乙型肝炎治疗性MVA载体疫苗的基础研究
发布时间：2018-09-09 17:01
【摘要】： 乙型肝炎是由HBV(Hepatitis B virus)感染导致的传染病，在我国1-59岁人群中乙型肝炎表面抗原携带率为7.18%，其中大约有15%-40%将会发展成为肝硬化或者肝癌。当前临床中主要用核苷类似物药物和干扰素进行治疗，但是并不能完全治愈乙型肝炎，，最主要的缺点是核苷类似物药物容易诱导HBV的基因突变，从而容易形成耐药性，并且对身体的副作用较大。国内已有三家机构的四种治疗性疫苗进入临床试验，但只有一种为DNA疫苗。目前认为只有通过诱导强烈的细胞免疫，打破机体的免疫耐受，才能达到治疗性效果。 本研究拟通过构建乙型肝炎治疗性MVA载体疫苗，探讨治疗慢性乙肝的途径。研究中选用了国内北方流行病毒株adr亚型的E4基因（即HBV的突变型preC/C基因），构建含有E4基因的重组MVA病毒疫苗：rMVA-E4。E4基因是通过在HBV preC/C基因的Furin蛋白酶水解位点上依次引入点突变使水解位点失活，表达生成分子量为P22的胞内型HBeAg前体，即E4抗原蛋白。突变点在编码151、154、164、167位氨基酸处，突变方式为：151CGA→GGA、154AGG→GGG、164CGC→GGC、167AGG→GGG，突变后表达的氨基酸由精氨酸变为甘氨酸。 研究中以Balb/c小鼠为动物模型，采用prime-boost免疫策略，结合IL-2、IL-18和IFN-γ等不同细胞因子免疫佐剂进行免疫试验。并与携带未突变preC/C基因和S2S基因的同类疫苗进行比较。通过ELISA检测体液免疫和ELISPOT、CTL检测细胞免疫来对重组病毒疫苗的免疫效果做初步评价。结果显示采用prime-boost免疫策略联合细胞因子IL-2佐剂可以诱导较高的细胞免疫。并且E4基因比未突变preC/C基因能诱导更强的细胞免疫，略好于S2S基因。目前正以转基因鼠为动物模型对疫苗的治疗性效果进行研究。
[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- 纬 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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.11
本文编号：2233025
[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- 纬 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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.11
【参考文献】
相关期刊论文 前2条
1 夏国良,Omana V Nainan,贾志远,刘洪斌,罗述斌,李荣成,曹慧霖,刘崇柏,HaroldS Margolis;乙型肝炎病毒基因型和血清亚型在我国部分地区的分布及其特点[J];中华流行病学杂志;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
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