金黄色葡萄球菌重组表皮剥脱毒素的表达及其抗体的免疫保护作用研究

发布时间：2018-03-22 10:51

本文选题：金黄色葡萄球菌　切入点：表皮剥脱毒素　出处：《重庆医科大学》2010年博士论文　论文类型：学位论文

【摘要】：目的通过基因工程技术获得大量纯化的重组表皮剥脱毒素A,为进一步研究表皮剥脱毒素的致病机理及机体的免疫保护作用提供实验材料,为更有效地防治葡萄球菌性烫伤样皮肤综合征奠定基础。方法根据Gene Bank公布的eta基因序列,用premier premier5.0软件设计引物,利用PCR技术从S. aureus基因组中扩增eta基因,插入原核表达载体pQE30中,转化表达受体菌大肠杆菌M15;SDS-PAGE分析其表达效果,优化诱导表达条件;Western blot鉴定其抗原性。重组蛋白经Ni-NTA2+树脂纯化后,注入BALB/C小鼠皮下鉴定其生物学活性。结果1.重组表达质粒pQE-ETA构建成功,序列分析显示插入的基因片段全长927bp,与基因文库中的eta基因完全吻合;2. SDS-PAGE显示表达产物相对分子量约为27 KD,重组表达质粒pQE-ETA在IPTG终浓度0.1mmol/L,诱导时间4h,可得到最大的表达量,表达量约为12%;3、Western blot法显示该蛋白具有良好的抗原性。4、Ni~(2+)-NTA树脂纯化可溶性的重组蛋白,可得到纯度90%以上的蛋白质。5、动物实验显示其有生物学活性。结论ETA毒素片段表达成功,获得高纯度重组蛋白并具有良好的抗原性及生物学活性,为诊断SSSS感染、研究其致病机理和制备抗体提供了可靠的材料。目的通过检测血清中抗ETA和抗ETB抗体的浓度以及利用新生小鼠SSSS模型来研究抗ET抗体的免疫保护作用,为临床上治疗和预防SSSS提供新的思路。方法1、间接ELISA法检测商品化IVIg及患者血清中抗ETA和抗ETB抗体的浓度。2、用纯化的重组ETA制备SSSS小鼠模型,观察给予抗ETA抗体小鼠组和未予抗体组间的差异。结果1、无论是在商品化IVIg中还是在人类血清中,抗ETA抗体的浓度大大高于抗ETB抗体的浓度。2、SSSS患儿组、AD患儿组以及正常儿童对照组血清抗ETA抗体的滴度没有显著差异。3、SSSS患儿组、AD患儿组以及正常儿童对照组血清抗ETB抗体的滴度有显著差异,SSSS患儿组明显低于AD患儿组以及正常儿童对照组。4、使用了抗ETA抗体组小鼠无论是大体体征、组织病理检查还是死亡率都明显优于未用抗体组。5、早期使用抗ETA抗体组的治疗效果明显好于6小时后再用抗体治疗组。结论1、临床上泛发型SSSS主要由ETB引起,这可能与人群血清中抗ETA抗体浓度大大高于抗ETB抗体浓度有关。2、SSSS患儿组血清抗ETB抗体的滴度明显低于AD患儿组以及正常儿童对照组,该组患儿没有足够抗体的保护可能是其患SSSS原因之一。3、通过小鼠SSSS模型证实抗ET抗体有免疫保护作用。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392.1

【参考文献】