【摘要】：金黃色葡萄球菌(Staphylococcus aureus)是引起人和動物多種疾病的重要致病菌。一直以來人們主要采用抗生素控制和治療S.aureus感染，但由于抗生素的廣泛使用，導(dǎo)致耐藥菌株不斷出現(xiàn)，，致使抗生素治療幾乎毫無效果。因此，研究和使用疫苗來防制S.aureus感染已成為熱點。過去的研究表明，金黃色葡萄球菌表面的TraP、IsdA、IsdB、GapC蛋白具有良好的免疫原性，為了比較金黃色葡萄球菌TRAP、IsdA、IsdB、GapC的免疫原性和免疫保護(hù)作用，應(yīng)用PCR的方法，分別擴(kuò)增出金黃色葡萄球菌Newman株的目的基因并進(jìn)行序列分析，再將目的基因分別插入到pQE-30載體上，導(dǎo)入宿主菌Escherichia coliM15(pREP4)并誘導(dǎo)表達(dá)和純化重組蛋白。用純化的重組蛋白分別免疫小鼠，檢測小鼠血清中抗體水平；在體外用相應(yīng)的抗原刺激淋巴細(xì)胞，ELSPOT檢測免疫脾淋巴細(xì)胞分泌IL-4和IFN-γ的細(xì)胞頻數(shù)，ELISA檢測脾細(xì)胞培養(yǎng)上清中IL-17的分泌水平。首次免疫后35d時用金黃色葡萄球菌Newman、Wod46、HLJ23-1株對小鼠攻毒，結(jié)果表明TraP、IsdA、IsdB、GapC重組蛋白在E．coli M15中均獲得表達(dá)；在首次免疫后35d時血清抗體水平，免疫脾臟淋巴細(xì)胞分泌的細(xì)胞因子與對照組相比，均顯著升高(P0.05)；攻毒結(jié)果表明，TraP、GapC、IsdB蛋白分別對Newman、Wood46、HLJ23-1菌株攻毒提供了較好的免疫保護(hù)作用。以上結(jié)果表明同一抗原對不同菌株攻毒所產(chǎn)生的免疫保護(hù)存在差異。
[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- 緯 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.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R392

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