本文關(guān)鍵詞：葡萄球菌腸毒素超抗原抑制性肽與減毒突變體的構(gòu)建和功能研究　出處：《第三軍醫(yī)大學(xué)》2008年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 葡萄球菌腸毒素A 葡萄球菌腸毒素B 葡萄球菌腸毒素C 葡萄球菌腸毒素D 超抗原 抑制性多肽 突變體

【摘要】： 超抗原是一種具有強(qiáng)大免疫刺激功能的新型蛋白質(zhì)抗原,業(yè)已證明超抗原參與多種疾病的發(fā)病過(guò)程。金黃色葡萄球菌(金葡菌)腸毒素超抗原家族涉及的疾病譜非常廣泛,并且也是一種失能型的生物戰(zhàn)劑,一直是比較活躍的研究領(lǐng)域。已取得的研究進(jìn)展提示我們,目前針對(duì)金葡菌腸毒素超抗原所致疾病除了臨床對(duì)癥支持治療外,尚無(wú)很好的針對(duì)超抗原的治療手段,因此,從其超抗原特性入手,尋找有效控制腸毒素毒性作用的方法,可以進(jìn)一步為相關(guān)疾病的防治研究和生物武器防護(hù)提供新的手段。 SEA、SEB、SEC和SED是常見(jiàn)的毒性作用較強(qiáng)的金葡菌腸毒素,國(guó)內(nèi)外對(duì)其研究?jī)?nèi)容大多數(shù)是利用其超抗原免疫活性進(jìn)行抗腫瘤臨床治療等,針對(duì)超抗原的活性本身來(lái)研究超抗原相關(guān)疾病的防治手段較少。目前的研究主要是針對(duì)腸毒素的免疫識(shí)別區(qū)域設(shè)計(jì)小分子多肽和構(gòu)建并篩選腸毒素的減毒突變體,從這兩方面研究抑制腸毒素超抗原的活性的手段。但是大多數(shù)研究都是對(duì)單一種類的腸毒素研究,針對(duì)多種腸毒素的廣譜抑制性手段的研究很少。 基于上述問(wèn)題,我們針對(duì)葡萄球菌腸毒素同源序列設(shè)計(jì)了9條小分子多肽,通過(guò)細(xì)胞模型篩選具有廣譜抑制性的多肽分子,并運(yùn)用動(dòng)物模型檢測(cè)篩選到的抑制性多肽對(duì)動(dòng)物的保護(hù)作用,研究抑制性肽與MHCⅡ類分子的親和力,預(yù)測(cè)抑制性肽的空間結(jié)構(gòu),探討其廣譜抑制活性的機(jī)制;同時(shí)構(gòu)建了SED減毒突變體,并檢測(cè)了該突變體的促人PBMC增殖活性、MHCⅡ類分子結(jié)合活性和TCRVβ特異性。主要研究?jī)?nèi)容和結(jié)果包括以下幾方面: 1.抑制性多肽的設(shè)計(jì)和合成,對(duì)金葡菌腸毒素家族的序列進(jìn)行比較,選擇具有同源性的保守序列設(shè)計(jì)多肽,共設(shè)計(jì)了9條多肽。 2.建立人外周血單個(gè)核細(xì)胞的細(xì)胞模型,檢測(cè)9條合成多肽對(duì)超抗原的抑制效應(yīng)。主要從多肽對(duì)SEs刺激人PBMC的增殖和分泌IL-2、IFN-γ、TNF-β的抑制效應(yīng)進(jìn)行檢測(cè),篩選具有廣譜抑制性效應(yīng)的多肽,試驗(yàn)結(jié)果顯示多肽P72對(duì)SEA、SEB和SEC超抗原活性具有明顯抑制作用,其他8條多肽對(duì)SEs的抑制作用不明顯。確證試驗(yàn)顯示P72對(duì)PHA、ConA的促PBMC增殖活性無(wú)抑制作用。 3.建立8周齡雌性BALB/c小鼠感染性休克的動(dòng)物模型,以脂多糖(LPS)和D-氨基半乳糖(D-GalN)致敏小鼠,再用不同劑量的SE進(jìn)行攻擊,獲得了此條件下SEA、SEB和SEC的最低致死劑量。利用該動(dòng)物模型檢測(cè)多肽P72對(duì)SEA、SEB和SEC攻擊小鼠的保護(hù)效果,結(jié)果顯示P72對(duì)SEA、SEB和SEC致休克效應(yīng)具有顯著的保護(hù)作用。 4.競(jìng)爭(zhēng)結(jié)合實(shí)驗(yàn)檢測(cè)抑制性多肽P72與MHCⅡ類分子的結(jié)合活性,結(jié)果顯示多肽P72不能與SEA、SEB和SEC有效競(jìng)爭(zhēng)結(jié)合Raji細(xì)胞上的MHCⅡ類分子,提示P72可能不是通過(guò)與MHCⅡ類分子結(jié)合產(chǎn)生的抑制活性。 5.對(duì)抑制性多肽P72的空間結(jié)構(gòu)進(jìn)行預(yù)測(cè),將P72在SEA、SEB、SEC的同源序列的空間結(jié)構(gòu)進(jìn)行對(duì)比,結(jié)果顯示3種超抗原與P72同源的序列在空間結(jié)構(gòu)上具有高度的相似性。 6.構(gòu)建了SEDN23A/H26R突變體。并對(duì)突變體蛋白進(jìn)行純化、定量和免疫印跡分析。檢測(cè)了SEDN23A/H26R突變體促T淋巴細(xì)胞增殖的活性,結(jié)果顯示其促增殖活性比SED顯著降低;以競(jìng)爭(zhēng)實(shí)驗(yàn)檢測(cè)SEDN23A/H26R突變體與MHCⅡ類分子的結(jié)合活性,并用流式細(xì)胞儀檢測(cè)突變體TCRVβ特異性變化。結(jié)果:突變體SEDN23A/H26R與MHCⅡ類分子的親合力未發(fā)生變化,但刺激人TCRVβ5+T細(xì)胞水平顯著降低。 綜上,本研究設(shè)計(jì)并篩選到了1條廣譜抑制性多肽P72,P72能夠在體外和體內(nèi)顯著抑制SEA、SEB、SEC的超抗原活性,證實(shí)了P72可能不是通過(guò)與MHCⅡ類分子結(jié)合對(duì)SEs產(chǎn)生的抑制作用;發(fā)現(xiàn)N23、H26位氨基酸殘基可能是SED與人TCRVβ5結(jié)合的關(guān)鍵位點(diǎn);研究結(jié)果豐富了對(duì)SEs超抗原免疫識(shí)別機(jī)制的認(rèn)識(shí),為細(xì)菌性超抗原相關(guān)疾病的防治以及生物武器防護(hù)技術(shù)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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

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