕赤酵母，分泌表达，培养基优化，晶体收集 Use recombinant pasteris to produce human insulin Name：Unknown Abstract：Use PCR synthesis of recombinant human insulin gene, join Kex2 cleavage sites between the A and B chains. The synthetic recombinant human insulin gene in the correct reading frame was inserted into the constitutive secretory expression vector pGAPZα-A α-factor signal peptide sequence Preparation the construct pGAPZα-A/Insulin recombinant secretory expression vector. The expression vector linearization Bln I treated electroporation competent cells of Pichia pastoris GS115 constructed expression of secretory Insulin engineered bacteria.The analysis SDS2PAGE and Native2PAGE showed that A and B chains have already been cut correctly or not to form the recombinant human insulin with correct advanced structure or not.WesternBlot: The results show that the recombinant protein was able to specifically react with mouse anti-human Insulin Antibodies having the same natural Insulin immunogenicity or not. By single factor analysis and orthogonal experiment method of high density fermentation of Pichia pastoris medium screening and optimization; optimized culture conditions of the high-level expression of human growth hormone; according flask fermentation optimization results the fed-batch fermentation; preliminary identification of the expression product. Key words：recombinant human insulin, Pichia pastoris, secretion of expression, media optimization, crystal collection 引言：利用重组酵母菌生产人类胰岛素在实验室中成功的案例已经被报道。通过分别构建重组人类胰岛素A链和B链的质粒来转染毕赤酵母的方法来获得分泌型表达人insulin的工程菌，并通过