热休克蛋白70与人乳头瘤病毒E7融合DNA疫苗的免疫效应研究

发布时间：2018-01-07 10:15

本文关键词：热休克蛋白70与人乳头瘤病毒E7融合DNA疫苗的免疫效应研究　出处：《武汉大学》2005年硕士论文　论文类型：学位论文

【摘要】：宫颈癌是常见的妇科恶性肿瘤之一,已成为危害女性身体健康的最主要的杀手。世界卫生组织统计资料表明,宫颈癌在全球妇女癌症死亡率中位居第二,在一些发展中国家甚至位居首位,每年全球大约有50万例新发宫颈癌病例,约20万人死于宫颈癌。据不完全统计,我国现有宫颈癌病人约13.8万,每年约有5万人死于宫颈癌。大量研究资料证明,约80%的宫颈癌与4种高危型(16、18、31型和45型)的HPV感染相关,其中约50%的宫颈癌与HPV16感染相关,根据分子流行病学资料的结果,HPV16是导致我国妇女宫颈癌的主要型别。但是,目前临床上尚无预防HPV16感染的措施,对中晚期宫颈癌的化疗和手术治疗效果也不理想。宫颈癌的复发率较高且治疗费用也高。因此,研制高效、安全和廉价的HPV DNA疫苗,采用特异性的免疫接种方式预防和治疗HPV感染及其所引起的恶性病变,具有重要的现实意义。大料文献资料证实,人乳头瘤病毒16型E7基因是重要的转化基因,在宫颈癌细胞中持续表达。大多数研究中靶基因主要集中于E7基因,然而由于E7基因本身具有转化活性,直接应用于人体有一定的危险性,去除E7的转化活性基团,保留其抗原性成为基因疫苗研制的前提。E7蛋白C末端锌指结构“Cys-X-X-Cys”对其稳定性有重要作用,锌指结构的突变可显著降低E7蛋白的稳定性和致癌性,提高DNA疫苗的安全性;改变E7C-末端锌指结构中的任何一个Cys(C58G、C61G、C91G、C94G),均能使E7蛋白完全失去永生化人上皮细胞的能力。尽管DNA疫苗有很多优点,但是裸DNA疫苗并不能诱导强烈的、足以保护动物抵抗HPV感染的、高水平的免疫应答,因此必须想方设法增强其免疫原性。近年来研究资料表明,热休克蛋白(heat shock protein,HSP)与抗原基因融合具有增强DNA疫苗免疫效应的潜能,HSP抗原肽复合物(来源于肿瘤或病毒感染细胞)能诱导较好的抗肿瘤和感染免疫应答。HSP介导的抗原呈递具有“cross-presentation”现象,即通过HSP介导进入体内的外源蛋白,可以诱导出抗原特异性的CTL。基于以上原理,我们将突变(91位点C→G)锌指结构的E7基因、野生型E7基因、热休克蛋白70基因分别克隆到真核表达载体pcDNA3.1~-质粒中,构建了pcd-mE7(突变)、pcd-wE7(野生)、pcd-HSP70三种重组质粒,经酶切和PCR鉴定构建成功。与嵌合型 pcd-HPVmE7-HSP70 DNA 疫苗作比较,进一步研究嵌合型pcd-HPVmE7-HSP70DNA疫苗的免疫增强效应。我们用这四种重组质粒、空载体及PBS
[Abstract]:Cervical cancer is one of the most common gynecological malignancies and has become the most important killer of women's health. The statistics of the World Health Organization show that cervical cancer is the second most common cancer death rate among women in the world. In some developing countries, there are about 500,000 new cervical cancer cases and 200,000 deaths from cervical cancer every year in the world. According to incomplete statistics, there are about 138,000 cervical cancer patients in China. About 50,000 people die of cervical cancer each year. A large number of studies have shown that about 80% of cervical cancer is associated with HPV infection in four high-risk types: type 161831 and type 45. About 50% of cervical cancer is associated with HPV16 infection. According to the results of molecular epidemiology, HPV16 is the main type of cervical cancer in Chinese women. At present, there is no clinical measures to prevent HPV16 infection, and the effect of chemotherapy and surgical treatment on advanced cervical cancer is not ideal. The recurrence rate of cervical cancer is high and the cost of treatment is also high. Therefore, the development of high efficiency. The safe and cheap HPV DNA vaccine has important practical significance to prevent and treat HPV infection and its malignant lesions by specific immunization. Large literature confirmed that human papillomavirus type 16 E7 gene is an important transformation gene, which is expressed in cervical cancer cells. The target gene is mainly focused on E7 gene in most studies. However, due to the transformation activity of E7 gene itself, it is dangerous to apply E7 gene directly to human body and remove the transformation active group of E7 gene. Preserving its antigenicity is a prerequisite for the development of gene vaccine. Zinc finger structure "Cys-X-X-Cys" at the C-terminal of E7 protein plays an important role in its stability. The mutation of zinc finger structure can significantly reduce the stability and carcinogenicity of E7 protein and improve the safety of DNA vaccine. Any of the CysC58GN C61GN C91GN C94 GG changes in the structure of E7C- terminal zinc finger can completely lose the ability of immortalized human epithelial cells. Despite the many advantages of the DNA vaccine, the naked DNA vaccine does not induce a high level of immune response that is strong enough to protect animals against HPV infection. Therefore, we must find ways to enhance its immunogenicity. Recent studies have shown that heat shock proteins (HSPs) are heat shock protein. The fusion of HSPand antigen gene has the potential to enhance the immune effect of DNA vaccine. HSP antigen peptide complex (derived from tumor or virus infected cells). HSP-mediated antigen presentation has the phenomenon of "cross-presentation". In other words, foreign proteins mediated by HSP can induce antigen-specific CTLs. On the basis of the above principle, we will change the mutation to locus C91. 鈫扵he E7 gene, wild type E7 gene and heat shock protein 70 gene of zinc finger structure were cloned into eukaryotic expression vector pcDNA3.1, respectively, and pcd-mE7 (mutagenesis) was constructed. Pcd-wE7 (wild pd-HSP70) recombinant plasmid. The results of restriction endonuclease digestion and PCR analysis were compared with the chimeric pcd-HPVmE7-HSP70 DNA vaccine. To further study the immune enhancement effect of chimeric pcd-HPVmE7-HSP70DNA vaccine. We use these four recombinant plasmids, empty vectors and PBS
【学位授予单位】：武汉大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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