葡萄球菌肠毒素超抗原抑制性肽与减毒突变体的构建和功能研究

发布时间：2017-12-31 02:03

本文关键词：葡萄球菌肠毒素超抗原抑制性肽与减毒突变体的构建和功能研究　出处：《第三军医大学》2008年博士论文　论文类型：学位论文

【摘要】： 超抗原是一种具有强大免疫刺激功能的新型蛋白质抗原,业已证明超抗原参与多种疾病的发病过程。金黄色葡萄球菌(金葡菌)肠毒素超抗原家族涉及的疾病谱非常广泛,并且也是一种失能型的生物战剂,一直是比较活跃的研究领域。已取得的研究进展提示我们,目前针对金葡菌肠毒素超抗原所致疾病除了临床对症支持治疗外,尚无很好的针对超抗原的治疗手段,因此,从其超抗原特性入手,寻找有效控制肠毒素毒性作用的方法,可以进一步为相关疾病的防治研究和生物武器防护提供新的手段。 SEA、SEB、SEC和SED是常见的毒性作用较强的金葡菌肠毒素,国内外对其研究内容大多数是利用其超抗原免疫活性进行抗肿瘤临床治疗等,针对超抗原的活性本身来研究超抗原相关疾病的防治手段较少。目前的研究主要是针对肠毒素的免疫识别区域设计小分子多肽和构建并筛选肠毒素的减毒突变体,从这两方面研究抑制肠毒素超抗原的活性的手段。但是大多数研究都是对单一种类的肠毒素研究,针对多种肠毒素的广谱抑制性手段的研究很少。基于上述问题,我们针对葡萄球菌肠毒素同源序列设计了9条小分子多肽,通过细胞模型筛选具有广谱抑制性的多肽分子,并运用动物模型检测筛选到的抑制性多肽对动物的保护作用,研究抑制性肽与MHCⅡ类分子的亲和力,预测抑制性肽的空间结构,探讨其广谱抑制活性的机制;同时构建了SED减毒突变体,并检测了该突变体的促人PBMC增殖活性、MHCⅡ类分子结合活性和TCRVβ特异性。主要研究内容和结果包括以下几方面: 1.抑制性多肽的设计和合成,对金葡菌肠毒素家族的序列进行比较,选择具有同源性的保守序列设计多肽,共设计了9条多肽。 2.建立人外周血单个核细胞的细胞模型,检测9条合成多肽对超抗原的抑制效应。主要从多肽对SEs刺激人PBMC的增殖和分泌IL-2、IFN-γ、TNF-β的抑制效应进行检测,筛选具有广谱抑制性效应的多肽,试验结果显示多肽P72对SEA、SEB和SEC超抗原活性具有明显抑制作用,其他8条多肽对SEs的抑制作用不明显。确证试验显示P72对PHA、ConA的促PBMC增殖活性无抑制作用。 3.建立8周龄雌性BALB/c小鼠感染性休克的动物模型,以脂多糖(LPS)和D-氨基半乳糖(D-GalN)致敏小鼠,再用不同剂量的SE进行攻击,获得了此条件下SEA、SEB和SEC的最低致死剂量。利用该动物模型检测多肽P72对SEA、SEB和SEC攻击小鼠的保护效果,结果显示P72对SEA、SEB和SEC致休克效应具有显著的保护作用。 4.竞争结合实验检测抑制性多肽P72与MHCⅡ类分子的结合活性,结果显示多肽P72不能与SEA、SEB和SEC有效竞争结合Raji细胞上的MHCⅡ类分子,提示P72可能不是通过与MHCⅡ类分子结合产生的抑制活性。 5.对抑制性多肽P72的空间结构进行预测,将P72在SEA、SEB、SEC的同源序列的空间结构进行对比,结果显示3种超抗原与P72同源的序列在空间结构上具有高度的相似性。 6.构建了SEDN23A/H26R突变体。并对突变体蛋白进行纯化、定量和免疫印迹分析。检测了SEDN23A/H26R突变体促T淋巴细胞增殖的活性,结果显示其促增殖活性比SED显著降低;以竞争实验检测SEDN23A/H26R突变体与MHCⅡ类分子的结合活性,并用流式细胞仪检测突变体TCRVβ特异性变化。结果:突变体SEDN23A/H26R与MHCⅡ类分子的亲合力未发生变化,但刺激人TCRVβ5+T细胞水平显著降低。综上,本研究设计并筛选到了1条广谱抑制性多肽P72,P72能够在体外和体内显著抑制SEA、SEB、SEC的超抗原活性,证实了P72可能不是通过与MHCⅡ类分子结合对SEs产生的抑制作用;发现N23、H26位氨基酸残基可能是SED与人TCRVβ5结合的关键位点;研究结果丰富了对SEs超抗原免疫识别机制的认识,为细菌性超抗原相关疾病的防治以及生物武器防护技术的研究提供了新的手段。
[Abstract]:Super antigen is a novel protein antigen has strong immunostimulatory function, it has been proved that the pathogenesis of superantigen in many diseases. Staphylococcus aureus enterotoxin superantigens (Jin Pujun) family involved in a wide spectrum of diseases, and is also a disability type biological agent research has been more active in the field of research. Progress has been made that we present for Staphylococcus aureus enterotoxins superantigen induced disease in addition to clinical symptomatic treatment, there is no good for superantigen treatment, therefore, the paper starts from the characteristics of the super antigen, method for effective control of enterotoxin toxicity, provide a new means of prevention research and protection of biological weapons some related diseases.
SEA, SEB, SEC and SED are common strong toxic effects of staphylococcal enterotoxin, the research on it is the most content of anti-tumor clinical treatment using the super antigen activity, the superantigen activity itself of superantigen related diseases prevention and treatment is less. The present research is poison the immune recognition area minus mutant enterotoxin design small molecule polypeptide and construction and screening of enterotoxin, from the two aspects of inhibition of enterotoxin superantigen activity means. But most of the studies are research on single species of enterotoxin, few studies on the inhibitory means for the broad spectrum of enterotoxin.
Based on the above problems, we focused on staphylococcal enterotoxin 9 homologous sequences of small molecular peptides designed by cell model for screening the broad-spectrum inhibitory polypeptide molecules, the protective effect of inhibitory polypeptide and the use of animal models to screening for animal research, inhibitory peptide and MHC class II molecules affinity structure prediction inhibition peptide, to explore the mechanism of broad-spectrum inhibitory activity; at the same time to construct the SED attenuated mutant, and detected the proliferation of PBMC in promoting activity of the mutant, MHC II binding activity and specificity of TCRV beta. The main research contents and results include the following:
1. design and synthesis of inhibitory peptides, compare the sequences of Staphylococcus aureus enterotoxin family, select homologous conservative sequences to design polypeptides, and design 9 polypeptides.
2. to establish a cell model of human peripheral blood mononuclear cells, detection of 9 synthetic peptides on the inhibitory effect of superantigen. Mainly from the proliferation of human PBMC polypeptide on the stimulation of SEs and secretion of IL-2, IFN- gamma, the inhibitory effect of TNF- beta testing, screening has broad-spectrum inhibitory effect of the peptide, the test results showed that polypeptide P72 of SEA, SEB and SEC can significantly inhibit the superantigen activity, inhibition of other 8 peptides on the SEs was not obvious. The confirmatory test showed P72 of PHA, PBMC proliferation activity of ConA had no inhibitory effect.
3. establish the animal model of 8 week old female BALB/c mice with septic shock, lipopolysaccharide (LPS) and D- galactosamine (D-GalN) sensitized mice with different doses of SE were obtained under the condition of this attack, SEA, SEB and the minimum lethal dose of SEC. Measurement of polypeptide P72 on SEA using the model. Animal protection, the effect of SEB and SEC against mice, the results showed that P72 of SEA, SEB and SEC induced shock effect has significant protective effect.
4. competitive binding assays were used to detect the binding activity of inhibitory peptides P72 to MHC class II molecules. The results showed that polypeptide P72 could not compete effectively with SEA, SEB and SEC to bind MHC class II molecules on Raji cells, suggesting that P72 might not be inhibited by binding with MHC class II molecules.
5., we predicted the spatial structure of inhibitory polypeptide P72, and compared the spatial structure of P72 in the homologous sequences of SEA, SEB and SEC. The results showed that the sequences of 3 superantigens and P72 homologous sequences were highly similar in spatial structure.
6. the SEDN23A/H26R mutant was constructed. And the mutant proteins were purified, quantitative analysis and Western blot detection. The SEDN23A/H26R mutant promote T lymphocyte proliferation activity, the results showed that the proliferation activity of SED was significantly lower than in competition assays; binding activity of mutant SEDN23A/H26R and MHC class II molecules, and mutant TCRV beta specific changes flow cytometry. Results: the mutant SEDN23A/H26R and MHC II molecule affinity did not change, but the stimulation of human TCRV beta 5+T cells decreased significantly.
In conclusion, this study designed and screened 1 broad-spectrum inhibitory peptide P72, P72 can significantly inhibit SEA in vitro and in vivo SEB superantigen activity of SEC, P72 may not be confirmed by combining the inhibitory effect on the SEs and MHC class II molecules; N23, H26 amino acid residue may is SED and TCRV beta 5 key sites combined; the results enrich the understanding of SEs super antigen immune recognition mechanism, provides a new means for the research of prevention and control of bacterial superantigen disease and biological weapons protection technology.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

【相似文献】