【作者】 郭雷；

【导师】 孔维； 貌盼勇； 于湘晖；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2010， 硕士

【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。更多还原

【Abstract】 Hepatitis B, an infectious diseases caused by HBV (Hepatitis B virus), hasbecome a serious public health problem. In our country, 7.18% of the 1-59 years oldpeople—about 1 million people—carried the hepatitis B surface antigen. Patients canrarely natural healing, after a long period’s treatment most patients even showresistance, and about 15%-40% of them will become included cirrhosis or liver cancer.In northern China, the most popular subspecies of HBV is the adr-type, while infectedpeople in the south mainly is adw subtype. Currently there is no specific treatment forchronic hepatitis B. At present, in order to treat hepatitis B we mainly use thenucleoside analog drugs and interferon treatment, but they can not cure hepatitis. Butthe main drawback is that nucleoside analog drugs easily induce HBV gene mutation,which tends to form a resistance and side effects on the body. Generally speaking，theonly way to clear HBV virus in vivo is to induce the effective cellular immuneresponse. The core of current research on therapeutic hepatitis B vaccine is how toinduce cellular immunity effectively under the safe situation. Therefore, in this study,the mainly work is how to improve therapeutic vaccine’s effect on the induction ofimmune responses, especially cellular immune response. If the chronic hepatitis Bpatients want to get rid of HBV, the best way is to improve their own cellular immuneagainst HBV.Therefore, the most important step in studying the therapeutic hepatitis B vaccineis to select the appropriate antigen protein and the carried vector. HBeAg is expressedby preC/C gene, which can be hydrolysed into P22, P20 and other types of intracellular protein intermediates, and eventually generate secretory HBeAg.The secretory HBeAg may induce immunological tolerance, but the P22 whichhas the similar specificly antigen as the HBcAg can be assembled into virus particleswithout HBV DNA .When the level of P22 is high, it is not only can induce specificcellular immunity, but also can inhibit the replication of HBV. Therefore, in theprevious studies of this issue, we got the E4 gene that can stability express P22. E4gene will be used as the vaccine to express antigen in this study. In order to obtain amore effectively immunity, we construct a rMVA-E4 therapeutic vaccine used MVAvector. MVA virus have many unique advantages as vaccine carrier. First of all, it canaccommodate multiple large fragments of exogenous genes. As non-replication virus,MVA virus can propagate only in to CEF, but not in the mammalian body. So it willnot be replicated and not appear atavism of pathogenic phenomenon. It has reliablesecurity as vaccine carriers; Secondly, MVA has a complicated structure, containing alarge number of the coat protein and enzyme. It is possible to have both a stimulus fora variety of immune response, at the same time it allows itself to possess thecapabilities of the adjuvant. It can be able to assist foreign gene inducing moreefficient immune protective response. Currently there has widely studied on MVAvirus vector in the word, and got a largely success.In order to obtain effective and high-quality cellular immune responses, we willuse the prime / boost immunization strategy, and select cytokines such as IL-18, IL-12,IFN-γas adjuvant for combined immunodeficiency. Prime refers to the first fewimmune with DNA vaccine; boost refers to immune by using a heterologous carrier-the MVA viral vectors - to enhance immunity. Besides, this strategy can eliminate theinfluence specific antibodies of smallpox vaccine which can reduce the effectivenessof rMVA-E4 vaccine.In this paper, the gene which expresses pre C/C with mutations in four differentnucleotide of HBV adr subtype was inserted into a shuttle vector - pSC11, which ishomologous with vaccinia Modified Vaccinia Ankara (MVA), to construct and pickrecombinant plasmid. Construct recombinant MVA vector vaccine by co-transfection.Then we confirmed that the recombinant MVA vector vaccine can express the right antigen by indirect immuno-fluorescence, Western-blot and other methods.In our study, we compared the immune response of prime-boost combinationvaccine strategy and individual use of DNA vaccine by animal experiment; Andcompared the immune response to vaccine impact of E4 and preC / C and the S2S asthe target genes ;The therapeutic effect of our combination vaccine IL-18, IL-12,IFN-γtested in Balb/c mice. Compare with the difference immunity effort of humoraland cellular immunity by ELISA, CTL, ELISPOT experiments. The results showedthat: In the context of humoral immunity there is no significant difference between theexperimental group, because all of them provoke a higher humoral immune response;but there is great difference in cell-mediated immunity. The result shows that theprime-boost strategy can induce more effitive cellular immunity than using DNAvaccine and rMVA-E4 lonely. At the same time, by comparison we show that E4 caninduce a better immune response, and in effect on the induction of cellular immunityis far better than preC/C gene, and slightly better than S2S gene induced immuneeffects. Based on the safety of E4 gene, E4 is the better choice for the treatment ofvaccine genes. By comparison, we prove that E4 can induce a better immune response,and a more reliable security; IL-2 can make the largest enhanced cellular immuneresponse to vaccine as the adjuvant followed by IFN-γand IL-18;We confirm thatDNA-E4 prime/rMVA-E4 boost with IL-2 as adjuvant can make optimal immuneeffectiveness, which is laid a foundation for further study.更多还原