金黄色葡萄球菌IsdB蛋白的表达与免疫原性研究

发布时间：2018-05-01 22:51

  本文选题：金黄色葡萄球菌 + 奶牛乳房炎　； 参考：《黑龙江八一农垦大学》2008年硕士论文

【摘要】： 金黄色葡萄球菌(S.aureus)是一种可以引起人和动物多种疾病的重要致病菌,其生长需要的铁来源于感染宿主的血红蛋白。IsdB蛋白是S.aureus铁利用过程中所必须的血红蛋白受体,在S.aureus中具有高度保守性。研究表明IsdB对小鼠和猕猴均有良好的免疫原性,且产生的抗体水平显著高于其它表面蛋白。因此,研究IsdB蛋白的免疫原性,利用IsdB的铁结合机制来发展人和动物抗S.aureus疫苗将具有重要的实践价值和理论意义。 本研究首先采用PCR方法扩增了50株奶牛乳房炎S. aureus分离株的isdB基因,确定isdB基因在我国牛源S.aureus中的保守性。然后对S. aureus菌株BMSA/855/23-1的isdB基因PCR扩增片段进行了纯化回收,克隆到pMD18-T载体上。经测序后,与GenBank已知序列对比分析。进一步克隆到pQE-30表达载体上,转化大肠杆菌XL1-Blue,IPTG诱导后,SDS-PAGE鉴定IsdB融合蛋白的表达。应用纯化的IsdB融合蛋白和全菌体灭活菌苗分别免疫小鼠,Western-Blot检测重组蛋白的特异性抗体结合活性。建立间接ELISA方法,检测蛋白组及全菌体组小鼠血清中IgG抗体水平;应用微量凝集试验检测IsdB蛋白免疫血清对13株奶牛乳房炎S.aureus不同地方分离株的交叉凝集反应;应用MTT和ELISPOT方法检测加强免疫后第10d蛋白组小鼠的T淋巴细胞转化效果及脾淋巴细胞中细胞因子IFN-γ的水平。在二免后第14d用不同S.aureus菌株对蛋白免疫组进行攻毒,用BMSA/855/23-1对全菌体免疫组进行攻毒,评价IsdB蛋白的免疫保护效果。 结果在50株S. aureus分离株中均扩增出了isdB基因,证明该基因在我国牛源S.aureus中是高度保守的。重组克隆pMD18-T-isdB经测序后,与GenBank上S. aureus MW2株的核苷酸及氨基酸序列一致性均为98.8%。SDS-PAGE结果表明,重组质粒pQE-30-isdB在E. coli XL1-Blue中成功表达。Western Blot检测表明重组蛋白具有较好的抗原性。蛋白组及全菌体组小鼠血清中抗IgG抗体水平在加强免疫第三周均达到最高值(1:64 000);微量凝集试验结果表明IsdB蛋白抗血清与12个菌株产生了特异性的凝集反应,证明IsdB蛋白抗血清具有与不同分离株产生抗原抗体反应的能力。T淋巴细胞转化试验结果显示免疫组与对照组相比差异显著(p0.05);ELISPOT检测蛋白组细胞因子IFN-γ的水平与对照组相比,显著升高(p0.05),表明IsdB蛋白能刺激小鼠产生较好的细胞免疫应答。攻毒试验结果表明蛋白免疫组对S.aureus菌株BMSA/855/23-1、SH-12和SH-16的保护率分别为80%、80%和50%,全菌体组对BMSA/855/23-1的保护率为80%。 综上所述,isdB基因在我国牛源金黄色葡萄球菌中具有高度保守性,表达的重组IsdB蛋白具有较好的免疫原性及免疫保护作用,可以作为疫苗的抗原应用于防制S. aureus感染。
[Abstract]:S. aureus is an important pathogen that can cause many diseases in human and animal. The iron needed for its growth comes from the hemoglobin. IsdB protein of infected host is the necessary hemoglobin receptor in the process of iron utilization of S.aureus. It is highly conservative in S.aureus. The results showed that IsdB had good immunogenicity in mice and rhesus monkeys, and the level of antibody produced was significantly higher than that of other surface proteins. Therefore, it is of great practical and theoretical significance to study the immunogenicity of IsdB protein and develop human and animal anti- vaccine by using the iron binding mechanism of IsdB. In this study, we first amplified the isdB gene of 50 strains of bovine mastitis isolated from S. aureus by PCR method, and confirmed the conserved nature of isdB gene in Chinese bovine S.aureus. Then the isdB gene PCR fragment of BMSA/855/23-1 of S. aureus strain was purified and recovered and cloned into pMD18-T vector. After sequencing, the results were compared with the known sequences of GenBank. The expression of IsdB fusion protein was identified by SDS-PAGE after induction by IPTG of Escherichia coli XL1-Blue. The specific antibody binding activity of the recombinant protein was detected by Western-Blot immunizing mice with purified IsdB fusion protein and whole-cell inactivated vaccine. Indirect ELISA method was established to detect the level of IgG antibody in the serum of proteoglycan group and whole cell group, and the cross-agglutination reaction of IsdB protein immunized serum to 13 isolates of S.aureus from dairy cow mastitis was detected by microagglutination test. The effect of T lymphocyte transformation and the level of cytokine IFN- 纬 in spleen lymphocytes of mice in the protein group on the 10th day after enhanced immunization were detected by MTT and ELISPOT methods. On the 14th day after the second immunization, different S.aureus strains were used to attack the protein-immunized group, and BMSA/855/23-1 was used to attack the whole cell immunized group to evaluate the immune protection effect of IsdB protein. Results the isdB gene was amplified from 50 S. aureus isolates, which proved that the gene was highly conserved in Chinese bovine S.aureus. After sequencing, the nucleotide and amino acid sequence of the recombinant pMD18-T-isdB was consistent with that of S. aureus MW2 strain on GenBank. The results showed that the recombinant plasmid pQE-30-isdB was successfully expressed in E. coli XL1-Blue. Western Blot analysis showed that the recombinant protein had good antigenicity. The level of anti IgG antibody in the serum of mice in protein group and whole cell group reached the highest value of 1: 64 000 in the third week of enhanced immunization. The results of microagglutination test showed that the antiserum of IsdB protein produced a specific agglutination reaction with 12 strains. The results of T lymphocyte transformation test showed that the level of cytokine IFN- 纬 in the immunized group was significantly different from that in the control group, and the level of IFN- 纬 in the protein group was higher than that in the control group. It was found that IsdB protein could stimulate a good cellular immune response in mice. The results showed that the protective rates of protein-immunized group against S.aureus strain BMSA / 855 / 23-1 SH-12 and SH-16 were 80% and 50%, respectively. The protective rate of whole cell group on BMSA/855/23-1 was 80% and 50% respectively. In conclusion, the recombinant IsdB gene expressed in Chinese bovine Staphylococcus aureus has a high degree of conservation and has good immunogenicity and protective effect. It can be used as an antigen of vaccine to prevent S. 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;S852.5

【引证文献】

相关硕士学位论文 前2条

1 马光学;金葡菌表面铁元素决定因子B蛋白免疫原性研究[D];吉林农业大学;2012年

2 陈莹;金黄色葡萄球菌抗原铁调节表面决定蛋白B及疫苗研究[D];吉林大学;2013年

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