乙肝表面抗原去糖基化核酸疫苗体液免疫应答的研究

发布时间：2018-01-25 14:07

本文关键词： 表面抗原中蛋白去糖基化 DNA疫苗体液免疫应答　出处：《南京医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 慢性乙型肝炎是我国流行最广、感染者最多的传染病之一，对人民身体健康造成了极大的威胁。HBsAg是乙型肝炎病毒表达的主要抗原之一，其引发的表面抗体(抗-HBs)是病毒的唯一的中和抗体，在机体完全清除病毒的过程中起着至关重要的作用。抗-HBs的出现是乙型肝炎痊愈的重要标志。然而，慢性乙型肝炎患者几乎都对HBsAg免疫耐受，不能产生抗-HBs，，其原因至今尚未完全明了，通常认为：机体早期特别是垂直传播的婴幼儿感染及儿童时期的感染，机体免疫系统尚未完全成熟时接触HBsAg，形成了免疫耐受。因而，打破机体对HBsAg的免疫耐受，促进机体产生抗-HBs，中和乙肝病毒，达到完全清除病毒的目的。表面抗原存在多个糖基位点，其糖基对体液免疫的影响尚不清楚。获得性免疫缺陷病毒(HIV)和猴免疫缺陷病毒(SIV)上的糖基减弱了病毒蛋白的免疫原性并且能够限制部分抗体和病毒颗粒表面的表位结合。去除HIV Gp120表面糖基发现抗体的中和活性明显提高。应用基因修饰方法构建译码去糖基化表面抗原中蛋白核酸疫苗，来研究糖基在表面抗原中蛋白体液免疫应答中的作用，对于设计更强免疫原性新型乙肝疫苗，诱导有效的保护性抗体有着重要的意义。目的探讨乙型肝炎病毒表面抗原中蛋白第146(NCT)和第59位(NHS)潜在糖基对小鼠体液免疫应答的影响。方法用本实验室已经构建好的去糖基MHBsag核酸疫苗(Dg23)和未去糖基野生株MHBsAg核酸疫苗(adr)免疫小鼠，ELISA法(enzyme linked immune sorbent array)检测小鼠血清抗-HBs，应用基于PCR的体外阻断试验分析去糖基化和非去糖基化疫苗免疫血清阻断效应。去糖基MHBs核疫酸苗(Dg)免疫小鼠八周后，应用细胞瘤融合技术和表位预测人工合成跨越去糖基位点线性表位短肽筛选并制备单克隆抗体，Westen-blot分析单克隆抗体(mAb)的抗原结合特异性，体外阻断试验检测核酸疫苗免疫小鼠血清和制备mAb阻断效应。结果Dg23、adr免疫小鼠血清之间体液免疫应答强度无明显差异，和对照组有明显差异，两种血清均可以有效中和病毒感染。获得两株(1-9A，2-5F)特异性较好、能稳定分泌mAb的杂交瘤细胞株；腹水效价分别为：1-9A：1：3.6×104，2-5F：1：1.2×104，1-9A＞2-5F，相对亲和力1-9A 2.6×10-5；2-5F 1.25×10-4。免疫印迹分析显示，在分子量为33KD去糖基化表面抗原中蛋白发生特异性结合，36KD糖基化表面抗原中蛋白位置不显示条带。中和实验显示1-9A株不能而2-5F能有效阻断病毒感染Hep G2细胞。结论去除HBV中蛋白S区两个潜在糖基不影响小鼠体液免疫强度，但可能影响抗体应答种类。两株抗体表现出不同的阻断效应，表明两者作用于不同的B细胞表位，推测不同单抗和不同的B细胞表位结合后引起抗原变构不同，从而导致阻断感染的能力差异。本研究获得的一株单抗具有良好的HBV感染阻断效应，可作为潜在的治疗性抗体。
[Abstract]:Chronic hepatitis B is the most popular in China, one of the most infectious disease infection, on people's health caused great threat to.HBsAg is one of the major antigen expression of hepatitis B virus surface antibody, the (anti -HBs) is the only virus neutralizing antibody, completely removed in the body plays a vital role in the process of anti virus. -HBs is an important marker of hepatitis B cured. However, almost all patients with chronic hepatitis B of HBsAg immune tolerance can produce anti -HBs, the reason is not yet fully understood, especially the body usually think that early vertical transmission of infection and childhood infant infection, the immune system has not yet when fully ripe contact HBsAg, formation of immune tolerance. Therefore, to break the immune tolerance of HBsAg, promote the body to produce anti -HBs and hepatitis B virus, to completely remove disease The purpose of the poison.
There are many surface antigen glycosylation sites, the effect of glycosylation on humoral immunity is unclear. Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) on the immunogenicity of glycosyl attenuated virus protein and can limit the part surface antibody and virus particles epitope binding. The removal of HIV Gp120 surface carbohydrate found antibody neutralizing activity was significantly improved. Application of genetic modification method to construct encoded deglycosylation surface antigen protein nucleic acid vaccine, to study the glycosylated protein in surface antigen in humoral immune response in the role for the design of more strong immunogenicity of hepatitis vaccine, plays an important role in inducing protective antibodies effective.
Objective to investigate the effect of hepatitis B virus surface antigen protein 146th (NCT) and fifty-ninth (NHS) potential glycosyl groups on the humoral immune response in mice.
This method constructed the deglycosylation of MHBsag nucleic acid vaccine (Dg23) and deglycosylation of wild strain MHBsAg nucleic acid vaccine (ADR) mice, ELISA (enzyme linked immune sorbent array) to detect anti -HBs serum, application test analysis to glycosylated and non glycosylated vaccine to immune serum blocking effect of PCR in vitro. Based on blocking deglycosylation of MHBs nuclear acid vaccine disease (Dg) mice eight weeks after the application of tumor cell fusion and epitope prediction of synthetic glycosylation sites across to linear epitope peptide screening and preparation of monoclonal antibody and analysis of monoclonal antibody Westen-blot (mAb) antigen binding. Specific blocking test in vitro detection of nucleic acid vaccines in mice serum and preparation of mAb blocking effect.
The results of Dg23, there is no significant difference between ADR in sera of mice immunized with humoral immune response intensity, and there are significant differences in the control group, two serum can effectively neutralize the virus infection. Two strains (1-9A, 2-5F) better specificity, hybridoma cell line stably secreting mAb; ascites titers were: 1-9A:1:3.6 * 104,2-5F:1:1.2 * 104,1-9A 2-5F, the relative affinity of 1-9A 2.6 * 2-5F 1.25 * 10-4. 10-5; Western blot analysis showed that the molecular weight of 33KD protein glycosylation in surface antigen specific binding protein, location of glycosylation of 36KD surface antigen does not show bands. Neutralization test indicated that 1-9A was not 2-5F can effectively block the virus Hep infected G2 cells.
Two potential glycosylation in HBV protein S region conclusion removal does not affect the humoral immune strength of mice, but may affect the antibody response. Two kinds of mAbs showed different inhibitory effect, indicating that both effects on different B cell epitopes, that different monoclonal antibody and different B cell epitope binding by different allosteric antigen thus, the result of blocking ability difference of infection. In this study a strain of monoclonal antibody has good blocking effect of HBV infection can be used as a potential therapeutic antibody.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

【参考文献】