細(xì)胞毒性T細(xì)胞抗原4胞外結(jié)構(gòu)域的高效表達(dá)與純化
發(fā)布時(shí)間：2018-09-02 08:14
【摘要】：背景針對(duì)自身免疫性疾病以及惡性腫瘤的治療尚無(wú)療效較好且副作用較少的方法或藥物,近年來(lái)的研究表明,在上述疾病的發(fā)生、發(fā)展過(guò)程中,抗原特異性T細(xì)胞起一定作用。CTLA4作為一種主要的共刺激信號(hào)負(fù)調(diào)節(jié)分子,具有阻斷T細(xì)胞活化的作用;作為CTLA4的單克隆抗體則具有激活T細(xì)胞的作用,二者通過(guò)對(duì)T細(xì)胞的調(diào)控作用,干預(yù)機(jī)體免疫微環(huán)境,開(kāi)辟了對(duì)上述疾病的新的治療途徑。目的本研究目的在于獲得高純度的細(xì)胞毒性T淋巴細(xì)胞相關(guān)分子4胞外結(jié)構(gòu)域(extracellular domain of cytotoxic T-lymphocyte-associated protein 4,ex CTLA4)。通過(guò)構(gòu)建ex CTLA4重組表達(dá)質(zhì)粒,并在大腸桿菌Transetta(DE3)中進(jìn)行重組蛋白的表達(dá)。以期獲得高純度的ex CTLA4重組蛋白,為研究其生物學(xué)功能或制備人源化的CTLA4單克隆抗體提供基礎(chǔ)。方法1.構(gòu)建原核表達(dá)質(zhì)粒p ET-28a-ex CTLA4。根據(jù)人源CTLA4基因序列由公司合成胞外域序列,以此序列為模板,擴(kuò)增后并構(gòu)建到p ET-28a載體的相應(yīng)酶切位點(diǎn)。2.誘導(dǎo)表達(dá)ex CTLA4重組蛋白。將測(cè)序正確的表達(dá)質(zhì)粒轉(zhuǎn)化至大腸桿菌Transetta(DE3)中,在誘導(dǎo)物IPTG濃度為0.5m M、誘導(dǎo)溫度為37°C時(shí),誘導(dǎo)菌體表達(dá)蛋白,培養(yǎng)4h后收集菌體,SDS-PAGE檢測(cè)菌體中的重組蛋白有無(wú)表達(dá)。3.優(yōu)化ex CTLA4重組蛋白表達(dá)條件。從誘導(dǎo)物濃度IPTG,誘導(dǎo)溫度,誘導(dǎo)時(shí)間三方面誘導(dǎo)條件進(jìn)行優(yōu)化。并在此基礎(chǔ)上,設(shè)計(jì)三水平三因素正交試驗(yàn),選擇最優(yōu)表達(dá)條件。4.純化ex CTLA4重組蛋白。選擇最優(yōu)條件誘導(dǎo)目的蛋白表達(dá),收集菌體后,超聲破碎菌體并收集包涵體,先用低濃度尿素(2M)洗去大部分背景蛋白后,再用高濃度尿素(8M)溶解包涵體,進(jìn)一步用鎳離子親和層析方法得到高純度ex CTLA4。5.梯度稀釋法復(fù)性ex CTLA4重組蛋白。用復(fù)性緩沖液梯度稀釋樣品,并經(jīng)過(guò)透析可將變性的重組蛋白復(fù)性。6.檢測(cè)重組蛋白ex CTLA4的生物學(xué)活性。利用CTLA4的配體B7-1以及外周血單核細(xì)胞(PBMC),通過(guò)相應(yīng)ELISA試劑盒檢測(cè)ex CTLA4的生物學(xué)活性。結(jié)果1.成功構(gòu)建表達(dá)質(zhì)粒p ET-28a-ex CTLA4,測(cè)序后序列與已知序列經(jīng)BLAST比對(duì)完全一致。2.成功誘導(dǎo)重組蛋白的表達(dá),獲得了ex CTLA4在Transetta(DE3)中的最佳誘導(dǎo)條件,即在0.75m M IPTG、25°C下,誘導(dǎo)6h能得到最佳表達(dá),但重組蛋白以包涵體形式存在。3.通過(guò)鎳離子親和層析純化及復(fù)性獲得高純度重組蛋白,經(jīng)Western blot鑒定為CTLA4。4.經(jīng)過(guò)ELISA檢測(cè)鑒定,純化的ex CTLA4具有生物學(xué)活性。結(jié)論本研究構(gòu)建了ex CTLA4重組表達(dá)質(zhì)粒,獲得了ex CTLA4重組蛋白在大腸桿菌中的最佳表達(dá)條件以及最佳優(yōu)化結(jié)果,得到了具有生物學(xué)活性的重組蛋白,為該蛋白的應(yīng)用以及后期的抗體篩選奠定了基礎(chǔ)。
[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 擄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 擄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.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R392
本文編號(hào)：2218783
[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 擄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 擄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.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R392
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