【摘要】： 乙型肝炎是由HBV(Hepatitis B virus)感染導(dǎo)致的傳染病，在我國1-59歲人群中乙型肝炎表面抗原攜帶率為7.18%，其中大約有15%-40%將會發(fā)展成為肝硬化或者肝癌。當(dāng)前臨床中主要用核苷類似物藥物和干擾素進(jìn)行治療，但是并不能完全治愈乙型肝炎，，最主要的缺點是核苷類似物藥物容易誘導(dǎo)HBV的基因突變，從而容易形成耐藥性，并且對身體的副作用較大。國內(nèi)已有三家機構(gòu)的四種治療性疫苗進(jìn)入臨床試驗，但只有一種為DNA疫苗。目前認(rèn)為只有通過誘導(dǎo)強烈的細(xì)胞免疫，打破機體的免疫耐受，才能達(dá)到治療性效果。 本研究擬通過構(gòu)建乙型肝炎治療性MVA載體疫苗，探討治療慢性乙肝的途徑。研究中選用了國內(nèi)北方流行病毒株adr亞型的E4基因（即HBV的突變型preC/C基因），構(gòu)建含有E4基因的重組MVA病毒疫苗：rMVA-E4。E4基因是通過在HBV preC/C基因的Furin蛋白酶水解位點上依次引入點突變使水解位點失活，表達(dá)生成分子量為P22的胞內(nèi)型HBeAg前體，即E4抗原蛋白。突變點在編碼151、154、164、167位氨基酸處，突變方式為：151CGA→GGA、154AGG→GGG、164CGC→GGC、167AGG→GGG，突變后表達(dá)的氨基酸由精氨酸變?yōu)楦拾彼帷?研究中以Balb/c小鼠為動物模型，采用prime-boost免疫策略，結(jié)合IL-2、IL-18和IFN-γ等不同細(xì)胞因子免疫佐劑進(jìn)行免疫試驗。并與攜帶未突變preC/C基因和S2S基因的同類疫苗進(jìn)行比較。通過ELISA檢測體液免疫和ELISPOT、CTL檢測細(xì)胞免疫來對重組病毒疫苗的免疫效果做初步評價。結(jié)果顯示采用prime-boost免疫策略聯(lián)合細(xì)胞因子IL-2佐劑可以誘導(dǎo)較高的細(xì)胞免疫。并且E4基因比未突變preC/C基因能誘導(dǎo)更強的細(xì)胞免疫，略好于S2S基因。目前正以轉(zhuǎn)基因鼠為動物模型對疫苗的治療性效果進(jìn)行研究。
[Abstract]:Hepatitis B is an infectious disease caused by HBV (Hepatitis B virus) infection. The carrying rate of hepatitis B surface antigen in Chinese population aged 1-59 years is 7.18. About 15% to 40% will develop into liver cirrhosis or liver cancer. At present, nucleoside analogue drugs and interferon are mainly used to treat hepatitis B, but they can not cure hepatitis B. the main disadvantage is that nucleoside analogues can easily induce gene mutation in HBV, thus forming drug resistance. And the side effect on the body is bigger. Four therapeutic vaccines from three institutions have entered clinical trials, but only one is DNA vaccine. It is believed that only by inducing strong cellular immunity and breaking the immune tolerance can therapeutic effect be achieved. The aim of this study was to explore the therapeutic approach to chronic hepatitis B by constructing therapeutic MVA vector vaccine. In this study, the E4 gene of adr subtype (HBV mutant preC/C gene) was selected to construct the recombinant MVA virus vaccine containing E4 gene. The recombinant MVA virus vaccine: rMVA-E4.E4 gene was constructed on the Furin proteolytic site of HBV preC/C gene. Point mutation was introduced in turn to inactivate the hydrolysis site. Expression of intracellular HBeAg precursor, called E4 antigen protein, with molecular weight P22. The mutation point was at the amino acid encoding 151154164167, and the mutation mode was that the amino acid expressed after the mutation of GGA,154AGG GGG,164CGC GGC,167AGG GGG, changed from arginine to glycine. In this study, Balb/c mice were used as animal models, prime-boost immunization strategy was used, and different cytokine adjuvants such as IL-2,IL-18 and IFN- 緯 were used to carry out immunological tests. The results were compared with those of the same vaccine carrying unmutated preC/C gene and S 2S gene. In order to evaluate the immune effect of recombinant virus vaccine, humoral immunity was detected by ELISA and cellular immunity was detected by ELISPOT,CTL. The results showed that prime-boost immunization strategy combined with cytokine IL-2 adjuvant could induce higher cellular immunity. And E4 gene can induce stronger cellular immunity than unmutated preC/C gene, which is slightly better than S2S gene. The therapeutic effect of the vaccine is being studied using transgenic mice as animal model.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392.11

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相關(guān)期刊論文 前2條

1 夏國良,Omana V Nainan,賈志遠(yuǎn),劉洪斌,羅述斌,李榮成,曹慧霖,劉崇柏,HaroldS Margolis;乙型肝炎病毒基因型和血清亞型在我國部分地區(qū)的分布及其特點[J];中華流行病學(xué)雜志;2001年05期

2 ;HBV DNA vaccine with adjuvant cytokines induced specific immune responses against HBV infection[J];World Journal of Gastroenterology;2003年01期

本文編號：2233025