金黄色葡萄球菌表面多糖偶联抗原的制备及其免疫原性分析

发布时间：2018-06-16 18:50

本文选题：金黄色葡萄球菌 + 奶牛乳房炎　；参考：《石河子大学》2011年硕士论文

【摘要】：金黄色葡萄球菌是奶牛乳房炎以及医院临床感染的主要病原,对该菌感染的防治主要采用抗生素进行治疗。然而,长期、大量的抗生素应用导致细菌耐药性的增强和蔓延,使防治难度不断加大。此外,用抗生素治疗奶牛乳房炎还存在奶产品抗生素残留,影响食品安全。研制和开发有效疫苗,通过免疫接种进行金黄色葡萄球菌感染的预防已成为近年来研究的热点。表面多糖(CP5、CP8和336PS)是金黄色葡萄球菌的主要共同性抗原成分和毒力因子,也是疫苗开发的主要靶抗原。该类多糖的分子量小,免疫原性差,通过技术方法改善其免疫原性对于研制有效疫苗至关重要。本研究分别对金黄色葡萄球菌的3种重要表面多糖进行了提取、纯化、蛋白载体偶联和偶联抗原的免疫原性研究。首先采用高压破碎法使荚膜多糖CP5和CP8从金黄色葡萄球菌菌体释放,选用不同的酶处理去除破碎产物中的核酸和蛋白,再通过离子交换层析获得一定纯度的CP5、CP8荚膜多糖。表面多糖336PS的获取则采用溶葡萄球菌酶消化法使其从菌体释放,梯度乙醇浸提和不同酶的消化进行粗提,再通过凝胶层析纯化,得到了一定纯度的336PS多糖。研究建立了金黄色葡萄球菌表面多糖的化学检定方法。检测结果表明,CP5、CP8、336PS的纯度分别为67.51%、63.83%和68.51%；用免疫琼脂双扩散试验检测,纯化的多糖仅与抗同型菌株免疫血清发生沉淀反应。研究制备了3种表面多糖的蛋白偶联抗原,并对偶联抗原的免疫原性进行了检测。将纯化表面多糖通过ADH间桥法和EDAC零距离交联法与BSA偶联,凝胶过滤层析纯化,紫外扫描鉴定,从检定波长判定偶联成功。分别制备了6种表面多糖偶联抗原的油佐剂亚单位疫苗免疫小鼠,并以同样的方式制备3种未偶联表面多糖、PBS疫苗做对照。分别在免疫前,免疫第14d和第28d采集各组免疫小鼠血,分离血清,用间接ELISA检测血清中针对抗表面多糖的抗体水平。检测结果表明：2种偶联方法制备的抗原都能刺激机体产生抗良好的免疫应答反应,而未偶联的多糖则无免疫原性。为了解该抗体的的免疫保护性,在第28 d加强免疫一次,7天后后腿外侧注射同型菌株,观察小鼠后腿外侧临床病理变化并进行临床指数评分。攻毒实验表明,偶联抗原组能够对免疫小鼠提供较好的免疫保护作用。经综合分析,ADH间桥法制备的CP5-BSA、CP8-BSA和336PS-BSA偶联抗原免疫效果优于EDAC零距离交联法。为克服载体蛋白BSA在奶牛免疫中免疫原性差的问题,采用基因工程方法,通过PCR扩增绿脓杆菌外毒素A基因(ETA),构建重组表达载体pET-28a-ETA,转化进DH5a感受态细胞,提取重组质粒pET-28a-ETA并进行双酶切、PCR鉴定及测序鉴定。用IPTG进行诱导表达,对其产物进行SDS-PAGE和western blotting分析,并对诱导表达条件进行优化,确定目的蛋白分布,最终批量进行表达。采用尿素对包涵体进行变性并确定最佳的复性条件和复性缓冲液,Ni-NTA树脂柱上复性目的蛋白。柱上复性后目的蛋白的纯度达到93%以上。采用ADH间桥法制备了表面多糖-rEPA偶联,经过动物实验表明,以上三种多糖-rEPA偶联抗原均能刺激产生免疫应答反应。且分离血清能与同型菌株发生血清凝集反应,具有较好的免疫原性。该研究首次在国内建立了金黄色葡萄球菌表面多糖CP8、336PS的分离纯化和与载体蛋白的偶联技术方法,并通过小鼠免疫和攻毒证明了偶联抗原的免疫原性和免疫保护作用,为金黄色葡萄球菌奶牛乳房炎亚单位疫苗的研制提供了技术储备。
[Abstract]:Staphylococcus aureus is the main pathogen of Dairy Mastitis and clinical infection in the hospital. Antibiotics are mainly used in the prevention and treatment of the infection. However, a large number of antibiotic applications have led to the enhancement and spread of bacterial resistance in the long term, which makes the prevention and control difficulty more and more. In addition, the use of antibiotics in the treatment of dairy cow mastitis is also milk production. The residue of antibiotics affects food safety. The development and development of effective vaccines and the prevention of Staphylococcus aureus infection through immunization have become a hot spot of research in recent years.
Surface polysaccharide (CP5, CP8 and 336PS) is the main common antigen component and virulence factor of Staphylococcus aureus. It is also the main target antigen of vaccine development. The molecular weight of this kind of polysaccharide is small and the immunogenicity is poor. It is very important to improve its immunogenicity by technical methods. This study on Staphylococcus aureus 3, respectively. Polysaccharides from important surface polysaccharides were extracted, purified, coupled with protein carriers and immunogenicity of conjugated antigens.
First, the capsular polysaccharide CP5 and CP8 were released from the Staphylococcus aureus bacteria by high pressure crushing. Different enzymes were used to remove the nucleic acid and protein in the broken products, and then a certain purity of CP5 and CP8 capsule polysaccharide was obtained by ion exchange chromatography. The acquisition of the surface polysaccharide 336PS was made by the digestion method of staphylococcal enzyme to make it from the bacteria. After release, gradient ethanol extraction and digestion of different enzymes, crude 336PS was extracted and purified by gel chromatography.
A chemical assay for the surface polysaccharide of Staphylococcus aureus was established. The results showed that the purity of CP5 and CP8336PS were 67.51%, 63.83% and 68.51%, respectively, and the purified polysaccharide was only reacted with the immune sera of the antihomo strain by immuno agar double diffusion test.
The protein coupled antigen of 3 kinds of surface polysaccharides was prepared and the immunogenicity of the coupled antigen was detected. The purified surface polysaccharide was coupled with BSA by ADH bridge method and EDAC zero distance cross-linking method, the gel filtration chromatography was purified and the UV scanning was identified. The coupling resistance of 6 kinds of surface polysaccharides was prepared. The original oil adjuvant subunit vaccine was immune to mice, and 3 kinds of uncoupled surface polysaccharides were prepared in the same way. PBS vaccine was used as control. Before immunization, the blood of mice immunized with immunization 14d and 28d were collected, serum was isolated and the level of antibody against surface polysaccharides in serum was detected by indirect ELISA. The results showed that 2 coupling methods were found. All antigens prepared can stimulate the body to produce a good immune response, while unconjugated polysaccharides have no immunogenicity.
In order to understand the immunological protection of the antibody, the immunization was strengthened at twenty-eighth D and the same type of strain was injected into the lateral leg 7 days later. The clinical pathological changes of the lateral hind leg of the mice were observed and the clinical index score was evaluated. The experiment showed that the coupling antigen group could provide better immune protection to the immune mice. After comprehensive analysis, the ADH bridge method was used. The CP5-BSA, CP8-BSA and 336PS-BSA conjugated antigen immunization effect is superior to EDAC zero distance crosslinking method.
In order to overcome the problem of poor immunogenicity of carrier protein BSA in cow immunity, the recombinant expression vector pET-28a-ETA was constructed by genetic engineering method and PCR amplification of ETA (Pseudomonas aeruginosa exotoxin A gene), and transformed into DH5a receptive cells. The recombinant plasmid pET-28a-ETA was extracted and analyzed by double enzyme digestion, PCR identification and sequencing identification. It was induced by IPTG for induction. SDS-PAGE and Western blotting analysis of the products were carried out, and the expression conditions were optimized to determine the distribution of the target protein, and the final batch was expressed. The urea was used to denaturate the inclusion bodies and to determine the best refolding conditions and the complex buffer solution. The target protein on the Ni-NTA resin column. The target protein after the refolding of the column was found. The purity is above 93%.
The surface polysaccharide -rEPA coupling was prepared by the ADH bridge method. Through animal experiments, the above three polysaccharides -rEPA coupling antigen could stimulate the immune response, and the isolated sera could react with the same type strain and have good immunogenicity.
In this study, the isolation and purification of the surface polysaccharide CP8336PS of Staphylococcus aureus and the coupling technique with the carrier protein were established in China for the first time. The immunogenicity and immune protection of the coupled antigen were proved by immunization and attack in mice, which provided a technical storage for the development of the yellowish yellowish yellowish unit vaccine of Staphylococcus aureus. Prepare.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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