本文選題：金黃色葡萄球菌 + 奶牛乳房炎　； 參考：《黑龍江八一農(nóng)墾大學(xué)》2008年碩士論文

【摘要】： 金黃色葡萄球菌(S.aureus)是一種可以引起人和動物多種疾病的重要致病菌,其生長需要的鐵來源于感染宿主的血紅蛋白。IsdB蛋白是S.aureus鐵利用過程中所必須的血紅蛋白受體,在S.aureus中具有高度保守性。研究表明IsdB對小鼠和獼猴均有良好的免疫原性,且產(chǎn)生的抗體水平顯著高于其它表面蛋白。因此,研究IsdB蛋白的免疫原性,利用IsdB的鐵結(jié)合機制來發(fā)展人和動物抗S.aureus疫苗將具有重要的實踐價值和理論意義。 本研究首先采用PCR方法擴增了50株奶牛乳房炎S. aureus分離株的isdB基因,確定isdB基因在我國牛源S.aureus中的保守性。然后對S. aureus菌株BMSA/855/23-1的isdB基因PCR擴增片段進(jìn)行了純化回收,克隆到pMD18-T載體上。經(jīng)測序后,與GenBank已知序列對比分析。進(jìn)一步克隆到pQE-30表達(dá)載體上,轉(zhuǎn)化大腸桿菌XL1-Blue,IPTG誘導(dǎo)后,SDS-PAGE鑒定IsdB融合蛋白的表達(dá)。應(yīng)用純化的IsdB融合蛋白和全菌體滅活菌苗分別免疫小鼠,Western-Blot檢測重組蛋白的特異性抗體結(jié)合活性。建立間接ELISA方法,檢測蛋白組及全菌體組小鼠血清中IgG抗體水平;應(yīng)用微量凝集試驗檢測IsdB蛋白免疫血清對13株奶牛乳房炎S.aureus不同地方分離株的交叉凝集反應(yīng);應(yīng)用MTT和ELISPOT方法檢測加強免疫后第10d蛋白組小鼠的T淋巴細(xì)胞轉(zhuǎn)化效果及脾淋巴細(xì)胞中細(xì)胞因子IFN-γ的水平。在二免后第14d用不同S.aureus菌株對蛋白免疫組進(jìn)行攻毒,用BMSA/855/23-1對全菌體免疫組進(jìn)行攻毒,評價IsdB蛋白的免疫保護(hù)效果。 結(jié)果在50株S. aureus分離株中均擴增出了isdB基因,證明該基因在我國牛源S.aureus中是高度保守的。重組克隆pMD18-T-isdB經(jīng)測序后,與GenBank上S. aureus MW2株的核苷酸及氨基酸序列一致性均為98.8%。SDS-PAGE結(jié)果表明,重組質(zhì)粒pQE-30-isdB在E. coli XL1-Blue中成功表達(dá)。Western Blot檢測表明重組蛋白具有較好的抗原性。蛋白組及全菌體組小鼠血清中抗IgG抗體水平在加強免疫第三周均達(dá)到最高值(1:64 000);微量凝集試驗結(jié)果表明IsdB蛋白抗血清與12個菌株產(chǎn)生了特異性的凝集反應(yīng),證明IsdB蛋白抗血清具有與不同分離株產(chǎn)生抗原抗體反應(yīng)的能力。T淋巴細(xì)胞轉(zhuǎn)化試驗結(jié)果顯示免疫組與對照組相比差異顯著(p0.05);ELISPOT檢測蛋白組細(xì)胞因子IFN-γ的水平與對照組相比,顯著升高(p0.05),表明IsdB蛋白能刺激小鼠產(chǎn)生較好的細(xì)胞免疫應(yīng)答。攻毒試驗結(jié)果表明蛋白免疫組對S.aureus菌株BMSA/855/23-1、SH-12和SH-16的保護(hù)率分別為80%、80%和50%,全菌體組對BMSA/855/23-1的保護(hù)率為80%。 綜上所述,isdB基因在我國牛源金黃色葡萄球菌中具有高度保守性,表達(dá)的重組IsdB蛋白具有較好的免疫原性及免疫保護(hù)作用,可以作為疫苗的抗原應(yīng)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- 緯 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- 緯 in the immunized group was significantly different from that in the control group, and the level of IFN- 緯 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.
【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】：2008
【分類號】：R378;S852.5

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前2條

1 馬光學(xué);金葡菌表面鐵元素決定因子B蛋白免疫原性研究[D];吉林農(nóng)業(yè)大學(xué);2012年

2 陳瑩;金黃色葡萄球菌抗原鐵調(diào)節(jié)表面決定蛋白B及疫苗研究[D];吉林大學(xué);2013年

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本文編號：1831348