人胰岛素原基因在大肠杆菌中的可溶性表达及分离纯化研究
发布时间：2018-12-13 06:02
【摘要】：根据NCBI中的人胰岛素原基因序列及大肠杆菌密码子偏爱性设计特异引物,PCR扩增得到人胰岛素原基因,构建该基因的原核表达质粒p ET32-PI,重组质粒转化大肠杆菌BL21(DE3)。对重组菌进行温度和IPTG浓度优化,发现重组菌在30℃和终浓度为0.6 mmol/L的IPTG条件下表达效果最好且没有包涵体,经SDS-PAGE和Westem blot检测,表达蛋白的相对分子质量与理论相对分子质量一致且具有胰岛素的免疫原性,证明胰岛素原基因得到了正确表达。对重组菌进行流加发酵,发酵液离心收集菌体,破菌后上清液过亲和层析柱和离子交换柱分离纯化目的蛋白,透析后的样品用肠激酶和羧肽酶酶切,利用亲和柱去除融合蛋白与His标签,最终分离胰岛素样品,利用免疫酶标法,l m L样品测得活性92μIU,证明实验所得样品具有人胰岛素活性。
[Abstract]:According to the sequence of human proinsulin gene in NCBI and the preference of Escherichia coli codon, a specific primer was designed, and the proinsulin gene was amplified by PCR. The prokaryotic expression plasmid p ET32-PI, recombinant plasmid was constructed and transformed into Escherichia coli BL21 (DE3). By optimizing the temperature and IPTG concentration of the recombinant bacteria, it was found that the recombinant strain expressed best at 30 鈩，
本文编号：2376028
[Abstract]:According to the sequence of human proinsulin gene in NCBI and the preference of Escherichia coli codon, a specific primer was designed, and the proinsulin gene was amplified by PCR. The prokaryotic expression plasmid p ET32-PI, recombinant plasmid was constructed and transformed into Escherichia coli BL21 (DE3). By optimizing the temperature and IPTG concentration of the recombinant bacteria, it was found that the recombinant strain expressed best at 30 鈩，
本文编号：2376028
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