本文選題：金黃色葡萄球菌　切入點(diǎn)：表皮剝脫毒素　出處：《重慶醫(yī)科大學(xué)》2010年博士論文　論文類型：學(xué)位論文

【摘要】：目的通過基因工程技術(shù)獲得大量純化的重組表皮剝脫毒素A,為進(jìn)一步研究表皮剝脫毒素的致病機(jī)理及機(jī)體的免疫保護(hù)作用提供實(shí)驗(yàn)材料,為更有效地防治葡萄球菌性燙傷樣皮膚綜合征奠定基礎(chǔ)。 方法根據(jù)Gene Bank公布的eta基因序列,用premier premier5.0軟件設(shè)計(jì)引物,利用PCR技術(shù)從S. aureus基因組中擴(kuò)增eta基因,插入原核表達(dá)載體pQE30中,轉(zhuǎn)化表達(dá)受體菌大腸桿菌M15;SDS-PAGE分析其表達(dá)效果,優(yōu)化誘導(dǎo)表達(dá)條件;Western blot鑒定其抗原性。重組蛋白經(jīng)Ni-NTA2+樹脂純化后,注入BALB/C小鼠皮下鑒定其生物學(xué)活性。 結(jié)果1.重組表達(dá)質(zhì)粒pQE-ETA構(gòu)建成功,序列分析顯示插入的基因片段全長927bp,與基因文庫中的eta基因完全吻合;2. SDS-PAGE顯示表達(dá)產(chǎn)物相對(duì)分子量約為27 KD,重組表達(dá)質(zhì)粒pQE-ETA在IPTG終濃度0.1mmol/L,誘導(dǎo)時(shí)間4h,可得到最大的表達(dá)量,表達(dá)量約為12%;3、Western blot法顯示該蛋白具有良好的抗原性。4、Ni~(2+)-NTA樹脂純化可溶性的重組蛋白,可得到純度90%以上的蛋白質(zhì)。5、動(dòng)物實(shí)驗(yàn)顯示其有生物學(xué)活性。 結(jié)論ETA毒素片段表達(dá)成功,獲得高純度重組蛋白并具有良好的抗原性及生物學(xué)活性,為診斷SSSS感染、研究其致病機(jī)理和制備抗體提供了可靠的材料。 目的通過檢測(cè)血清中抗ETA和抗ETB抗體的濃度以及利用新生小鼠SSSS模型來研究抗ET抗體的免疫保護(hù)作用,為臨床上治療和預(yù)防SSSS提供新的思路。 方法1、間接ELISA法檢測(cè)商品化IVIg及患者血清中抗ETA和抗ETB抗體的濃度。2、用純化的重組ETA制備SSSS小鼠模型,觀察給予抗ETA抗體小鼠組和未予抗體組間的差異。 結(jié)果1、無論是在商品化IVIg中還是在人類血清中,抗ETA抗體的濃度大大高于抗ETB抗體的濃度。2、SSSS患兒組、AD患兒組以及正常兒童對(duì)照組血清抗ETA抗體的滴度沒有顯著差異。3、SSSS患兒組、AD患兒組以及正常兒童對(duì)照組血清抗ETB抗體的滴度有顯著差異,SSSS患兒組明顯低于AD患兒組以及正常兒童對(duì)照組。4、使用了抗ETA抗體組小鼠無論是大體體征、組織病理檢查還是死亡率都明顯優(yōu)于未用抗體組。5、早期使用抗ETA抗體組的治療效果明顯好于6小時(shí)后再用抗體治療組。 結(jié)論1、臨床上泛發(fā)型SSSS主要由ETB引起,這可能與人群血清中抗ETA抗體濃度大大高于抗ETB抗體濃度有關(guān)。2、SSSS患兒組血清抗ETB抗體的滴度明顯低于AD患兒組以及正常兒童對(duì)照組,該組患兒沒有足夠抗體的保護(hù)可能是其患SSSS原因之一。3、通過小鼠SSSS模型證實(shí)抗ET抗體有免疫保護(hù)作用。
[Abstract]:Objective to obtain a large number of purified recombinant epidermal exfoliating toxin A by genetic engineering, and to provide experimental materials for further study on the pathogenesis of epidermal exfoliating toxin and the immune protection of the body. To lay a foundation for more effective prevention and treatment of staphylococcal scalded skin syndrome. Methods according to the sequence of eta gene published by Gene Bank, primers were designed by premier premier5.0 software. Eta gene was amplified from the genome of S.#en6# by PCR technique and inserted into the prokaryotic expression vector pQE30. The recombinant protein was purified by Ni-NTA2 resin and subcutaneously injected into BALB/C mice to identify its biological activity. Results 1.Recombinant expression plasmid pQE-ETA was successfully constructed. Sequence analysis showed that the inserted gene fragment was 927bp, which was completely consistent with the eta gene in the gene library. The relative molecular weight of the expressed product was about 27kD. the final concentration of the recombinant expression plasmid pQE-ETA was 0.1 mmol / L in IPTG, and the induction time was 4 h, and the maximum amount of expression was obtained. Western blot assay showed that the protein had a good antigenicity. 4NTA resin purified the soluble recombinant protein. The purity of the protein was more than 90%. The animal experiment showed that the protein had biological activity. Conclusion the ETA toxin fragment was successfully expressed, the recombinant protein with high purity and good antigenicity and biological activity were obtained, which provided a reliable material for the diagnosis of SSSS infection, the study of its pathogenic mechanism and the preparation of antibodies. Objective to detect the concentration of anti ETA and anti ETB antibodies in serum and to study the immunoprotective effect of anti et antibody by using SSSS model of newborn mice to provide a new idea for clinical treatment and prevention of SSSS. Methods 1. Indirect ELISA assay was used to detect the concentration of anti ETA and anti ETB antibodies in commercial IVIg and patients' serum. The SSSS mouse model was established by purified recombinant ETA. The difference between the anti ETA antibody group and the non anti ETA antibody group was observed. Results 1, whether in commercial IVIg or in human serum, There was no significant difference in the titer of anti ETA antibody between AD group and normal control group. 3Serum anti ETA antibody level in AD group and normal control group was significantly higher than that in anti ETB antibody group. The titer of ETB antibody was significantly lower in the ETB group than that in the AD group and the normal control group. Histopathological examination and mortality were significantly better than that of untreated antibody group. The effect of early use of anti ETA antibody group was better than that of anti ETA antibody group after 6 hours. Conclusion 1. The clinical prevalence of SSSS is mainly caused by ETB, which may be related to the fact that the titer of anti ETA antibody in serum is much higher than that in anti ETB antibody group. The titer of anti ETB antibody in children with ETA is significantly lower than that in AD group and normal children control group. The lack of adequate antibody protection may be one of the causes of SSSS. The immunoprotective effect of anti et antibody was confirmed by mouse SSSS model.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392.1

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