本文選題：金黃色葡萄球菌 + 奶牛乳房炎��； 參考：《石河子大學(xué)》2011年碩士論文

【摘要】：金黃色葡萄球菌是奶牛乳房炎以及醫(yī)院臨床感染的主要病原,對該菌感染的防治主要采用抗生素進(jìn)行治療。然而,長期、大量的抗生素應(yīng)用導(dǎo)致細(xì)菌耐藥性的增強(qiáng)和蔓延,使防治難度不斷加大。此外,用抗生素治療奶牛乳房炎還存在奶產(chǎn)品抗生素殘留,影響食品安全。研制和開發(fā)有效疫苗,通過免疫接種進(jìn)行金黃色葡萄球菌感染的預(yù)防已成為近年來研究的熱點(diǎn)。 表面多糖(CP5、CP8和336PS)是金黃色葡萄球菌的主要共同性抗原成分和毒力因子,也是疫苗開發(fā)的主要靶抗原。該類多糖的分子量小,免疫原性差,通過技術(shù)方法改善其免疫原性對于研制有效疫苗至關(guān)重要。本研究分別對金黃色葡萄球菌的3種重要表面多糖進(jìn)行了提取、純化、蛋白載體偶聯(lián)和偶聯(lián)抗原的免疫原性研究。 首先采用高壓破碎法使莢膜多糖CP5和CP8從金黃色葡萄球菌菌體釋放,選用不同的酶處理去除破碎產(chǎn)物中的核酸和蛋白,再通過離子交換層析獲得一定純度的CP5、CP8莢膜多糖。表面多糖336PS的獲取則采用溶葡萄球菌酶消化法使其從菌體釋放,梯度乙醇浸提和不同酶的消化進(jìn)行粗提,再通過凝膠層析純化,得到了一定純度的336PS多糖。 研究建立了金黃色葡萄球菌表面多糖的化學(xué)檢定方法。檢測結(jié)果表明,CP5、CP8、336PS的純度分別為67.51%、63.83%和68.51%；用免疫瓊脂雙擴(kuò)散試驗(yàn)檢測,純化的多糖僅與抗同型菌株免疫血清發(fā)生沉淀反應(yīng)。 研究制備了3種表面多糖的蛋白偶聯(lián)抗原,并對偶聯(lián)抗原的免疫原性進(jìn)行了檢測。將純化表面多糖通過ADH間橋法和EDAC零距離交聯(lián)法與BSA偶聯(lián),凝膠過濾層析純化,紫外掃描鑒定,從檢定波長判定偶聯(lián)成功。分別制備了6種表面多糖偶聯(lián)抗原的油佐劑亞單位疫苗免疫小鼠,并以同樣的方式制備3種未偶聯(lián)表面多糖、PBS疫苗做對照。分別在免疫前,免疫第14d和第28d采集各組免疫小鼠血,分離血清,用間接ELISA檢測血清中針對抗表面多糖的抗體水平。檢測結(jié)果表明：2種偶聯(lián)方法制備的抗原都能刺激機(jī)體產(chǎn)生抗良好的免疫應(yīng)答反應(yīng),而未偶聯(lián)的多糖則無免疫原性。 為了解該抗體的的免疫保護(hù)性,在第28 d加強(qiáng)免疫一次,7天后后腿外側(cè)注射同型菌株,觀察小鼠后腿外側(cè)臨床病理變化并進(jìn)行臨床指數(shù)評分。攻毒實(shí)驗(yàn)表明,偶聯(lián)抗原組能夠?qū)γ庖咝∈筇峁┹^好的免疫保護(hù)作用。經(jīng)綜合分析,ADH間橋法制備的CP5-BSA、CP8-BSA和336PS-BSA偶聯(lián)抗原免疫效果優(yōu)于EDAC零距離交聯(lián)法。 為克服載體蛋白BSA在奶牛免疫中免疫原性差的問題,采用基因工程方法,通過PCR擴(kuò)增綠膿桿菌外毒素A基因(ETA),構(gòu)建重組表達(dá)載體pET-28a-ETA,轉(zhuǎn)化進(jìn)DH5a感受態(tài)細(xì)胞,提取重組質(zhì)粒pET-28a-ETA并進(jìn)行雙酶切、PCR鑒定及測序鑒定。用IPTG進(jìn)行誘導(dǎo)表達(dá),對其產(chǎn)物進(jìn)行SDS-PAGE和western blotting分析,并對誘導(dǎo)表達(dá)條件進(jìn)行優(yōu)化,確定目的蛋白分布,最終批量進(jìn)行表達(dá)。采用尿素對包涵體進(jìn)行變性并確定最佳的復(fù)性條件和復(fù)性緩沖液,Ni-NTA樹脂柱上復(fù)性目的蛋白。柱上復(fù)性后目的蛋白的純度達(dá)到93%以上。 采用ADH間橋法制備了表面多糖-rEPA偶聯(lián),經(jīng)過動物實(shí)驗(yàn)表明,以上三種多糖-rEPA偶聯(lián)抗原均能刺激產(chǎn)生免疫應(yīng)答反應(yīng)。且分離血清能與同型菌株發(fā)生血清凝集反應(yīng),具有較好的免疫原性。 該研究首次在國內(nèi)建立了金黃色葡萄球菌表面多糖CP8、336PS的分離純化和與載體蛋白的偶聯(lián)技術(shù)方法,并通過小鼠免疫和攻毒證明了偶聯(lián)抗原的免疫原性和免疫保護(hù)作用,為金黃色葡萄球菌奶牛乳房炎亞單位疫苗的研制提供了技術(shù)儲備。
[Abstract]:Staphylococcus aureus is the main pathogen of Dairy Mastitis and clinical infection in the hospital. Antibiotics are mainly used in the prevention and treatment of the infection. However, a large number of antibiotic applications have led to the enhancement and spread of bacterial resistance in the long term, which makes the prevention and control difficulty more and more. In addition, the use of antibiotics in the treatment of dairy cow mastitis is also milk production. The residue of antibiotics affects food safety. The development and development of effective vaccines and the prevention of Staphylococcus aureus infection through immunization have become a hot spot of research in recent years.
Surface polysaccharide (CP5, CP8 and 336PS) is the main common antigen component and virulence factor of Staphylococcus aureus. It is also the main target antigen of vaccine development. The molecular weight of this kind of polysaccharide is small and the immunogenicity is poor. It is very important to improve its immunogenicity by technical methods. This study on Staphylococcus aureus 3, respectively. Polysaccharides from important surface polysaccharides were extracted, purified, coupled with protein carriers and immunogenicity of conjugated antigens.
First, the capsular polysaccharide CP5 and CP8 were released from the Staphylococcus aureus bacteria by high pressure crushing. Different enzymes were used to remove the nucleic acid and protein in the broken products, and then a certain purity of CP5 and CP8 capsule polysaccharide was obtained by ion exchange chromatography. The acquisition of the surface polysaccharide 336PS was made by the digestion method of staphylococcal enzyme to make it from the bacteria. After release, gradient ethanol extraction and digestion of different enzymes, crude 336PS was extracted and purified by gel chromatography.
A chemical assay for the surface polysaccharide of Staphylococcus aureus was established. The results showed that the purity of CP5 and CP8336PS were 67.51%, 63.83% and 68.51%, respectively, and the purified polysaccharide was only reacted with the immune sera of the antihomo strain by immuno agar double diffusion test.
The protein coupled antigen of 3 kinds of surface polysaccharides was prepared and the immunogenicity of the coupled antigen was detected. The purified surface polysaccharide was coupled with BSA by ADH bridge method and EDAC zero distance cross-linking method, the gel filtration chromatography was purified and the UV scanning was identified. The coupling resistance of 6 kinds of surface polysaccharides was prepared. The original oil adjuvant subunit vaccine was immune to mice, and 3 kinds of uncoupled surface polysaccharides were prepared in the same way. PBS vaccine was used as control. Before immunization, the blood of mice immunized with immunization 14d and 28d were collected, serum was isolated and the level of antibody against surface polysaccharides in serum was detected by indirect ELISA. The results showed that 2 coupling methods were found. All antigens prepared can stimulate the body to produce a good immune response, while unconjugated polysaccharides have no immunogenicity.
In order to understand the immunological protection of the antibody, the immunization was strengthened at twenty-eighth D and the same type of strain was injected into the lateral leg 7 days later. The clinical pathological changes of the lateral hind leg of the mice were observed and the clinical index score was evaluated. The experiment showed that the coupling antigen group could provide better immune protection to the immune mice. After comprehensive analysis, the ADH bridge method was used. The CP5-BSA, CP8-BSA and 336PS-BSA conjugated antigen immunization effect is superior to EDAC zero distance crosslinking method.
In order to overcome the problem of poor immunogenicity of carrier protein BSA in cow immunity, the recombinant expression vector pET-28a-ETA was constructed by genetic engineering method and PCR amplification of ETA (Pseudomonas aeruginosa exotoxin A gene), and transformed into DH5a receptive cells. The recombinant plasmid pET-28a-ETA was extracted and analyzed by double enzyme digestion, PCR identification and sequencing identification. It was induced by IPTG for induction. SDS-PAGE and Western blotting analysis of the products were carried out, and the expression conditions were optimized to determine the distribution of the target protein, and the final batch was expressed. The urea was used to denaturate the inclusion bodies and to determine the best refolding conditions and the complex buffer solution. The target protein on the Ni-NTA resin column. The target protein after the refolding of the column was found. The purity is above 93%.
The surface polysaccharide -rEPA coupling was prepared by the ADH bridge method. Through animal experiments, the above three polysaccharides -rEPA coupling antigen could stimulate the immune response, and the isolated sera could react with the same type strain and have good immunogenicity.
In this study, the isolation and purification of the surface polysaccharide CP8336PS of Staphylococcus aureus and the coupling technique with the carrier protein were established in China for the first time. The immunogenicity and immune protection of the coupled antigen were proved by immunization and attack in mice, which provided a technical storage for the development of the yellowish yellowish yellowish unit vaccine of Staphylococcus aureus. Prepare.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392

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