细胞毒性T细胞抗原4胞外结构域的高效表达与纯化

发布时间：2018-09-02 08:14

【摘要】：背景针对自身免疫性疾病以及恶性肿瘤的治疗尚无疗效较好且副作用较少的方法或药物,近年来的研究表明,在上述疾病的发生、发展过程中,抗原特异性T细胞起一定作用。CTLA4作为一种主要的共刺激信号负调节分子,具有阻断T细胞活化的作用;作为CTLA4的单克隆抗体则具有激活T细胞的作用,二者通过对T细胞的调控作用,干预机体免疫微环境,开辟了对上述疾病的新的治疗途径。目的本研究目的在于获得高纯度的细胞毒性T淋巴细胞相关分子4胞外结构域(extracellular domain of cytotoxic T-lymphocyte-associated protein 4,ex CTLA4)。通过构建ex CTLA4重组表达质粒,并在大肠杆菌Transetta(DE3)中进行重组蛋白的表达。以期获得高纯度的ex CTLA4重组蛋白,为研究其生物学功能或制备人源化的CTLA4单克隆抗体提供基础。方法1.构建原核表达质粒p ET-28a-ex CTLA4。根据人源CTLA4基因序列由公司合成胞外域序列,以此序列为模板,扩增后并构建到p ET-28a载体的相应酶切位点。2.诱导表达ex CTLA4重组蛋白。将测序正确的表达质粒转化至大肠杆菌Transetta(DE3)中,在诱导物IPTG浓度为0.5m M、诱导温度为37°C时,诱导菌体表达蛋白,培养4h后收集菌体,SDS-PAGE检测菌体中的重组蛋白有无表达。3.优化ex CTLA4重组蛋白表达条件。从诱导物浓度IPTG,诱导温度,诱导时间三方面诱导条件进行优化。并在此基础上,设计三水平三因素正交试验,选择最优表达条件。4.纯化ex CTLA4重组蛋白。选择最优条件诱导目的蛋白表达,收集菌体后,超声破碎菌体并收集包涵体,先用低浓度尿素(2M)洗去大部分背景蛋白后,再用高浓度尿素(8M)溶解包涵体,进一步用镍离子亲和层析方法得到高纯度ex CTLA4。5.梯度稀释法复性ex CTLA4重组蛋白。用复性缓冲液梯度稀释样品,并经过透析可将变性的重组蛋白复性。6.检测重组蛋白ex CTLA4的生物学活性。利用CTLA4的配体B7-1以及外周血单核细胞(PBMC),通过相应ELISA试剂盒检测ex CTLA4的生物学活性。结果1.成功构建表达质粒p ET-28a-ex CTLA4,测序后序列与已知序列经BLAST比对完全一致。2.成功诱导重组蛋白的表达,获得了ex CTLA4在Transetta(DE3)中的最佳诱导条件,即在0.75m M IPTG、25°C下,诱导6h能得到最佳表达,但重组蛋白以包涵体形式存在。3.通过镍离子亲和层析纯化及复性获得高纯度重组蛋白,经Western blot鉴定为CTLA4。4.经过ELISA检测鉴定,纯化的ex CTLA4具有生物学活性。结论本研究构建了ex CTLA4重组表达质粒,获得了ex CTLA4重组蛋白在大肠杆菌中的最佳表达条件以及最佳优化结果,得到了具有生物学活性的重组蛋白,为该蛋白的应用以及后期的抗体筛选奠定了基础。
[Abstract]:Background there are no effective methods or drugs for the treatment of autoimmune diseases and malignant tumors. Recent studies have shown that, in the process of occurrence and development of these diseases, Antigen-specific T cells play a certain role. CTLA4, as a major negative regulation molecule of costimulatory signal, can block the activation of T cells, and as a monoclonal antibody of CTLA4, it can activate T cells. Through the regulation of T cells and the intervention of immune microenvironment, they have opened up a new way to treat these diseases. Objective to obtain high purity cytotoxic T lymphocyte associated molecule 4 (extracellular domain of cytotoxic T-lymphocyte-associated protein 4 ex CTLA4). The recombinant expression plasmid of ex CTLA4 was constructed and the recombinant protein was expressed in E. coli Transetta (DE3). The aim of this study was to obtain high purity ex CTLA4 recombinant protein and to provide a basis for the study of its biological function and the preparation of humanized CTLA4 monoclonal antibodies. Method 1. Construction of prokaryotic expression plasmid p ET-28a-ex CTLA4. According to the human CTLA4 gene sequence, the extracellular domain sequence was synthesized from the company, which was used as a template to amplify and construct the corresponding restriction site of p ET-28a vector. Ex CTLA4 recombinant protein was induced to express. The correctly sequenced expression plasmid was transformed into E. coli Transetta (DE3). When the concentration of IPTG was 0.5 mm and the induction temperature was 37 掳C, the expressed protein was induced. After 4 hours of culture, SDS-PAGE was collected to detect the expression of recombinant protein in E. coli. The expression conditions of ex CTLA4 recombinant protein were optimized. The induction conditions were optimized from three aspects: induction temperature and induction time of inducer concentration (IPTG,). And on this basis, design three levels and three factors orthogonal experiment, select the optimal expression conditions. 4. Ex CTLA4 recombinant protein was purified. The optimal conditions were selected to induce the expression of the target protein. After collecting the bacteria, the bacteria were broken by ultrasound and the inclusion bodies were collected. After washing most of the background proteins with low concentration urea (2m), the inclusion bodies were dissolved with high concentration urea (8M). Further preparation of High Purity ex CTLA4.5. by Nickel Ion Affinity Chromatography Gradient dilution refolding of ex CTLA4 recombinant protein. The denatured recombinant protein could be renatured by gradient dilution with refolding buffer and dialysis. The biological activity of recombinant protein ex CTLA4 was determined. The biological activity of ex CTLA4 was detected by using the ligand B7-1 of CTLA4 and (PBMC), of peripheral blood monocytes by using the corresponding ELISA kit. Result 1. The sequence of the expressed plasmid p ET-28a-ex CTLA4, was identical with the known sequence by BLAST alignment. The expression of recombinant protein was successfully induced, and the optimal induction conditions of ex CTLA4 in Transetta (DE3) were obtained, that is, the best expression could be obtained at 0.75m IPTG,25 掳C for 6 h, but the recombinant protein existed in the form of inclusion body. High purity recombinant protein was obtained by purification and renaturation by nickel ion affinity chromatography. The recombinant protein was identified as CTLA4.4. by Western blot. The purified ex CTLA4 showed biological activity by ELISA detection. Conclusion in this study, the recombinant expression plasmid of ex CTLA4 was constructed, the optimal expression conditions and results of ex CTLA4 recombinant protein in E. coli were obtained, and the recombinant protein with biological activity was obtained. It lays a foundation for the application of the protein and the screening of antibody in the later stage.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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