基于反向疫苗学理论筛选猪链球菌2型保护性抗原

发布时间：2018-07-31 11:48

【摘要】：猪链球菌2型是一种人畜共患病原。该病原不仅给全球的养猪业造成了非常大的经济损失,而且危害人类的公众健康安全。目前人们对猪链球菌2型的致病机理研究并不透彻。针对该病的预防,我国所生产的疫苗均为灭活疫苗。随着生物信息学技术的不断发展和完善,利用反向疫苗学开发猪链球菌2型基因工程亚单位疫苗便具备了理论基础。本研究通过对GenBank数据库中的猪链球菌2型P1/7株全基因序列进行生物信息学分析,从1883多个开放性阅读框(ORF)中筛选出39个候选保护性抗原基因,但是MRP基因在实验室所分离的毒株中检出率低,因此对剩余的38个候选抗原进行了基因克隆。其中编号为23、24、26、28、33、34、35、36、38、39所对应的抗原分子量较大,因此进行了节段表达。共构建了58个重组质粒。经过SDS-PAGE电泳鉴定后,共表达了43个。将所纯化到的35个候选抗原免疫小鼠,以本实验室分离鉴定的猪链球菌2型CZ-XXL200毒株进行攻毒,并通过检测各组之间第36h血液中细菌含量,第72h体重变化,第5天脾脏与体重的比值进行免疫效果评估。通过重复实验最终获得5个保护效果优良的保护性抗原,其名称与编号分别为：Sao (Z-29)、Surface protein (Z-35)、hypothetical protein (Z-39)、hypothetical protein (Z-21)、ABC transporter protein (Z-13)。本研究进一步证实了SAO对猪链球菌2型具有较好的保护效果,同时也首次发现了另外4个具有一定保护效果的保护性抗原,其中Z-39和Z-21均一直被认为是假定蛋白。通过对5个保护性抗原的基因序列进行Blast分析,发现保护效果最好的SAO和Surface protein (Z-35)基因存在于猪链球菌多个血清型全基因组中,这两个保护性抗原不仅适用于猪链球菌2型基因工程亚单位疫苗的抗原,而且可能对多个血清型具有交叉保护效果。该研究为猪链球菌2型基因工程亚单位疫苗的开发奠定基础。
[Abstract]:Streptococcus suis type 2 is a zoonotic pathogen. The pathogen not only causes great economic loss to the pig industry in the world, but also endangers the public health and safety of human beings. At present, the pathogenesis of Streptococcus suis type 2 is not well studied. For the prevention of the disease, all the vaccines produced in China are inactivated vaccines. With the development and improvement of bioinformatics technology, the development of Streptococcus suis type 2 gene engineering subunit vaccine by reverse vaccinology has a theoretical foundation. In this study, 39 candidate protective antigen genes were screened from more than 1883 open reading frames (ORF) by bioinformatics analysis of the whole gene sequence of Streptococcus suis type 2 P1 / 7 strain in GenBank database. However, the detection rate of MRP gene was low in the strains isolated in laboratory, so the remaining 38 candidate antigens were cloned. The corresponding molecular weight of the antigens numbered 23 ~ (24) ~ (26) ~ (28) ~ (3) ~ (3) ~ (3) ~ (3) ~ (3) ~ (3) ~ (3) ~ (39) ~ (3) ~ (35) ~ (35) ~ (36) ~ ( A total of 58 recombinant plasmids were constructed. A total of 43 were identified by SDS-PAGE electrophoresis. 35 candidate antigens were purified to immunize mice. The strains of Streptococcus suis type 2 CZ-XXL200 were isolated and identified in our laboratory. The bacterial content in blood and body weight at the 36th hour and 72h were detected. The immune effect was evaluated by the ratio of spleen to body weight on day 5. Five protective antigens with good protective effect were obtained by repeated experiments. The names and numbers of the protective antigens were: protein (Z-35) and hypothetical protein (Z-39) / hypothetical protein (Z-21) / ABC transporter protein (Z-13). This study further confirmed that SAO has a good protective effect on Streptococcus suis type 2. At the same time, four other protective antigens have been found for the first time, among which Z-39 and Z-21 have been considered as hypothetical proteins. By Blast analysis of five protective antigens, it was found that the best protective SAO and Surface protein (Z-35 genes were found in the whole genome of several serotypes of Streptococcus suis. These two protective antigens are not only suitable for the antigen of Streptococcus suis type 2 genetic engineering subunit vaccine, but also may have cross-protective effect on multiple serotypes. This study laid a foundation for the development of genetic engineering subunit vaccine of Streptococcus suis type 2.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.611

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