乙型肝炎治疗性疫苗的研究

发布时间：2018-05-07 06:09

本文选题：乙型肝炎 + 核酸疫苗　；参考：《第二军医大学》2005年博士论文

【摘要】：乙型肝炎是一种严重危险人类健康的全球性疾病,疫苗是预防和治疗乙肝的重要手段。本研究构建了表达HBV(中国流行株)表面抗原的DNA疫苗,并在表达载体中引入了人特异的CpG免疫刺激序列,摸索了一套适合于人用DNA疫苗中试生产的工艺路线。经过两步纯化,有效地去除菌液中的蛋白质和杂质核酸,降低了内毒素含量,使得到的DNA疫苗的质量符合有关标准。从体液免疫、细胞免疫全面地考察了乙肝DNA疫苗的免疫原性,发现DNA疫苗能在免疫小鼠体内表达目的抗原,能诱导出抗原特异性的体液反应和细胞免疫,诱导的抗体的IgG亚型疫IgG_(2(?))为主。进一步考察了乙肝DNA疫苗对HBV转基因小鼠的治疗作用。DNA疫苗免疫后,转基因小鼠血清中HBsAg水平均有不同程度地下降,部分小鼠HBsAg完全转阴;DNA疫苗能诱导出特异性的抗体反应,说明DNA疫苗能打破转基因小鼠对乙肝病毒的免疫耐受。鉴于DNA疫苗的免疫原性较低,本课题分别通过改善疫苗的免疫方案、改善疫苗的剂型,来提高乙肝DNA疫苗的免疫效果。采用DNA疫苗、蛋白质疫苗联合免疫的策略,从不同免疫组合、不同免疫次序、不同免疫次数的8种免疫方案中筛选得到的一种最佳联合方案(2次DNA疫苗初免和1次蛋白质疫苗加强免疫的组合),能明显地增强体液免疫和细胞免疫。本课题采用PLGA微球制剂的两种剂型分别包裹或吸附乙肝DNA疫苗,系统地考察了疫苗新剂型的免疫效果,并探讨了其增效的分子机制。结果表明两种微球给药系统明显地增强DNA疫苗的免疫原性,PLGA包裹的乙肝DNA口服疫苗缓慢释放质粒DNA,不仅诱导了系统的抗原特异性免疫,还能诱导高效的消化道粘膜免疫反应。增强的免疫原性与微球制剂的缓释、靶向给药的特点有关。研究发现乙肝DNA疫苗PLGA微球制剂延长目的基因的转录和表达,还能靶向性向抗原提呈细胞递送DNA疫苗。 HBx是一种多功能蛋白,为病毒基因组转录所必需。HBx与许多共刺激因子、转录因子相互作用,调控靶基因的转录,从而影响HBV感染的细胞的功能。由于HBx在乙肝以及乙肝引起的肝硬化、肝癌的肝组织上较HBV其他亚基有更高的表达率,所以本研究将其作为靶抗原,联合应用超基序法、延展基序法、量化基序法等生物信息学的表位预测方案,并结合生物学功能实验加以验证,筛选到3个来源于HBx、并能与HLA-A*0201分子高亲和性结合的抗原九肽VLHKRTLGL(92-100),
[Abstract]:Hepatitis B is a serious global disease which is dangerous to human health. Vaccine is an important method to prevent and treat hepatitis B. In this study, DNA vaccine expressing surface antigen of hepatitis B virus (Chinese epidemic strain) was constructed, and human specific CpG immunostimulatory sequence was introduced into the expression vector, and a set of technological route suitable for pilot production of human DNA vaccine was explored. After two steps of purification, the protein and impurity nucleic acid were removed effectively, the endotoxin content was reduced, and the quality of the obtained DNA vaccine was up to the relevant standard. The immunogenicity of hepatitis B DNA vaccine was investigated comprehensively from humoral and cellular immunity. It was found that DNA vaccine could express the target antigen in mice and induce antigen-specific humoral reaction and cellular immunity. IgG subtype of induced antibody Mainly. The therapeutic effect of hepatitis B DNA vaccine on HBV transgenic mice was further investigated. The serum HBsAg level of transgenic mice was decreased to some extent after immunization with HBV transgenic mice. Some mice could induce a specific antibody response by transforming HBsAg completely into DNA vaccine. The results showed that DNA vaccine could break the immune tolerance of transgenic mice to hepatitis B virus. In view of the low immunogenicity of DNA vaccine, the immune effect of hepatitis B DNA vaccine was improved by improving the immunization scheme and the dosage form of the vaccine. DNA vaccine, protein vaccine combined immunization strategy, from different immunization combinations, different immunization order, The best combination of two DNA vaccines and one protein vaccine was selected from 8 immunization schemes with different immunization times, which could significantly enhance humoral immunity and cellular immunity. In this paper, two kinds of PLGA microspheres were used to encapsulate or adsorb hepatitis B DNA vaccine respectively. The immune effect of the new formulation was investigated systematically, and the molecular mechanism of its synergism was discussed. The results showed that the two microsphere delivery systems could significantly enhance the immunogenicity of DNA vaccine and slow release of plasmid DNA-PLGA-encapsulated hepatitis B DNA oral vaccine, which not only induced the antigen-specific immunity of the system, but also induced a highly effective mucosal immune response in digestive tract. The enhanced immunogenicity is related to the characteristics of sustained release and targeted delivery of microspheres. It was found that hepatitis B DNA vaccine PLGA microspheres could prolong the transcription and expression of the target gene and deliver DNA vaccine to antigen presenting cells. HBx is a multifunctional protein, which is necessary for viral genome transcription. HBX interacts with many costimulatory factors and transcription factors to regulate the transcription of target genes, thus affecting the function of HBV infected cells. Because HBx has a higher expression rate in liver tissues of hepatitis B and liver cirrhosis caused by hepatitis B than other subunits of HBV, this study used HBx as a target antigen, combined with supermotif method and extended motif method. Quantitative motif method and other bioinformatics epitope prediction schemes were verified by biological functional experiments. Three antigenic peptides VLHKRTL GLN 92-100, derived from HBxand binding to HLA-A*0201 molecules with high affinity, were screened.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392.1

【引证文献】