猪脑心肌炎病毒NJ08株感染性cDNA克隆构建及拯救病毒生物学特性

发布时间：2018-12-15 11:57

【摘要】：脑心肌炎病毒(Encephalomyocarditis virus,EMCV)属于微RNA病毒科心病毒属成员,是一种无囊膜、单股正链的RNA病毒,是一种重要的人畜共患病病原体。EMCV感染后可造成仔猪急性致死性心肌炎,致死率可高达100%。本研究以我国中东地区EMCV NJ08分离株为材料,采用体内转录的方法成功建立了此毒株的反向遗传操作系统,成功拯救到病毒,并对该病毒的生物学特性进行鉴定;拯救病毒的免疫原性也得到初步的研究,为今后深入研究脑心肌炎病毒的分子致病机制、基因功能以及新型疫苗奠定了基础。具体研究内容如下:1猪脑心肌炎病毒感染性克隆的构建及鉴定本研究采用RT-PCR方法获得覆盖EMCV NJ08株全基因组(GenBank HM641897)的A、B和C三个基因片段,其中C片段第6243位的T突变为A,引入EcoRI标记位点,分别克隆至pEasy simple Blunt载体,基因测序正确。采用PacI、XhoI、NheI和SpeI将3个片段依次克隆至CMY-β质粒,获得含EMCV全基因组cDNA的重组质粒pCMV-NJ08。采用脂质体将其转染至BHK-21细胞,结果出现明显细胞病变,经RT-PCR产物EcoRI酶切和间接免疫荧光试验鉴定,证明获得重组EMCV rNJ08;拯救病毒rNJ08株在BHK21细胞上的TCID_(50)、一步生长曲线和空斑形态与亲本毒NJ08相似,但对小鼠致病性明显降低,为该病毒致病机制及疫苗研制奠定了重要基础。2重组脑心肌炎病毒灭活疫苗小鼠免疫特性本研究使用终浓度为0.002mM的BEI将重组病毒(rNJ08)和亲本病毒(NJ08)进行灭活处理,加入ISA15A和ISA206佐剂分别配制成灭活疫苗。将6周龄ICR小鼠随机分成8组,每组5只。第1、2组分别皮下注射由ISA15A佐剂配制的NJ08和rNJ08灭活疫苗,第3、4组分别皮下注射由ISA206佐剂配制的NJ08和rNJ08灭活疫苗(抗原浓度均为10~7 TCID_(50)/0.2ml)。第5-7组依次皮下多点注射等体积的由ISA15A佐剂配制的梯度抗原浓度(10~6TCID_(50)/0.2ml、10~5 TCID_(50)/0.2ml、10~4TCID_(50)/0.2ml)的 rNJ08灭活疫苗;第8组皮下注射2%DMEM作为阴性对照。间隔三周进行加强免疫。首免后3周采集小鼠血清测其ELISA抗体水平;二免后3周采集血清测定ELISA抗体效价和中和抗体效价,同时分离脾脏淋巴细胞进行淋巴细胞增殖转化试验。结果为:rNJ08疫苗免疫组ELISA和中和抗体水平具有明显的抗原剂量依赖性关系;各免疫组小鼠血清ELISA和中和抗体水平在加强免疫后显著提高(1:16以上)·淋巴细胞增殖试验结果显示,接种相同剂量的各免疫组刺激指数均在3.0左右。结果表明rNJ08与NJ08具有相似的免疫原性,均能有效地刺激机体产生良好的免疫反应,并能产生中和抗体。由于该重组病毒毒力较弱,因此,安全性更高。
[Abstract]:Encephalitis virus (Encephalomyocarditis virus,EMCV), a member of the family RNA virus family, is a non-encapsulated, single-stranded RNA virus, which is an important zoonotic pathogen. EMCV infection can cause acute fatal myocarditis in piglets. Mortality can be as high as 100. In this study, the reverse genetic operating system of EMCV NJ08 isolate from the Middle East region of China was successfully established by in vivo transcription method, and the virus was successfully saved, and the biological characteristics of the virus were identified. The immunogenicity of the rescue virus has also been preliminarily studied, which will lay a foundation for further study on the molecular pathogenesis, gene function and new vaccine of encephalitis virus. The specific contents of the study were as follows: 1 Construction and identification of infectious clone of porcine encephalomyocarditis virus. In this study, three gene fragments, Agna B and C, covering the whole genome (GenBank HM641897) of EMCV NJ08 strain were obtained by RT-PCR method. The T mutation at position 6243 of the C fragment was A. the EcoRI marker site was introduced and cloned into the pEasy simple Blunt vector, and the gene was sequenced correctly. The three fragments were cloned into CMY- 尾 plasmid by PacI,XhoI,NheI and SpeI, and the recombinant plasmid pCMV-NJ08. containing EMCV genomic cDNA was obtained. After transfection with liposome into BHK-21 cells, obvious cytopathic changes were observed. The recombinant EMCV rNJ08; was confirmed by EcoRI digestion and indirect immunofluorescence assay. The one-step growth curve and plaque morphology of TCID_ (50) on BHK21 cells were similar to those of parent virus NJ08, but the pathogenicity of TCID_ (50) on BHK21 cells was significantly decreased. 2 the immune characteristics of recombinant encephalitis virus inactivated vaccine in mice. In this study, the recombinant virus (rNJ08) and parental virus (NJ08) were inactivated by BEI with the final concentration of 0.002mM. Inactivated vaccine was prepared by adding ISA15A and ISA206 adjuvant respectively. ICR mice aged 6 weeks were randomly divided into 8 groups with 5 mice in each group. NJ08 and rNJ08 inactivated vaccine prepared by ISA15A adjuvant were injected subcutaneously into the first group and NJ08 and rNJ08 inactivated vaccine prepared by ISA206 adjuvant were injected subcutaneously in the third group (antigen concentration was 10 7 TCID_ (50) / 0.2ml). In group 5-7, the gradient antigen concentration (10 ~ 6TCID _ (50) / 0.2ml of CID _ (50) / 0.2ml ~ 104TCID _ (50) / 0.2ml) was injected subcutaneously with the same volume of rNJ08 inactivated vaccine, and the dose of gradient antigen (10 ~ 6TCID _ (50) / 0.2ml) was 104TCID _ (50) / 0.2ml. Group 8 was subcutaneously injected with 2%DMEM as negative control. The immunization was strengthened at intervals of three weeks. The levels of ELISA antibody in serum of mice were measured 3 weeks after the first immunization, and the titers of ELISA antibody and neutralizing antibody were measured 3 weeks after the second immunization, and lymphocyte proliferation and transformation tests were performed by isolating spleen lymphocytes at the same time. The results showed that the levels of ELISA and neutralizing antibody in rNJ08 vaccine immunized group were in a dose-dependent manner. The levels of serum ELISA and neutralizing antibody in each immunized group were significantly increased (more than 1:16) after enhanced immunization. The results of lymphocyte proliferation test showed that the stimulating index of the same dose of each immunization group was about 3. 0. The results showed that rNJ08 and NJ08 had similar immunogenicity, which could effectively stimulate the body to produce a good immune response and produce neutralizing antibodies. Because of the weak virulence of the recombinant virus, the safety of the recombinant virus is higher.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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