金黄色葡萄球菌Trap、IsdA、IsdB、GapC蛋白免疫保护作用比较研究
发布时间：2018-10-26 18:16
【摘要】：金黄色葡萄球菌(Staphylococcus aureus)是引起人和动物多种疾病的重要致病菌。一直以来人们主要采用抗生素控制和治疗S.aureus感染，但由于抗生素的广泛使用，导致耐药菌株不断出现，，致使抗生素治疗几乎毫无效果。因此，研究和使用疫苗来防制S.aureus感染已成为热点。过去的研究表明，金黄色葡萄球菌表面的TraP、IsdA、IsdB、GapC蛋白具有良好的免疫原性，为了比较金黄色葡萄球菌TRAP、IsdA、IsdB、GapC的免疫原性和免疫保护作用，应用PCR的方法，分别扩增出金黄色葡萄球菌Newman株的目的基因并进行序列分析，再将目的基因分别插入到pQE-30载体上，导入宿主菌Escherichia coliM15(pREP4)并诱导表达和纯化重组蛋白。用纯化的重组蛋白分别免疫小鼠，检测小鼠血清中抗体水平；在体外用相应的抗原刺激淋巴细胞，ELSPOT检测免疫脾淋巴细胞分泌IL-4和IFN-γ的细胞频数，ELISA检测脾细胞培养上清中IL-17的分泌水平。首次免疫后35d时用金黄色葡萄球菌Newman、Wod46、HLJ23-1株对小鼠攻毒，结果表明TraP、IsdA、IsdB、GapC重组蛋白在E．coli M15中均获得表达；在首次免疫后35d时血清抗体水平，免疫脾脏淋巴细胞分泌的细胞因子与对照组相比，均显著升高(P0.05)；攻毒结果表明，TraP、GapC、IsdB蛋白分别对Newman、Wood46、HLJ23-1菌株攻毒提供了较好的免疫保护作用。以上结果表明同一抗原对不同菌株攻毒所产生的免疫保护存在差异。
[Abstract]:Staphylococcus aureus (Staphylococcus aureus) is an important pathogen causing many diseases in humans and animals. Antibiotics have been widely used to control and treat S.aureus infection, but because of the widespread use of antibiotics, drug-resistant strains continue to appear, resulting in almost no effect of antibiotic treatment. Therefore, the research and use of vaccines to prevent S.aureus infection has become a hot spot. Previous studies have shown that the TraP,IsdA,IsdB,GapC protein on the surface of Staphylococcus aureus has good immunogenicity. In order to compare the immunogenicity and immunogenicity of Staphylococcus aureus TRAP,IsdA,IsdB,GapC, the method of PCR was used. The target gene of Staphylococcus aureus Newman strain was amplified and sequenced. Then the target gene was inserted into the pQE-30 vector respectively. The target gene was introduced into the host strain Escherichia coliM15 (pREP4) and induced to express and purify the recombinant protein. Mice were immunized with purified recombinant protein to detect the level of antibody in serum. Lymphocytes were stimulated by corresponding antigens in vitro. ELSPOT was used to detect the frequency of IL-4 and IFN- 纬 secreted by immune splenic lymphocytes, and ELISA was used to detect the secretion of IL-17 in the supernatant of splenocyte culture. The mice were inoculated with Staphylococcus aureus Newman,Wod46,HLJ23-1 strain 35 days after the first immunization. The results showed that the recombinant TraP,IsdA,IsdB,GapC protein was expressed in E.coli M15. At the 35d after the first immunization, the levels of serum antibody and cytokines secreted by immunized spleen lymphocytes were significantly higher than those in the control group (P0.05). The results showed that the TraP,GapC,IsdB protein provided better immune protection to Newman,Wood46,HLJ23-1 strain. The above results indicated that the same antigen had different immune protection to different strains.
【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】：2013
【分类号】：R392
本文编号：2296562
[Abstract]:Staphylococcus aureus (Staphylococcus aureus) is an important pathogen causing many diseases in humans and animals. Antibiotics have been widely used to control and treat S.aureus infection, but because of the widespread use of antibiotics, drug-resistant strains continue to appear, resulting in almost no effect of antibiotic treatment. Therefore, the research and use of vaccines to prevent S.aureus infection has become a hot spot. Previous studies have shown that the TraP,IsdA,IsdB,GapC protein on the surface of Staphylococcus aureus has good immunogenicity. In order to compare the immunogenicity and immunogenicity of Staphylococcus aureus TRAP,IsdA,IsdB,GapC, the method of PCR was used. The target gene of Staphylococcus aureus Newman strain was amplified and sequenced. Then the target gene was inserted into the pQE-30 vector respectively. The target gene was introduced into the host strain Escherichia coliM15 (pREP4) and induced to express and purify the recombinant protein. Mice were immunized with purified recombinant protein to detect the level of antibody in serum. Lymphocytes were stimulated by corresponding antigens in vitro. ELSPOT was used to detect the frequency of IL-4 and IFN- 纬 secreted by immune splenic lymphocytes, and ELISA was used to detect the secretion of IL-17 in the supernatant of splenocyte culture. The mice were inoculated with Staphylococcus aureus Newman,Wod46,HLJ23-1 strain 35 days after the first immunization. The results showed that the recombinant TraP,IsdA,IsdB,GapC protein was expressed in E.coli M15. At the 35d after the first immunization, the levels of serum antibody and cytokines secreted by immunized spleen lymphocytes were significantly higher than those in the control group (P0.05). The results showed that the TraP,GapC,IsdB protein provided better immune protection to Newman,Wood46,HLJ23-1 strain. The above results indicated that the same antigen had different immune protection to different strains.
【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】：2013
【分类号】：R392
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