【摘要】：目的:腫瘤是全世界發(fā)病率和死亡率增長最快,對人類健康和生命威脅較大的疾病,從免疫學角度對其進行研究和治療是一條很有希望的途徑。而尋找和研究新的有效的腫瘤抗原是這一途徑的核心問題。近年研究發(fā)現(xiàn)CT抗原呈高度的組織限制性表達、在腫瘤患者體內(nèi)具有免疫原性、可以誘導(dǎo)體液免疫與細胞免疫等特性,為腫瘤免疫治療提供了新的機遇。而NY-ESO-1是其中免疫原性最強的分子,本文以分子生物學實驗和免疫學實驗為基礎(chǔ),克隆腫瘤特異性抗原NY-ESO-1基因,構(gòu)建表達載體并在大腸桿菌中高效表達NY-ESO-1全蛋白,制備多抗,并研究該分子在不同類型腫瘤組織中的表達情況,為深入探討NY-ESO-1表達機理及其在腫瘤診斷、免疫治療方面的應(yīng)用奠定基礎(chǔ)。 方法:用RT-PCR方法從A375細胞中獲得人NY-ESO-1基因全長,構(gòu)建重組表達質(zhì)粒,用IPTG誘導(dǎo)表達NY-ESO-1蛋白,親和層析進行純化,用純化的GST融合蛋白免疫動物,獲得多克隆抗體。并通過NY-ESO-1及其他CT抗原在腫瘤組織中的表達譜初步觀察NY-ESO-1與腫瘤發(fā)生發(fā)展的關(guān)系,分析CT抗原多價疫苗制備的意義。 結(jié)果:成功地克隆了NY-ESO-1基因,構(gòu)建了重組表達質(zhì)粒pGEX-4T-2/NY-ESO-1,成功優(yōu)化GST-NY-ESO-1蛋白的原核表達條件,得到高效表達的NY-ESO-1蛋白,經(jīng)SDS-PAGE分析可觀察到一分子量與理論值相符的誘導(dǎo)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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R392

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