重组CS1联合HBsAg治疗性乙型肝炎疫苗的研究

发布时间：2018-06-12 22:55

本文选题：慢性乙型肝炎 + 治疗性疫苗　；参考：《兰州大学》2006年硕士论文

【摘要】：慢性乙型肝炎是一种严重危害人类健康的全球性疾病,目前尚无有效的药物进行治疗,疫苗仍然是预防乙肝的重要手段。目前普遍使用的乙型肝炎疫苗是以乙肝表面抗原蛋白为基础的,此类疫苗可诱导人体产生保护性抗体,从而起到预防乙型肝炎的作用。但此类疫苗最大的缺点是不能诱导有效的细胞免疫,而细胞免疫在清除细胞内病毒,以及打破慢性乙肝患者持续的免疫耐受状态具有极其重要的作用。本研究对由中国预防医学科学院病毒学研究所构建的HBV(中国流行株)核心抗原与PreS1融合重组质粒(CS1)进行了转导、培养、诱导表达,并对诱导表达的条件进行了优化。通过表达产物的SDS-PAGE电泳、western检测和抗原性鉴定,确定该蛋白理化、生物学特性与理论结果一致。对表达的HBV融合蛋白CS1使用DEAE-Sepharose 4FF离子交换和Sephadex-G75分子筛进行了两步层析纯化得到了纯度达到98%以上,抗原性较好,免疫原性较强的蛋白。将纯化后的HBV融合蛋白CS1与兰州生物制品研究所疫苗三室生产的CHO-乙型肝炎表面抗原(HBsAg)联合,以不同剂量免疫BALB/C小鼠,并用两疫苗单独免疫作为参照,以生理盐水作为对照。免疫后于不同时间采血ELISA方法检测小鼠血清抗体;制备脾脏单个核细胞(MNC),做MTT检测;用血清抗体效价和淋巴细胞增殖情况两种标准确定最优剂量和最佳免疫程序。用合适剂量的HBcAg与HBsAg联合(CS1+S)后,以终浓度为1mg/ml Al(OH)3作为佐剂与不含佐剂的CS1+S抗原组同时免疫BALB/C小鼠,并用两种疫苗单独免疫(含和不含Al(OH)3)作为参照,以生理盐水作为对照。免疫后用ELISA方法测定抗体产生的滴度;制备脾脏单个核细胞(MNC),ELISA法检测细胞因子IFN-γ分泌水平;应用流式细胞仪分析T细胞亚群;最后应用DTH试验检测抗原引起的BALB/C小鼠迟发型超敏反应。最后的结果显示HBcAg与HBsAg联合免疫小鼠产生的IFN-γ量与二者单独免疫相比有显著性差异(P0.05),CD~(3+)CD~(4+)水平与后者相比显著增高(P0.05);含Al(OH)_3佐剂的联合疫苗CD8~+水平比空白对照组有显著性差异(P0.05);联合疫苗产生的保护性抗体anti-HBs与2倍剂量的HBsAg单独免疫相比无显著性差异(P0.05);含Al(OH)_3佐剂与不含佐剂的抗原免疫效果差异不显著(P0.05)。总之,本课题工作表明,用CS1与HBsAg联合,具有良好的体液免疫与细胞免疫效果,有助于清除HBV病毒并有希望打破HBV感染引起的慢性乙型肝炎病人持续
[Abstract]:Chronic hepatitis B (CHB) is a global disease that seriously endangers human health. There are no effective drugs to treat chronic hepatitis B. Vaccine is still an important method to prevent hepatitis B. Hepatitis B vaccine, which is widely used at present, is based on hepatitis B surface antigen (HBsAg) protein, which can induce human body to produce protective antibody and thus play a role in the prevention of hepatitis B. But the biggest drawback of this kind of vaccine is that it can not induce effective cellular immunity which plays an extremely important role in clearing the intracellular virus and breaking the persistent immune tolerance in patients with chronic hepatitis B. In this study, the recombinant plasmid of HBV core antigen and PreS1 fusion recombinant plasmid constructed by Institute of Virology, Chinese Academy of Preventive Medical Sciences, was transduced, cultured and expressed, and the conditions of induced expression were optimized. The protein was determined by SDS-PAGE electrophoresis and antigenicity identification. The physicochemical and biological properties of the protein were in agreement with the theoretical results. The expressed HBV fusion protein CS1 was purified by DEAE-Sepharose 4FF ion exchange and Sephadex-G75 molecular sieve by two-step chromatography. The purified HBV fusion protein CS1 was combined with CHO- hepatitis B surface antigen HBsAg produced by Lanzhou Biological products Research Institute. BALB / C mice were immunized with different doses. After immunization, serum antibodies were detected by Elisa method at different time points, MNCX was prepared from spleen mononuclear cells, and MTT assay was performed. The optimal dose and the best immune procedure were determined by two criteria: serum antibody titer and lymphocyte proliferation. BALB / C mice were immunized with appropriate dose of HBcAg and HBsAg combined with CS1 S with final concentration of 1mg/ml Albumin OH3 as adjuvant and CS1 S antigen without adjuvant. The mice were immunized with two kinds of vaccines alone (including and without Albumin OH3) as control and physiological saline as control. After immunization, the titer of antibody was determined by Elisa, the level of cytokine IFN- 纬 was detected by Elisa, the T cell subsets were analyzed by flow cytometry. Finally, DTH test was used to detect delayed type hypersensitivity induced by antigen in BALB / C mice. The results showed that the level of IFN- 纬 produced in mice immunized with HBcAg and HBsAg was significantly higher than that in mice immunized with HBcAg and HBsAg alone. There was no significant difference between the protective antibody anti-HBs produced by the combined vaccine and the 2 times dose of HBsAg alone (P 0.05), and there was no significant difference in the immune effect between the adjuvant containing Albumin and the antigen without the adjuvant (P 0.05). In conclusion, the work of this study shows that the combination of CS1 and HBsAg has good humoral and cellular immune effects, which is helpful to eliminate HBV virus and hopefully break the persistence of chronic hepatitis B patients caused by HBV infection.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392;Q789

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