NY-ESO-1的克

发布时间：2018-08-09 09:06

【摘要】：目的:肿瘤是全世界发病率和死亡率增长最快,对人类健康和生命威胁较大的疾病,从免疫学角度对其进行研究和治疗是一条很有希望的途径。而寻找和研究新的有效的肿瘤抗原是这一途径的核心问题。近年研究发现CT抗原呈高度的组织限制性表达、在肿瘤患者体内具有免疫原性、可以诱导体液免疫与细胞免疫等特性,为肿瘤免疫治疗提供了新的机遇。而NY-ESO-1是其中免疫原性最强的分子,本文以分子生物学实验和免疫学实验为基础,克隆肿瘤特异性抗原NY-ESO-1基因,构建表达载体并在大肠杆菌中高效表达NY-ESO-1全蛋白,制备多抗,并研究该分子在不同类型肿瘤组织中的表达情况,为深入探讨NY-ESO-1表达机理及其在肿瘤诊断、免疫治疗方面的应用奠定基础。方法:用RT-PCR方法从A375细胞中获得人NY-ESO-1基因全长,构建重组表达质粒,用IPTG诱导表达NY-ESO-1蛋白,亲和层析进行纯化,用纯化的GST融合蛋白免疫动物,获得多克隆抗体。并通过NY-ESO-1及其他CT抗原在肿瘤组织中的表达谱初步观察NY-ESO-1与肿瘤发生发展的关系,分析CT抗原多价疫苗制备的意义。结果:成功地克隆了NY-ESO-1基因,构建了重组表达质粒pGEX-4T-2/NY-ESO-1,成功优化GST-NY-ESO-1蛋白的原核表达条件,得到高效表达的NY-ESO-1蛋白,经SDS-PAGE分析可观察到一分子量与理论值相符的诱导表
[Abstract]:Objective: cancer is a disease with the fastest increasing morbidity and mortality in the world. It is a promising way to study and treat cancer from the immunological point of view, which is a great threat to human health and life. Finding and studying new and effective tumor antigens is the core of this approach. In recent years, it has been found that CT antigen is highly tissue restricted expression and has immunogenicity in tumor patients, which can induce humoral immunity and cellular immunity, which provides a new opportunity for tumor immunotherapy. NY-ESO-1 is the most immunogenicity molecule. Based on the molecular biological and immunological experiments, the tumor specific antigen (NY-ESO-1) gene was cloned, the expression vector was constructed and the whole NY-ESO-1 protein was highly expressed in Escherichia coli. The expression of NY-ESO-1 in different types of tumor tissues was studied, which laid a foundation for the further study of the mechanism of NY-ESO-1 expression and its application in tumor diagnosis and immunotherapy. Methods: the full length of human NY-ESO-1 gene was obtained from A375 cells by RT-PCR method. The recombinant expression plasmid was constructed. The NY-ESO-1 protein was induced by IPTG and purified by affinity chromatography. The polyclonal antibody was obtained by immunizing animals with the purified GST fusion protein. The expression profiles of NY-ESO-1 and other CT antigens in tumor tissues were used to observe the relationship between NY-ESO-1 and tumorigenesis and development, and the significance of preparation of multivalent vaccine against CT antigen was analyzed. Results: the NY-ESO-1 gene was cloned successfully, and the recombinant expression plasmid pGEX-4T-2 / NY-ESO-1 was constructed. The prokaryotic expression conditions of GST-NY-ESO-1 protein were optimized successfully, and the highly expressed NY-ESO-1 protein was obtained. A inducing surface with molecular weight consistent with the theoretical value was observed by SDS-PAGE analysis.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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