重组人胰岛素原在大肠杆菌中的可溶性表达及分离纯化研究

发布时间：2018-10-26 07:16

【摘要】： 糖尿病是胰岛素分泌的相对或绝对不足引起的一种代谢障碍性疾病,部分患者需要依靠注射外源胰岛素来维持生命。外源胰岛素以前主要从动物胰脏中提取,但动物胰岛素由于本身和人胰岛素氨基酸组成上的差异,有很大的副作用,给患者带来极大痛苦。随着分子生物学及基因工程技术的迅猛发展,利用生物工程技术生产副作用小、更高效的人胰岛素以及人胰岛素类似物,成为了一大热点。但在利用大肠杆菌等微生物表达生产胰岛素的过程中,表达产物常常形成包涵体,要通过变性和复性过程才能得到有活性的人胰岛素,这给后续分离和纯化带来很大不便,同时也影响目的蛋白的最终产量。本研究希望通过合理的设计、表达系统的选择及发酵条件的优化使胰岛素原基因以可溶形式表达,同时建立方便有效的纯化工艺。本实验选用pET32-a载体,BL21大肠杆菌表达系统。根据大肠杆菌的密码子偏嗜性,在胰岛素原基因N端设计肠激酶位点,合成人胰岛素原基因寡核苷酸片段,通过片段连接和PCR扩增得到胰岛素原基因。采用亚克隆的方式先将胰岛素原基因连接到pMD 18-T载体,经测序,将测序正确的人胰岛素原基因整合到pET32-a载体,构建表达载体,用测序正确的重组质粒转化BL21,构建基因工程菌,使胰岛素原基因和硫氧还蛋白融合表达,同时带有6XHis标签,可以利用亲和层析对表达的目的蛋白进行分离纯化。实验中对工程菌的培养温度和IPTG诱导浓度进行优化。通过实验,工程菌在300C条件下培养和在终浓度0.6mM IPTG条件下诱导,表达产物破菌后在上清。证明目的蛋白是以可溶形式进行表达。经SDS-PAGE和Westen blotting检测,表达的蛋白分子量与理论分子量一致,并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。对表达的重组人胰岛素原的分离纯化步骤研究得到了优化的分离步骤组合和分离条件。发酵液离心收集菌体,破菌后上清液过金属螯合层析柱,再过离子交换柱分离纯化目的蛋白。透析后样品用肠激酶和羧肽酶酶切,过亲和柱,除去融合蛋白、6XHis标签,凝胶层析分离胰岛素与连接肽,收集胰岛素样品液。利用免疫酶标法,lmL样品测得活性102μIU,证明实验所得样品具有人胰岛素活性。
[Abstract]:Diabetes mellitus is a metabolic disorder caused by relative or absolute insufficiency of insulin secretion. Exogenous insulin was mainly extracted from animal pancreas before, but the difference of amino acid composition between animal insulin and human insulin caused great side effects, which caused great pain to patients. With the rapid development of molecular biology and genetic engineering technology, the production of human insulin and its analogues by bioengineering technology has become a hot topic. However, in the process of using E. coli and other microorganisms to express and produce insulin, the expression products often form inclusion bodies, and the active human insulin can only be obtained through denaturation and renaturation, which brings great inconvenience to the subsequent separation and purification. It also affects the final yield of the target protein. The aim of this study was to make proinsulin gene expressed in soluble form by reasonable design, selection of expression system and optimization of fermentation conditions, and to establish a convenient and effective purification process. In this experiment, pET32-a vector and BL21 Escherichia coli expression system were selected. According to the codon bias of Escherichia coli, the enterokinase site was designed at the N-terminal of proinsulin gene, and the oligonucleotide fragment of human proinsulin gene was synthesized. The proinsulin gene was obtained by ligation and PCR amplification. The proinsulin gene was first ligated to pMD 18-T vector by subcloning. After sequencing, the correctly sequenced human proinsulin gene was integrated into the pET32-a vector, and the expression vector was constructed. The recombinant plasmid was transformed into BL21, by sequencing the correct recombinant plasmid. The recombinant bacteria were constructed to express proinsulin gene and thioredoxin with 6XHis tag. The expressed target protein could be separated and purified by affinity chromatography. In the experiment, the culture temperature and IPTG induction concentration of engineering bacteria were optimized. The engineering bacteria were cultured at 300C and induced under the final concentration of 0.6mM IPTG. The expression product was expressed in the supernatant after breaking the bacteria. It is proved that the target protein is expressed in soluble form. SDS-PAGE and Westen blotting analysis showed that the molecular weight of the expressed protein was consistent with the theoretical molecular weight and had the immunogenicity of insulin, which proved that the proinsulin gene was correctly expressed. The separation and purification of recombinant human proinsulin were studied. The bacteria were collected by centrifugation, the supernatant was separated by metal chelate chromatography column, and the target protein was purified by ion exchange column. After dialysis, the samples were digested with enterokinase and carboxypeptidase, the affinity column was used to remove the fusion protein, the 6XHis label was removed, and the insulin and conjugated peptides were separated by gel chromatography, and the insulin sample solution was collected. The activity of lmL samples was 102 渭 IU, which showed that the samples had human insulin activity.
【学位授予单位】：四川师范大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R587.1;R341

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