金黃色葡萄球菌凝集因子A的表達(dá)及免疫原性研究
發(fā)布時(shí)間：2018-07-17 06:31
【摘要】： 金黃色葡萄球菌(Staphylococcus.aureus, S. aureus)是一種重要的致病菌,能夠引起廣泛的感染。在感染早期,金黃色葡萄球菌通過(guò)表面黏附素的入侵、定殖寄主細(xì)胞。在這個(gè)過(guò)程中,表面黏附素成份之一凝集因子A(Clumping factor A, ClfA)發(fā)揮至關(guān)重要作用。以往研究表明,ClfA可能是一種很好的免疫原,為了解ClfA蛋白對(duì)S. aureus不同菌株感染的免疫保護(hù)作用,本試驗(yàn)表達(dá)了ClfA蛋白,以其制備抗原免疫小鼠,用S. aureus不同菌株攻擊,并檢測(cè)體液免疫和細(xì)胞免疫應(yīng)答。 通過(guò)PCR方法擴(kuò)增S. aureus菌株Newman的clfa基因,隨后將其克隆到pMD18-T載體上。重組克隆pMD-T/clfa經(jīng)測(cè)序后,與GenBank上已發(fā)表的基因序列進(jìn)行對(duì)比分析,同時(shí)對(duì)金黃色葡萄球菌Wood46、BMSA/855/23-1也進(jìn)行了序列分析。然后,將clfa基因插入到pQE-30載體上,構(gòu)建重組質(zhì)粒pQE30/clfa,并將其轉(zhuǎn)化大腸桿菌E.coli M15 (pREP4),用IPTG誘導(dǎo)表達(dá),經(jīng)SDS-PAGE分析,鑒定ClfA融合蛋白。然后用蛋白和全菌體免疫血清進(jìn)行Western blot檢測(cè),確定其抗原性。實(shí)驗(yàn)進(jìn)一步將純化的蛋白分別以50μg、100μg免疫小白鼠,同時(shí)以全菌體滅活苗和對(duì)照劑免疫小鼠作為對(duì)照。初次免疫后三周進(jìn)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)對(duì)免疫小鼠攻毒,觀察一周并紀(jì)錄結(jié)果,與此同時(shí),從初免后每隔一周各組取3只小鼠采血,用ELISA方法檢測(cè)血清中抗體水平及免疫血清中IFN-γ、IL-4、IL-2細(xì)胞因子濃度。 結(jié)果,試驗(yàn)擴(kuò)增獲得并克隆了S.aureus的clfa基因,clfa基因序列分析結(jié)果與GenBank上S. aureus Newman株的核苷酸序列一致性為99.8%,與S. aureus株Wood46、BMSA/855/23-1的同源性均為99.6%,說(shuō)明ClfA基因高度保守;SDS-PAGE結(jié)果證實(shí),重組質(zhì)粒pQE30/clfa在E. coli M15(pREP4)中正確表達(dá);Western Blot檢測(cè)證實(shí)重組蛋白具有較好抗原性;ClfA蛋白50μg組、100μg組及全菌體組小鼠在二次免疫后二周,血清中抗體水平分別達(dá)到1:65000、1:150000和1:4200。血清中細(xì)胞因子IFN-γ、IL-2、IL-4濃度與對(duì)照組相比顯著升高(p0.05),而全菌體免疫組細(xì)胞因子濃度升高不明顯(p0.05)。攻毒一周后,Wood46攻毒組結(jié)果:100μg蛋白、50μg蛋白和全菌體免疫組存活率分別是50%、12.5%和0;BMSA/855/23-1攻毒組結(jié)果:100μg、50μg蛋白免疫組和全菌體免疫組存活率分別是37.5%、0、0。 以上結(jié)果表明,表達(dá)的重組ClfA蛋白具有免疫原性及較好的免疫保護(hù)力,為金黃色葡萄球菌引起的感染的新型疫苗的研究提供參考。
[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- 緯 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.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：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- 緯 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.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R378.11
【引證文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 王U，
本文編號(hào)：2129410
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