鸡传染性支气管炎病毒多表位核酸疫苗免疫效力研究

发布时间：2018-11-26 18:02

【摘要】：鸡传染性支气管炎(Infectious bronchitis,IB)是由鸡传染性支气管炎病毒(Infectious bronchitis virus,IBV)引起的,严重危害养鸡业的重要的病毒性传染病之一。因为IBV血清型众多,且不同的血清型之间的交叉保护力弱,给疫病的防控带来了极大的困难。目前IB的预防措施主要还是依赖于疫苗接种,生产中常采用的是弱毒苗和弱毒苗,弱毒苗存在毒力返祖和自然散毒的隐患,灭活疫苗虽然安全性好,但是其免疫保护效果维持时间短,需要多次注射免疫,所需的成本较高。核酸疫苗具有安全性好和诱导全方位的免疫应答等优点,而IBV的表面纤突(S)蛋白基因是最重要的免疫原性蛋白,能诱导良好的保护性免疫,它又分为S1和S2 2个亚单位,其中S1蛋白是决定IBV特异性抗原决定簇的主要蛋白。M基因相对而言保守性较高,与病毒复制有关,且其可能存在具有免疫作用的抗原决定簇。因此,研制IBV表位核酸疫苗这种新型基因工程疫苗,对防制IBV具有重要的意义。本研究应用筛选出的IBV毒株的结构蛋白基因S1基因的T细胞抗原表位盒和B细胞抗原表位盒,酶切连接入真核表达载体pVAX,构建7组表位核酸疫苗,研究其免疫保护效力。分别利用前期筛选出的IBV的结构蛋白基因S1全基因、S1基因的T细胞抗原表位盒和结构蛋白M全基因,构建3组真核表达质粒:pVAX-S1、pVAX-S1T+M、pVAX-S1+S1T+M;IBV的结构蛋白基因S1基因的T细胞抗原表位盒、Australia T株和M41株的S1基因的B细胞抗原表位,构建4组真核表达质粒pVAX-S1B(M41)、pVAX-S1B(T)、pVAX-S1T+S1B(M41)、pVAX-S1T+S1B(T);将原有的质粒S1B(T)、S1T重新设计引物,改造酶切位点重新连接构成pVAX-S1BT+S1T。将每组提取制备的质粒溶液通过腿部肌肉多点注射的方式免疫7日龄SPF雏鸡,28日龄加强免疫一次。同时设立PBS组作为对照组。分别在首免前及免疫后的7d、14d、21d以及二免后的7d每组随机抽取3只鸡,翅静脉采血并分离血清,用间接ELISA方法检测特异性抗体水平。二免后第10d用剂量10~6ELD_(50)/只的IBV病毒液滴鼻点眼攻毒,通过保护率和排毒量检测的结果这两个指标来评价疫苗的保护效果。抗体效价结果显示:pVAX-S1、pVAX-S1T+M组与PBS组相比差异显著(p0.05);pVAX-S1B(T)、pVAX-S1T+S1B(M41)和pVAX-S1T+S1B(T)组与PBS组相比差异均显著(p0.05);pVAX-S1B(T)、pVAX-S1B+S1T组与PBS组相比差异均显著(p0.05)。攻毒结果表明,疫苗免疫组pVAX-S1和pVAX-S1T+M免疫后,有效诱导了鸡体的免疫反应,分别能提供80%(8/10)和70%(7/10)的保护率;疫苗组pVAX-S1、pVAX-S1T+M、pVAX-S1B(T)免疫后,其保护率明显增高,分别为75%(6/8)、62.5(5/8)和75%(6/8);疫苗组pVAX-S1B(T)、pVAX-S1B+S1T免疫后,提供的保护效力分别为84.7%(11/13)和69.3%(9/13)。综上所述,本实验表明筛选的IBV表位抗原成分能够有效发挥保护效力,为研制安全有效的新型IBV疫苗提供了新的途径。
[Abstract]:Avian infectious bronchitis (Infectious bronchitis,IB) is one of the most important viral infectious diseases, which is caused by (Infectious bronchitis virus,IBV (avian infectious bronchitis virus). Because of the numerous serotypes of IBV and the weak cross-protection between different serotypes, it is very difficult to prevent and control epidemic diseases. At present, the preventive measures of IB mainly depend on vaccine inoculation. The attenuated vaccine and attenuated vaccine are often used in production. The attenuated vaccine has the hidden danger of virulence atavism and natural dispersal, although the inactivated vaccine is safe, However, the protective effect of immune protection is short and requires multiple injections of immunization, which requires high cost. Nucleic acid vaccine has the advantages of good safety and comprehensive immune response. The surface fibrin (S) protein gene of IBV is the most important immunogenicity protein, it can induce good protective immunity, and it can be divided into S1 and S2 subunits. S1 protein is the main protein that determines the specific antigen determinant of IBV. M gene is relatively conservative and related to virus replication, and it may have an immunological determinant. Therefore, the development of IBV epitope nucleic acid vaccine, a new genetic engineering vaccine, is of great significance to the control of IBV. In this study, the T cell epitope box and B cell antigen epitope box of S1 gene of structural protein gene of IBV strain were selected and ligated into eukaryotic expression vector pVAX, to construct 7 groups of epitope nucleic acid vaccine. Three groups of eukaryotic expression plasmids were constructed by using the whole S1 gene of IBV, the T cell epitope box of S1 gene and the whole gene of structural protein M, respectively. Three groups of eukaryotic expression plasmids were constructed: pVAX-S1,pVAX-S1T MVAX-S1 S1T M; Four groups of eukaryotic expression plasmids, pVAX-S1B (M41), pVAX-S1B (T), pVAX-S1T S1B (M41), were constructed from T cell epitope box, Australia T strain of S1 gene of IBV and B cell epitope of S1 gene from M41 strain. PVAX-S1T S1B (T); The primer was redesigned from the original plasmid S1B (T), S1T, and the restriction site was reconnected to form pVAX-S1BT S1T. SPF chicks of 7 days old were immunized with plasmid solution extracted from each group by multi-point injection of leg muscle, and the chickens were immunized once at 28 days of age. At the same time, PBS group was established as control group. Three chickens were randomly selected from each group on day 21 and day 7 respectively before immunization and 7 days after immunization. Blood samples were collected from wing vein and serum was isolated. Specific antibody levels were detected by indirect ELISA method. On the 10th day after the second immunization, the protective effect of the vaccine was evaluated by the protective rate and the results of the detoxification test with the dose of 10 ~ 6ELD _ (50) / mouse IBV. The results of antibody titer showed that there were significant differences between pVAX-S1,pVAX-S1T M group and PBS group (p0.05), pVAX-S1B (T), pVAX-S1T S1B (M41) and pVAX-S1T S1B (T) group compared with PBS group (p0.05). There were significant differences between pVAX-S1B (T), pVAX-S1B S 1 T group and PBS group (p 0. 05). The results showed that pVAX-S1 and pVAX-S1T M could effectively induce the immune response of chicken body, which could provide 80% (8 / 10) and 70% (7 / 10) protective rates, respectively. The protective rate of pVAX-S1,pVAX-S1T MVAX-S1B (T) was 75% (6 / 8), 62.5% (5 / 8) and 75% (6 / 8) respectively. The protective effects of pVAX-S1B (T), pVAX-S1B S1T were 84.7% (11 / 13) and 69.3% (9 / 13), respectively. In conclusion, this study shows that the selected IBV epitope antigen can effectively exert the protective effect, and provide a new way for the development of safe and effective new IBV vaccine.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31

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