罗非鱼无乳链球菌CBP和CPS的免疫保护性研究

发布时间：2018-07-26 11:38

【摘要】：罗非鱼作为我国南方一种重要的养殖鱼类,拥有生长繁殖快、环境适应能力强的优点。然而,近几年爆发的无乳链球菌病威胁着罗非鱼养殖业,很大程度上影响着罗非鱼产业的健康发展。免疫防控被证实是罗非鱼无乳链球菌病防治的有效手段,因而相关疫苗的研制越来越受到人们的重视。无乳链球菌的表面成分在病原粘附宿主过程中发挥着重要作用,且其免疫原性通常较好。因此,可用于研制疫苗的候选抗原。本论文选择罗非鱼无乳链球菌的胆碱结合蛋白(Choline-binding protein,CBP)和荚膜多糖(Capsular polysaccharide,CPS)为抗原,研究其作为疫苗对罗非鱼抗无乳链球菌感染的免疫保护效果。主要研究内容如下:1.无乳链球菌CBP基因的克隆、序列分析和原核表达对CBP基因进行分子克隆,获得了大小为1515 bp的核苷酸序列,编码504个氨基酸。CBP蛋白在无乳链球菌中有高度的保守性,高达98%-100%。CBP含有两种结构域,分别是GBS Bsp-like repeat domain和Src Homology 3(SH3)domains。CBP蛋白没有信号肽及跨膜结构域,是细胞的表面蛋白。构建了pET28a-CBP质粒并转入大肠杆菌BL21(DE3),获得CBP表达菌株。在IPTG的作用下,成功诱导了目的蛋白的表达。使用镍柱纯化目的蛋白,得到了纯度较高的目的蛋白。2.无乳链球菌CPS的提取、检测和鉴定使用CTAB有机溶剂沉淀法提取罗非鱼源无乳链球菌的CPS,使用蒽酮法检测浓度,并对单糖组成和分子量进行检测。结果显示,本实验提取的CPS是由半乳糖、葡萄糖构成的杂多糖;提取的CPS是相对均一组分,其分子量分布于40000 D-600000 D之间;本实验提取的CPS中未检出蛋白质。本实验成功提取到了浓度、纯度均较高的CPS。3.无乳链球菌免疫保护效果研究制备出CBP亚单位疫苗和荚膜多糖疫苗,腹腔注射免疫罗非鱼,免疫两次即初次免疫和加强免疫,并实施攻毒试验。结果显示,CBP组、CPS-TT组、CPS-CBP组、CPS组的相对保护率分别是:56.58%、69.03%、57.21%、42.79%。ELISA结果显示,免疫后鱼血清中的抗体水平出现不同程度的升高,CBP疫苗诱导的抗体滴度显著高于CPS疫苗,表明疫苗可以诱导特异性抗体的出现。免疫保护性试验结果显示,CBP亚单位疫苗和荚膜多糖疫苗都能对罗非鱼抗无乳链球菌的感染起到免疫保护效果,提高了罗非鱼的抗菌能力。
[Abstract]:As an important breed of fish in the south of China, tilapia has the advantages of fast growth and reproduction and strong adaptability to the environment. However, the outbreak of Streptococcus lactis disease in recent years threatens the breeding of tilapia and affects the healthy development of the tilapia industry to a great extent. As a result, more and more attention has been paid to the development of the related vaccines. The surface composition of Streptococcus lactis plays an important role in the pathogenesis of the pathogen adhesion to host, and its immunogenicity is usually better. Therefore, it can be used to develop the candidate antigen of the vaccine. This paper selects the cholinergic binding protein (Choline-bindi) of Streptococcus niloticus. Ng protein, CBP) and capsule polysaccharide (Capsular polysaccharide, CPS) were used as antigens to study the immune protection effect of vaccine against Streptococcus nactis free streptococcus infection. The main contents are as follows: 1. cloning of CBP gene of Streptococcus nactis, sequence analysis and prokaryotic expression of molecular cloning of CBP gene, and obtained a nucleus of 1515 BP size. The 504 amino acid.CBP protein encoded by the glycoside sequence is highly conserved in Streptococcus free Streptococcus, and up to 98%-100%.CBP contains two domains, which are GBS Bsp-like repeat domain and Src Homology 3 (SH3) domains.CBP protein without signal peptide and transmembrane domain. It is the surface protein of the cell. Enterobacteriaceae BL21 (DE3) obtained the CBP expression strain. Under the action of IPTG, the expression of the target protein was successfully induced. The purify protein was purified by the nickel column and the purity of the target protein,.2. Lactococcus lactis CPS, was extracted, and the CTAB organic solvent precipitation method was used to extract CPS of Streptococcus nilacae from the source of tilapia, and the anthrone method was used. The concentration was detected and the monosaccharide composition and molecular weight were detected. The results showed that the CPS extracted by this experiment was a heteropolysaccharide composed of galactose and glucose; the extracted CPS was a relatively homogeneous component, its molecular weight was between 40000 D-600000 D, and the protein extracted from the experiment was not detected. The concentration and purity of the experiment were successfully extracted. High CPS.3. Streptococcus free Streptococcus immune protection effect study to prepare CBP subunit vaccine and capsule polysaccharide vaccine, intraperitoneal injection of immunized tilapia, immunization two times, primary immunization and enhanced immunization, and carrying out the attack test. The results showed that the relative protection rate of group CBP, CPS-TT group, CPS-CBP group and CPS group were respectively: 56.58%, 69.03%, 57.21%, 42.79%., respectively. The ELISA results showed that the antibody level in the fish serum was elevated in varying degrees after immunization. The antibody titer induced by the CBP vaccine was significantly higher than that of the CPS vaccine, indicating that the vaccine could induce the emergence of specific antibodies. The protective test results showed that the CBP subunit vaccine and the capsular polysaccharide vaccine were all susceptible to the anti Streptococcus mutans of tilapia. The effect of immune protection was enhanced, and the antibacterial ability of tilapia was improved.
【学位授予单位】：中山大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S943

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