【作者】 潘海学；

【导师】 关怡新；

【作者基本信息】 浙江大学 ， 生物化工， 2005， 硕士

【摘要】 γ-干扰素(IFN-γ)是一种细胞因子,由两个相同的亚基组成同型二聚体结构,它在细胞对病毒入侵和分枝杆菌感染的免疫响应机制中扮演着重要的角色,在临床上IFN-γ可用于治疗类风湿性关节炎、结核分枝杆菌感染、黑热病等疾病。基因重组人γ-干扰素(rhIFN-γ)的大量生产,解决了天然γ-干扰素来源稀少,价格昂贵的难题。但是利用E.coli表达的重组人γ-干扰素,常以包涵体形式存在,需要经过体外重折叠复性后才能恢复其生物活性。 本文采用层析复性方法,对重组人γ-干扰素的体外重折叠复性进行了研究,寻求较优的复性条件,以期为蛋白质复性提供一种简便且适宜大规模应用的方法。 用3.7L发酵罐制备得到了重组人γ-干扰素包涵体,目标蛋白的表达量占总蛋白的60%,经过破胞离心,lL发酵液可以得到1.6g粗制包涵体。重组人γ-干扰素包涵体经过含0.5%Triton X-100的50 mmol/L Tris-HCl,pH 8.0洗涤纯化后,溶于含8M尿素的溶解液,蛋白纯度可达85%左右。 以尿素梯度离子交换层析(IEC)作为蛋白质复性系统,能有效地复性重组人γ-干扰素包涵体,实现复性和纯化的过程集成化。在蛋白上样量2.5mg、流速0.5mL/min、尿素梯度长度3.4CV、尿素终浓度为2 mol/L的条件下,复性后人γ-干扰素的比活为7.5×10~5 IU/mg,蛋白收率为54%,经SDS-PAGE分析纯度大于95%。 利用疏水作用层析(HIC)法对重组人γ-干扰素进行了复性,采用等尿素梯度和线性尿素梯度两种复性方法,考察了线性尿素梯度下尿素梯度长度、尿素终浓度、流速和上样量对蛋白质复性的影响。在优化的线性尿素梯度复性条件下,重组人γ-干扰素活性收率比稀释复性法提高6.5倍,蛋白质量回收率为36%。 在以上实验的基础上,对固定化小分子伴侣复合柱法复性重组人γ-干扰素进行了探索性研究,将本实验室生产的小分子伴侣sht GroEL191-345固定化在Ni-NTA亲和凝胶柱中,并与体积排阻层析柱(SEC)串联使用。结果表明,该复性方法中SEC柱能避免上样样品中高浓度变性剂对小分子伴侣的变性作用,比单独使用小分子伴侣sht GroEL191-345介导的复性方法,效果有了较大的改善,复性后重组人γ-干扰素的活性收率比稀释复性提高了4.2倍,蛋白质量收率为77%。更多还原

【Abstract】 As a cytokine, interferon-gamma (IFN-γ) is a homodimeric protein that plays an essential role in cell-mediated immune responses to viral and mycobacterial infections. Recombinant human IFN-γ (rhIFN-γ) is a good approach to meet the requirement of clinical therapy in large amounts. However, rhIFN-γ is expressed as IBs in E.coli and the renaturation of the protein is a bottleneck of the process.Here, we reported some on-column chromatographic processes for the refolding and purification of rhIFN-y produced in E.coli. SDS-PAGE was performed for protein analysis and rhIFN-γ activity was determined by cytopathic effect (CPE) method as well as ABS-ELISA method.The rhIFN-γ produced in the E.coli host after induction at 42℃ and accumulated as inclusion bodies (IBs). Yield of the rhIFN-γ IBs was approximately 1.6g/L culture. After washed with the detergent buffer including 50 mmol/L Tris-HCl and 0.5% Triton X-100, the purity of IBs was up to 85%.Ion exchange chromatography (IEC) with urea gradient was performed as protein refolding system. The process of refolding and purification could be integrated together by IEC. The specific activity of rhIFN-γ was 7.5 ×105 IU/mg and the protein yield was 54% under such experiment conditions: protein loading 2.5mg, flow rate 0.5mL/min, urea gradient length 3.4CV and final urea concentration 2mol/L. The purity of rhIFN-γ determined by SES-PAGE was above 95%.Refolding processes by hydrophobic interaction chromatography (HIC) with no urea gradient and linear decreased urea gradient were proposed. Under the optimum conditions in the refolding with linear decreased urea gradient, the activity yield of rhIFN-γ was 6.5 times of that in the dilution refolding process and the protein mass yield was up to 36%.Minichaperone (sht GroEL191-345) was produced in 3.7L agitated bioreactor. The rhIFN-y IBs were refolded effectively by a combined column with immobilized minichaperone at Ni-NTA agarose, which was connected with a short SEC column. As a result, the activity yield of rhIFN-γ was 4.2 times of that in the dilution refolding and the protein yield was 77%.更多还原