金黄色葡萄球菌凝集因子A的表达及免疫原性研究
发布时间：2018-07-17 06:31
【摘要】： 金黄色葡萄球菌(Staphylococcus.aureus, S. aureus)是一种重要的致病菌,能够引起广泛的感染。在感染早期,金黄色葡萄球菌通过表面黏附素的入侵、定殖寄主细胞。在这个过程中,表面黏附素成份之一凝集因子A(Clumping factor A, ClfA)发挥至关重要作用。以往研究表明,ClfA可能是一种很好的免疫原,为了解ClfA蛋白对S. aureus不同菌株感染的免疫保护作用,本试验表达了ClfA蛋白,以其制备抗原免疫小鼠,用S. aureus不同菌株攻击,并检测体液免疫和细胞免疫应答。 通过PCR方法扩增S. aureus菌株Newman的clfa基因,随后将其克隆到pMD18-T载体上。重组克隆pMD-T/clfa经测序后,与GenBank上已发表的基因序列进行对比分析,同时对金黄色葡萄球菌Wood46、BMSA/855/23-1也进行了序列分析。然后,将clfa基因插入到pQE-30载体上,构建重组质粒pQE30/clfa,并将其转化大肠杆菌E.coli M15 (pREP4),用IPTG诱导表达,经SDS-PAGE分析,鉴定ClfA融合蛋白。然后用蛋白和全菌体免疫血清进行Western blot检测,确定其抗原性。实验进一步将纯化的蛋白分别以50μg、100μg免疫小白鼠,同时以全菌体灭活苗和对照剂免疫小鼠作为对照。初次免疫后三周进行二次免疫,二次免疫二周后,用S. aureus菌株Wood46(3×109CFU/只)、23-1(1×109CFU/只)对免疫小鼠攻毒,观察一周并纪录结果,与此同时,从初免后每隔一周各组取3只小鼠采血,用ELISA方法检测血清中抗体水平及免疫血清中IFN-γ、IL-4、IL-2细胞因子浓度。 结果,试验扩增获得并克隆了S.aureus的clfa基因,clfa基因序列分析结果与GenBank上S. aureus Newman株的核苷酸序列一致性为99.8%,与S. aureus株Wood46、BMSA/855/23-1的同源性均为99.6%,说明ClfA基因高度保守;SDS-PAGE结果证实,重组质粒pQE30/clfa在E. coli M15(pREP4)中正确表达;Western Blot检测证实重组蛋白具有较好抗原性;ClfA蛋白50μg组、100μg组及全菌体组小鼠在二次免疫后二周,血清中抗体水平分别达到1:65000、1:150000和1:4200。血清中细胞因子IFN-γ、IL-2、IL-4浓度与对照组相比显著升高(p0.05),而全菌体免疫组细胞因子浓度升高不明显(p0.05)。攻毒一周后,Wood46攻毒组结果:100μg蛋白、50μg蛋白和全菌体免疫组存活率分别是50%、12.5%和0;BMSA/855/23-1攻毒组结果:100μg、50μg蛋白免疫组和全菌体免疫组存活率分别是37.5%、0、0。 以上结果表明,表达的重组ClfA蛋白具有免疫原性及较好的免疫保护力,为金黄色葡萄球菌引起的感染的新型疫苗的研究提供参考。
[Abstract]:Staphylococcus aureus (S. aureus) is an important pathogen that can cause a wide range of infections. In the early stage of infection, Staphylococcus aureus colonizes host cells through the invasion of surface adhesion. In this process, surface adhesion factor A (CLFA), one of the components of surface adhesion, plays an important role. Previous studies have shown that ClfA may be a good immunogen. In order to understand the protective effect of ClfA protein on the infection of different strains of S. aureus, we expressed ClfA protein to immunize mice with antigens and attacked with different strains of S. aureus. Humoral and cellular immune responses were detected. The clfa gene of S. aureus strain Newman was amplified by PCR and cloned into pMD18-T vector. The recombinant clone pMD-Trclfa was sequenced and compared with the published gene sequence in GenBank. The sequence analysis was also carried out on Wood46 BMSA / 855 / 23-1 of Staphylococcus aureus. Then, the clfa gene was inserted into pQE-30 vector to construct the recombinant plasmid pQE30 / clfa. the recombinant plasmid was transformed into E. coli M15 (pREP4) and expressed by IPTG. The fusion protein was identified by SDS-PAGE. Then the antigenicity of protein and whole cell immune serum was determined by Western blot. The purified protein was further immunized with 50 渭 g / 100 渭 g of the protein, and the mice were immunized with the whole bacterial inactivated vaccine and the control group respectively. After three weeks of primary immunization, the mice were immunized with S.aureus strain Wood46 (3 脳 10 9 CFU / mouse) for one week and the results were recorded. At the same time, three mice were collected from each group every other week after the first immunization. Elisa was used to detect the level of antibody in serum and the level of IL-2 in immune serum. The results showed that the nucleotide sequence of S. aureus Newman strain was 99.8, and the homology with that of S.aureus strain Wood46 BMSA / 855 / 23-1 was 99.6%, which indicated that the ClfA gene was highly conserved by SDS-PAGE. The recombinant plasmid pQE30 / clfa was correctly expressed in E. coli M15 (pREP4). The results of Western blot analysis showed that the recombinant protein had good antigenicity in 50 渭 g / g group and 100 渭 g / 100 渭 g / g mice, and the antibody levels in serum reached 1: 650001w / 150000 and 1: 4200 respectively two weeks after the second immunization of the recombinant plasmid pQE30 / clfa. The concentration of IL-4 in serum IFN- 纬 and IL-2 was significantly higher than that in the control group (p0.05), but the concentration of cytokine in the whole cell immunized group was not significantly increased (p0.05). One week after attack, the survival rate of 50 渭 g protein of 100 渭 g protein and 50 渭 g protein of whole cell immunized group were 50% and 12.5%, respectively, and the survival rate of 100 渭 g protein 50 渭 g protein immunization group and the whole cell immunization group were 37.50.0.The survival rates of BMSA / 855 / 23-1 group were 37.5% and 37.5% respectively. These results suggest that the expressed recombinant ClfA protein has immunogenicity and good immune protection, which provides a reference for the study of the new vaccine caused by Staphylococcus aureus infection.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378.11
[Abstract]:Staphylococcus aureus (S. aureus) is an important pathogen that can cause a wide range of infections. In the early stage of infection, Staphylococcus aureus colonizes host cells through the invasion of surface adhesion. In this process, surface adhesion factor A (CLFA), one of the components of surface adhesion, plays an important role. Previous studies have shown that ClfA may be a good immunogen. In order to understand the protective effect of ClfA protein on the infection of different strains of S. aureus, we expressed ClfA protein to immunize mice with antigens and attacked with different strains of S. aureus. Humoral and cellular immune responses were detected. The clfa gene of S. aureus strain Newman was amplified by PCR and cloned into pMD18-T vector. The recombinant clone pMD-Trclfa was sequenced and compared with the published gene sequence in GenBank. The sequence analysis was also carried out on Wood46 BMSA / 855 / 23-1 of Staphylococcus aureus. Then, the clfa gene was inserted into pQE-30 vector to construct the recombinant plasmid pQE30 / clfa. the recombinant plasmid was transformed into E. coli M15 (pREP4) and expressed by IPTG. The fusion protein was identified by SDS-PAGE. Then the antigenicity of protein and whole cell immune serum was determined by Western blot. The purified protein was further immunized with 50 渭 g / 100 渭 g of the protein, and the mice were immunized with the whole bacterial inactivated vaccine and the control group respectively. After three weeks of primary immunization, the mice were immunized with S.aureus strain Wood46 (3 脳 10 9 CFU / mouse) for one week and the results were recorded. At the same time, three mice were collected from each group every other week after the first immunization. Elisa was used to detect the level of antibody in serum and the level of IL-2 in immune serum. The results showed that the nucleotide sequence of S. aureus Newman strain was 99.8, and the homology with that of S.aureus strain Wood46 BMSA / 855 / 23-1 was 99.6%, which indicated that the ClfA gene was highly conserved by SDS-PAGE. The recombinant plasmid pQE30 / clfa was correctly expressed in E. coli M15 (pREP4). The results of Western blot analysis showed that the recombinant protein had good antigenicity in 50 渭 g / g group and 100 渭 g / 100 渭 g / g mice, and the antibody levels in serum reached 1: 650001w / 150000 and 1: 4200 respectively two weeks after the second immunization of the recombinant plasmid pQE30 / clfa. The concentration of IL-4 in serum IFN- 纬 and IL-2 was significantly higher than that in the control group (p0.05), but the concentration of cytokine in the whole cell immunized group was not significantly increased (p0.05). One week after attack, the survival rate of 50 渭 g protein of 100 渭 g protein and 50 渭 g protein of whole cell immunized group were 50% and 12.5%, respectively, and the survival rate of 100 渭 g protein 50 渭 g protein immunization group and the whole cell immunization group were 37.50.0.The survival rates of BMSA / 855 / 23-1 group were 37.5% and 37.5% respectively. These results suggest that the expressed recombinant ClfA protein has immunogenicity and good immune protection, which provides a reference for the study of the new vaccine caused by Staphylococcus aureus infection.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378.11
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