【作者】 李鹏；

【导师】 陈溥言；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2008， 博士

【摘要】 日本乙型脑炎病毒(Japanese encephalitis virus, JEV),又称乙型脑炎病毒,简称乙脑病毒,属于黄病毒科(Flaviviridae)黄病毒属,主要引起人类或马的中枢神经系统感染以及猪的流产,能通过持续感染的蚊子传播给人,并且主要在脊椎动物宿主内存在,特别是具有较高病毒血症的猪和鸟类,在人类乙脑主要引起致死率较高的脑炎,致死率从20-50%,但是绝大多数的幸存者都具有中枢神经系统的后遗症；猪是主要中间宿主和扩散宿主,也是主要的传染源。许多研究表明,猪流行性乙型脑炎与人流行性乙型脑炎密切相关,因此预防猪感染本病是防止人患乙脑的重要措施。同时,乙脑也是猪的重大疫病之一,能引起怀孕母猪流产、死胎及弱仔,公猪睾丸炎,仔猪呈神经症状,其暴发常给养猪业带来巨大的经济损失。目前用于JEV预防的疫苗主要是传统的灭活苗和弱毒苗,乙脑弱毒疫苗和灭活疫苗等常规疫苗都具有良好的免疫原性,在预防和控制乙脑的过程中发挥着重要作用,但是鼠脑来源的乙脑灭活疫苗在过敏问题上越来越突出,而且使用灭活疫苗的一个主要问题是缺乏长效免疫,由于需要多次重复免疫,使得免疫程序繁琐。弱毒疫苗则存在病毒毒力返强的不安全因素,因此寻求稳定、安全以及新型的JE疫苗已经成为近年JE感染防治的主要研究方向。近年来,随着分子生物学技术的飞速发展,JE基因工程疫苗等多种新型的疫苗都在不断的研究开发中。本研究首次构建了乙脑E蛋白多表位串联基因(TEP),包括E蛋白上四个重要的B细胞表位327aa～333aa,337aa～345aa,373aa～399aa,397aa～403aa和两个T细胞表位60～68aa,436aa～445aa。为了提高免疫效力,在六个表位串联的基础上引入了组织酶原激活物信号肽序列(tissue plasminogen activator, TPA)、CpG基序和通用型辅助性T细胞表位。本研究分别以原核表达体系、真核表达体系及腺病毒表达系统表达了TEP基因,并比较了它们的免疫效力。论文的主要研究内容主要包括以下几个方面：1.乙脑E蛋白多表位基因串联真核质粒的构建人工合成含有JEV E基因的T,B细胞表位的多表位基因,插入真核表达质粒pCDNA3.0中构建重组真核质粒pCDNA-TEP。重组质粒转染293A细胞,利用间接免疫荧光证明pCDNA-TEP在真核细胞内能有效的转录和表达；Western-blot检测表明重组真核质粒的表达产物能与JEV阳性血清特异性结合,证明其具有一定的生物学活性和抗原性。2.乙脑E蛋白多表位基因在大肠杆菌中的融合表达以重组质粒pUC-TEP为模板,扩增不含TPA信号肽序列的基因片段,将其克隆至原核表达载体pET-32a(+),命名为pET-rEP。将重组质粒转化大肠杆菌(E. coli)BL21(DE3)感受态细胞,酶切鉴定的阳性菌经1mM IPTG诱导表达4h后,将大肠杆菌以6000rpm离心10min,用PBS洗两次,收集细菌,将其悬浮在PBS中,进行超声波破碎,以10,000 rpm离心20min,将上清和沉淀分别进行SDS-PAGE电泳分析,结果表明这种蛋白以包涵体形式存在。将包涵体在8M的尿素中过夜溶解,然后将上清过His Bind树亲和层析柱对重组表达产物进行纯化。结果显示融合蛋白的分子量为37kDa,Western blot表明重组蛋白具有抗JEV的特异抗原性。3.多表位DNA疫苗和重组蛋白疫苗对Balb/c小鼠的特异性免疫影响为了评价所构建的DNA疫苗和重组蛋白质疫苗的免疫效力,将6周龄Balb/c小鼠随机分成4组,每组8只,设pcDNA-TEP (1), pET-rEP (2), pcDNA-TEP+pET-rEP (3),灭活疫苗(4)共4个免疫组,同时设pcDNA3.0空载体对照组(5)。肌肉注射免疫,重组质粒的免疫剂量为50μg/只,重组蛋白的免疫剂量为200 pmol/只,灭活苗免疫组按照使用说明进行。利用微量中和试验检测免疫后的中和抗体,利用商品化的ELISA试剂盒检测免疫小鼠脾脏淋巴细胞分泌IFN-γ、IL-2及IL-4的能力,并与灭活苗进行比较。结果表明,试验构建的DNA疫苗和重组蛋白质疫苗都能诱导产生针对JEV的特异性体液免疫和细胞免疫应答。灭活苗免疫组产生了最高的中和抗体,其次由高到低依次为,pcDNA-TEP+pET-rEP, pET-rEP, pcDNA-TEP。pcDNA3.0空载体对照组没有检测到特异性针对JEV的中和抗体。通过对抗体亚型及细胞因子的测定,pET- rEP和灭活苗免疫组主要是增强Th2免疫反应,pcDNA-TEP增强Thl免疫反应,而pcDNA-TEP+pET-rEP则产生Thl/Th2混合型的免疫反应。这为研究能够更好地防制JEV感染的新型疫苗提供新的思路。4.表达乙脑E蛋白多表位基因重组腺病毒的构建将人工合成多表位基因克隆到腺病毒的穿梭载体pAdeno Vator-CMV5-IRES-GFP中。得到的阳性穿梭质粒经Pme I酶线性化后,利用电转化技术将线性化的质粒与腺病毒骨架载体pAdeno Vator△E1/E3共转化大肠杆菌BJ5183感受态细胞,使其在大肠杆菌中进行同源重组。将筛选到的重组病毒质粒经Pac I酶线性化后暴露出包装信号,通过脂质体LipofectamineTM2000转染293A细胞后得到重组病毒。利用报告基因GFP检测病毒滴度,通过PCR和wes tern-blot检测JEV多表位基因得到成功的表达并有很好的免疫原性,重组病毒rAd5-TEP的滴度为1010.59个TCID５0/mL。重组病毒经过连续五代传代后效价稳定。5.重组腺病毒对小鼠特异性免疫的影响6周龄小鼠随机分为4组,每组8只。将重组病毒rAd5-TEP、灭活疫苗分别以腹腔接种的方式给小鼠进行3次免疫,同时设对照组小鼠,免疫PBS。然后通过测定IgGl、IgG2a、中和抗体和IL-4、IFN-γ水平来衡量重组病毒的免疫效果。结果表明重组病毒rAd5-TEP既能诱导细胞免疫应答又能诱导体液免疫应答,抗体亚型测定结果显示,重组病毒rAd5-TEP既能诱导IgGl的分泌又能诱导IgG2a的分泌,但以IgG2a为主。同时细胞因子ELISA试验结果进一步表明重组病毒主要刺激Thl分化(IFN-y),但与灭活苗相比,也能刺激Th2(IL-4)的分化。pcDNA-TEP+rAd5-TEP免疫组能产生最高水平的IFN-γ和IL-4,而诱导产生的中和抗体滴度略低于灭活苗免疫组。6.不同疫苗联合应用对猪的免疫效力研究为了比较pcDNA-TEP+pET-rEP和pcDNA-TEP+rAd5-TEP对猪的免疫效力,将4周龄的健康仔猪随机分4组,每组5头,设pcDNA-TEP+pET-rEP (I)、pcDNA-TEP+rAd5-TEP(Ⅱ、)与灭活疫苗(Ⅲ)3个免疫组,同时PBS空白对照组(Ⅳ)。肌肉注射免疫,重组质粒的免疫剂量为200μg/头,重组蛋白的免疫剂量为800 pmo1/头,重组病毒免疫组免疫剂量为1×1010TCID５0/头,灭活苗免疫组按照使用说明进行,空白对照组每头注射2mL高压灭菌的PBS,4周后进行加强免疫。初次免疫后每隔2周采血分离血清,利用空斑抑制中和试验(PRNT)检测JEV特异性中和抗体水平；同时,在初次免疫后30、45与60天,无菌采血,分离血血清,采用ELISA定量检测细胞因子的水平。结果表明,pcDNA-TEP+rAd5-TEP免疫组在加强免疫后2周产生了可检测到的中和抗体,pcDNA-TEP+pET-rEP和灭活疫苗免疫组在加强免疫后4周检测到中和抗体,在整个试验过程中,灭活疫苗免疫组产生的中和抗体平均滴度最高；就细胞免疫而言,初次免疫后30天,各实验组细胞因子的产量没有明显差异；在初次免疫后45天,3个试验组IFN-y与IL-2的产量明显增多,pcDNA-TEP+rAd5-TEP产生最高水平的IFN-γ、IL-2和IL-4,在免疫后70d,各组IFN-γ与IL-2的产量没有明显变化,说明加强免疫后Thl型细胞因子的分泌至少可以维持3周,灭活苗免疫后,IFN-γ、IL-2都不明显,主要是IL-4的产量增加。上述研究结果表明以pcDNA-TEP作基础免疫,以rAd5-TEP加强免疫的‘"prime-boost"疫苗是一个值得深入研究的JE新型疫苗。综上所述：构建的重组疫苗pcDNA-TEP.pET-rEP. rAd5-TEP者能诱导产生针对JEV的特异性体液免疫和细胞免疫应答,采用"prime-boost"的免疫策略能明显提高其诱导的中和抗体水平和细胞免疫应答,以pcDNA-TEP作基础免疫,以rAd5-TEP加强免疫试验组在小鼠和猪体内获得了较高的特异性针对JEV的中和抗体和细胞免疫应答,从而为研究安全有效的乙脑新型疫苗提供了新的思路和方法。更多还原

【Abstract】 The Japanese encephalitis virus (JEV), which belongs to the family Flaviviridae,infects the central nervous system of humans and equines, and causes stillbirthsin swine. Japanese encephalitis virus (JEV) is transmitted to humans by persistently-infected mosquitoes and. is maintained in infected vertebrate reservoirs, especially in viremia-amplifying hosts such as swine and avian. In humans, JEV causes acute encephalitis with fatality rates ranging from 20% to as high as 50% and most of the survivors display persistent neurological or psychological sequelae.Swine is an important amplifier of Japanese encephalitis virus (JEV) in the paradomestic environmen.Thus mass vaccination of swine can prevent the disease in swine and help to prevent JE epidemics in humans, In the view of economics, the pig industry suffered serious losses from the viral infection as a result of the reproduce failure in sows and death in piglets. For vaccination, both inactivated and living-attenuated JEVs have been widely used with significant success in providing first-generation vaccines in many Asian countries. However, the former is limited by poor availability, high production cost, lack of long-term immunity, and the possibility of allergic reactions. Living-attenuated vaccine has the possible risk of reverting to virulence. Thus there is an urgent need to develop more effective vaccines and to alter the strategies against this viral disease. In recent years,many studies have demonstrated the efficacy of genetically engineered vaccine for JEV.In this study, we first constructed synthetic multi-epitope by synthesizing E epitopes (six amino acid residues 60-68,327-333,337-345,373-399,397-403 and 436-445 in E,designated TEP).The TEP were expressed in various systems:prokaryoti、eukaryotic and Adenovirus, then evaluated the abilities to induce immune responses in mice and swine.The contents of the paper contain five parts as following:1. Construction and eukaryotic expression for the multiple-epitope gene of Japanese encephalitis virusA synthetic multiple-epitope gene containing the critical epitopes of the Japanese encephalitis virus (JEV) envelope (E) gene was cloned into eukaryotic expression vector pcDNA3.0, named pcDNA-TEP.293 cells were then transfected with this recombinant plasmid. Recombinant protein was showed to transcript and express effectively by indirect immunofluorescence. Collect the cell supernatant after 48h transfection for Western blot, it showed the protein can interact with JEV positive anit-serum, proved its biological activity and antigenicity.2. The prokaryotic expression for the multiple-epitope gene of Japanese encephalitis virusA fragment without TPA signal peptide was amplified by PCR from plasmid pUC-TEP and cloned into pET-32a(+), named pET-rEP. Then the recombinant plasmid was transducted into BL21 Ecoli cells. Recombinant protein was purified as follow:Ecoli cells were induced by IPTG, after 4h, cells were collected by centrifugation,6000rpm 10min, washed twice by PBS, and suspended in PBS. Cells were lysed by ultrasonic, then centrifuged 10000rpm 20min. Both the supernatant and pellet was analyzed by electrophoresis. It showed that the protein was existed in form of inclusion. Dissolved the inclusion with 8M urea and purified by His-Band affinity chromatography, we got the 370Kd recombinant protein. Western blot showed the specific anti-JEV activity.3. The Immunogenic Efficacy for the recombinant DNA vaccine and recombinant protein vaccine on miceIn order to investigate the immunogenic efficiency of the pcDNA-TEP and pET-rEP, forty of JEV negative mice (4-week-old, female) were randomly divided into four groups with eight of them each group. Mice in groupⅠwere inoculated intramuscularly pcDNA-TEP at a dose of 50μg; Mice in groupⅡwere injected with purified pET-rEP at a dose of 200 pmol, Mice in groupⅢwere injected with pcDNA-TEP+pET-rEP; The blank eukaryotic expression plasmid pcDNA3.0 and inactivated vaccine were used as controls. JEV specific neutralizing antibody titer was evaluated with virus neutralization test assay, furthermore, the production of JEV specific IFN-γ, IL-2 and IL-4 in splenocytes was detected with commercial ELISA Kit. Datas showed that both the recombinant protein and the DNA vaccine can induce humoral and cellular immune responses. The rank for neutralizing antibodies titers is inactive vaccine, pcDNA-TEP+pET-rEP, pET-rEP, pcDNA-TEP, and pcDNA3.0 from high to low. No neutralizing antibody was found in the pcDNA3.0 inoculation group throughout the experiment. The production of gamma interferon (IFN-γ), interleukin-2 (IL-2) and interleukin-4 (IL-4) was detected from spleen lympholeukocyte of the immunized mice at 45 and 60days post primary immunization to evaluate the cellular immune response. Results showed that the IgG1 isotype was induced by the inactivated vaccine and rEP alone. In contrast, mice immunized with pcDNA-TEP alone elicited predominantly IgG2a.These results suggest that a mixed Th1/Th2 immune response was induced by DNA priming/protein boosting vaccine regimen. Therefore, it can be an attractive candidate vaccine for preventing JEV infection.4.Construction of Recombinnat Adenovirus Expression multiple-epitope gene of the Japanese encephalitis virusA synthetic multi-epitope of Japanese encephalitis virus was expressed in adenovirus. The multi-epitope gene was cloned into pAdeno Vator-CMV5-IRES-GFP, named as pA5-TEP. pA5-TEP was Linearized by Pme I, and then Linearized pA5-TEP and pAdeno VatorAEl/E3 co-transformed into E.coli BJ5183 competent cells by electroporation. The homologous recombinant plasmids pA5-TEP was selected and Linearized by Pac I to expose the encapsidation signal. Linearized virus plasmid was transfected into 293A cells by LipofectamineTM-2000, and recombinant virus was selected and named as rAd5-TEP. Infection titer and rate were evaluated through monitoring green fluorescent protein (GFP) expression. Expression of the multi-epitopes of JEV was identified by PCR and western-blot. Results indicated that expression products had specific antigenicity. The titer of rAd5-TEP is 1010..59 TCID50/mL, and the biological characteristics for recombinant virus were stable after passaged for 5 times.5. Recombinant Adenoviruses on Specific Immunization in Mice6 weeks old female BALB/c mice were randomly separated into 4 groups,8 mice per group, mice were celiac immunized with recombinant viruses(rAd5-TEP、inactivated vaccines) three times at 2 weeks intervals, and with PBS as control. Level of IgG1、IgG2a、IL-4、IFN-y and Antibody neutralization test were detected to evaluate humoral immune and cellular immune responses of recombinant viruses. ELISA test of antibody sub-type results indicated that recombinant viruses induced secretion both of IgGl and IgG2a, partial to IgG2a.The seem result elicited from cytokines ELISA test, secretion of both of Thl(y-IFN) and Th2(IL-4) were induced by recombinant viruses, partial to Thl. Furthermore, pcDNA-TEP+rAd5-TEP induces the highest level of the IFN-y and IL-4 and a stronger humoral response against JEV. the highest level of neutralizing antibodies was observed in the group inoculated with the inactive vaccine, which was slightly higher than the pcDNA-TEP+rAd5-TEP group(P>0.05). This promising candidate can be for instance in viral vectors vaccine studies. Thus provide valuable support for further development of JEV genetic engineering vaccines. 6. The Immunogenic Efficacy on Piglets for the Combination application with various vaccines of Japanese encephalitis virusIn order to test the immunogenic efficiency of the pcDNA-TEP+pET-rEP and pcDNA-TEP+rAd5-TEP which was constructed in the fifth chapter and the seventh chapter, twenty of JEV negative piglets (fourth-month-old) were randomly into four groups with ten of them in each group. Piglets in group one were injected (i.m.) with purified recombinant plasmid pcDNA-TEP; piglets in group two were injected with purified recombinant plasmid pcDNA-TEP, piglets in group three were injected with inactivated vaccine; piglets in group four were injected with PBS as control. Injections were done with 200μg purified plasmid via intramuscular (i.m.) route. Four weeks post the primary vaccination, the piglets were boosted with same dose of correspondence pET-rEP, rAd5-TEP, inactivated vaccine and PBS.Neutralizing antibody titers for each group were detected with virus neutralization test assay fortnightly interval after the primary vaccination for six times to evaluate the humoral immune response. The production of gamma interferon (IFN-y), interleukin-2 (IL-2) and interleukin-4 (IL-4) was detected with lympholeukocyte from blood of the immunized piglets at 30,45 and 60 days post immunization to evaluate the cellular immune response. Results showed, both pcDNA-TEP+pET-rEP and pcDNA-TEP+rAd5-TEP can induce both humoral and cellular immune responses, the inactivated vaccine obtained the highest neutralizing antibody titer,however the pcDNA-TEP+rAd5-TEP inoculation group got the earliest immune responses and the most vigorous Thl type response. The strategy of priming with the DNA vaccine and and then boosting the recombinant rAd5-TEP might be a promising JEV vaccine candidate for further.In summary, all the recombinant vaccines can induce humoral and cellular immune responses. The strategy of priming with the DNA vaccine and then boosting the recombinant rAd5-TEP can obtain the higher neutralizing antibody titer and induce the highest level of the IFN-γ、IL-2 and IL-4 production, which suggested that this DNA priming-recombinant rAd5-TEP boosting vaccine can produce high levels of antibody and result in significant T-cell responses both in mices and swine. It might be an attractive candidate vaccine to be tested for preventing JEV infection and the research may lead to a new approach for the generation of JE vaccines.更多还原