發(fā)布時(shí)間：2018-01-25 14:07

  本文關(guān)鍵詞： 表面抗原中蛋白 去糖基化 DNA疫苗 體液免疫應(yīng)答　出處：《南京醫(yī)科大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 慢性乙型肝炎是我國流行最廣、感染者最多的傳染病之一，對(duì)人民身體健康造成了極大的威脅。HBsAg是乙型肝炎病毒表達(dá)的主要抗原之一，其引發(fā)的表面抗體(抗-HBs)是病毒的唯一的中和抗體，在機(jī)體完全清除病毒的過程中起著至關(guān)重要的作用�？�-HBs的出現(xiàn)是乙型肝炎痊愈的重要標(biāo)志。然而，慢性乙型肝炎患者幾乎都對(duì)HBsAg免疫耐受，不能產(chǎn)生抗-HBs，，其原因至今尚未完全明了，通常認(rèn)為：機(jī)體早期特別是垂直傳播的嬰幼兒感染及兒童時(shí)期的感染，機(jī)體免疫系統(tǒng)尚未完全成熟時(shí)接觸HBsAg，形成了免疫耐受。因而，打破機(jī)體對(duì)HBsAg的免疫耐受，促進(jìn)機(jī)體產(chǎn)生抗-HBs，中和乙肝病毒，達(dá)到完全清除病毒的目的。 表面抗原存在多個(gè)糖基位點(diǎn)，其糖基對(duì)體液免疫的影響尚不清楚。獲得性免疫缺陷病毒(HIV)和猴免疫缺陷病毒(SIV)上的糖基減弱了病毒蛋白的免疫原性并且能夠限制部分抗體和病毒顆粒表面的表位結(jié)合。去除HIV Gp120表面糖基發(fā)現(xiàn)抗體的中和活性明顯提高。應(yīng)用基因修飾方法構(gòu)建譯碼去糖基化表面抗原中蛋白核酸疫苗，來研究糖基在表面抗原中蛋白體液免疫應(yīng)答中的作用，對(duì)于設(shè)計(jì)更強(qiáng)免疫原性新型乙肝疫苗，誘導(dǎo)有效的保護(hù)性抗體有著重要的意義。 目的探討乙型肝炎病毒表面抗原中蛋白第146(NCT)和第59位(NHS)潛在糖基對(duì)小鼠體液免疫應(yīng)答的影響。 方法用本實(shí)驗(yàn)室已經(jīng)構(gòu)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)檢測(cè)小鼠血清抗-HBs，應(yīng)用基于PCR的體外阻斷試驗(yàn)分析去糖基化和非去糖基化疫苗免疫血清阻斷效應(yīng)。去糖基MHBs核疫酸苗(Dg)免疫小鼠八周后，應(yīng)用細(xì)胞瘤融合技術(shù)和表位預(yù)測(cè)人工合成跨越去糖基位點(diǎn)線性表位短肽篩選并制備單克隆抗體，Westen-blot分析單克隆抗體(mAb)的抗原結(jié)合特異性，體外阻斷試驗(yàn)檢測(cè)核酸疫苗免疫小鼠血清和制備mAb阻斷效應(yīng)。 結(jié)果Dg23、adr免疫小鼠血清之間體液免疫應(yīng)答強(qiáng)度無明顯差異，和對(duì)照組有明顯差異，兩種血清均可以有效中和病毒感染。獲得兩株(1-9A，2-5F)特異性較好、能穩(wěn)定分泌mAb的雜交瘤細(xì)胞株；腹水效價(jià)分別為：1-9A：1：3.6×104，2-5F：1：1.2×104，1-9A＞2-5F，相對(duì)親和力1-9A 2.6×10-5；2-5F 1.25×10-4。免疫印跡分析顯示，在分子量為33KD去糖基化表面抗原中蛋白發(fā)生特異性結(jié)合，36KD糖基化表面抗原中蛋白位置不顯示條帶。中和實(shí)驗(yàn)顯示1-9A株不能而2-5F能有效阻斷病毒感染Hep G2細(xì)胞。 結(jié)論去除HBV中蛋白S區(qū)兩個(gè)潛在糖基不影響小鼠體液免疫強(qiáng)度，但可能影響抗體應(yīng)答種類。兩株抗體表現(xiàn)出不同的阻斷效應(yīng)，表明兩者作用于不同的B細(xì)胞表位，推測(cè)不同單抗和不同的B細(xì)胞表位結(jié)合后引起抗原變構(gòu)不同，從而導(dǎo)致阻斷感染的能力差異。本研究獲得的一株單抗具有良好的HBV感染阻斷效應(yīng)，可作為潛在的治療性抗體。
[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.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 吳興柳,蔡潔,劉雁雁,黃祖瑚;HBsAg核酸疫苗誘導(dǎo)H-2~b小鼠體液免疫應(yīng)答的初步研究[J];南京醫(yī)科大學(xué)學(xué)報(bào);2001年03期

2 林海,孟繼鴻;基于PCR的乙型肝炎病毒體外中和試驗(yàn)[J];中華實(shí)驗(yàn)和臨床病毒學(xué)雜志;2005年02期

本文編號(hào)：1463016