本文選題：乙型肝炎 + 核酸疫苗��； 參考：《第二軍醫(yī)大學》2005年博士論文

【摘要】：乙型肝炎是一種嚴重危險人類健康的全球性疾病,疫苗是預防和治療乙肝的重要手段。本研究構建了表達HBV(中國流行株)表面抗原的DNA疫苗,并在表達載體中引入了人特異的CpG免疫刺激序列,摸索了一套適合于人用DNA疫苗中試生產(chǎn)的工藝路線。經(jīng)過兩步純化,有效地去除菌液中的蛋白質和雜質核酸,降低了內毒素含量,使得到的DNA疫苗的質量符合有關標準。從體液免疫、細胞免疫全面地考察了乙肝DNA疫苗的免疫原性,發(fā)現(xiàn)DNA疫苗能在免疫小鼠體內表達目的抗原,能誘導出抗原特異性的體液反應和細胞免疫,誘導的抗體的IgG亞型疫IgG_(2(?))為主。進一步考察了乙肝DNA疫苗對HBV轉基因小鼠的治療作用。DNA疫苗免疫后,轉基因小鼠血清中HBsAg水平均有不同程度地下降,部分小鼠HBsAg完全轉陰;DNA疫苗能誘導出特異性的抗體反應,說明DNA疫苗能打破轉基因小鼠對乙肝病毒的免疫耐受。 鑒于DNA疫苗的免疫原性較低,本課題分別通過改善疫苗的免疫方案、改善疫苗的劑型,來提高乙肝DNA疫苗的免疫效果。采用DNA疫苗、蛋白質疫苗聯(lián)合免疫的策略,從不同免疫組合、不同免疫次序、不同免疫次數(shù)的8種免疫方案中篩選得到的一種最佳聯(lián)合方案(2次DNA疫苗初免和1次蛋白質疫苗加強免疫的組合),能明顯地增強體液免疫和細胞免疫。本課題采用PLGA微球制劑的兩種劑型分別包裹或吸附乙肝DNA疫苗,系統(tǒng)地考察了疫苗新劑型的免疫效果,并探討了其增效的分子機制。結果表明兩種微球給藥系統(tǒng)明顯地增強DNA疫苗的免疫原性,PLGA包裹的乙肝DNA口服疫苗緩慢釋放質粒DNA,不僅誘導了系統(tǒng)的抗原特異性免疫,還能誘導高效的消化道粘膜免疫反應。增強的免疫原性與微球制劑的緩釋、靶向給藥的特點有關。研究發(fā)現(xiàn)乙肝DNA疫苗PLGA微球制劑延長目的基因的轉錄和表達,還能靶向性向抗原提呈細胞遞送DNA疫苗。 HBx是一種多功能蛋白,為病毒基因組轉錄所必需。HBx與許多共刺激因子、轉錄因子相互作用,調控靶基因的轉錄,從而影響HBV感染的細胞的功能。由于HBx在乙肝以及乙肝引起的肝硬化、肝癌的肝組織上較HBV其他亞基有更高的表達率,所以本研究將其作為靶抗原,聯(lián)合應用超基序法、延展基序法、量化基序法等生物信息學的表位預測方案,并結合生物學功能實驗加以驗證,篩選到3個來源于HBx、并能與HLA-A*0201分子高親和性結合的抗原九肽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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2005
【分類號】：R392.1

【引證文獻】

相關碩士學位論文 前2條

1 秦永華;抗日本血吸蟲病天然分子靶基因pVAX1/SjRPS4·CB雙價疫苗與聯(lián)合免疫的研究[D];中南大學;2008年

2 吳平;日本血吸蟲核糖體蛋白SjRPS4基因及蛋白聯(lián)合免疫保護性價值的研究[D];南昌大學;2010年

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本文編號：1855711