本文關(guān)鍵詞： 葡萄球菌腸毒素 免疫相關(guān)表面分子 重組蛋白 腸道上皮細(xì)胞 趨化因子及其受體 相對(duì)熒光定量PCR　出處：《天津大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：葡萄球菌腸毒素(Staphyloccucal enterotoxins,SEs)是由葡萄球菌產(chǎn)生的一類耐熱性好、穩(wěn)定度高的可溶性胞外毒蛋白,可引發(fā)人類多種免疫疾病。葡萄球菌腸毒素通過(guò)潛在的胃腸毒素及超抗原兩種機(jī)制發(fā)揮其毒性作用,誘導(dǎo)炎性反應(yīng),但其致炎機(jī)制的分子細(xì)節(jié)尚不清楚。論文克隆表達(dá)了幾種免疫相關(guān)表面分子,并分析了其分子特性及結(jié)構(gòu),研究了幾種重組新型腸毒素對(duì)免疫細(xì)胞表達(dá)相關(guān)免疫分子的影響,評(píng)價(jià)了這些分子表達(dá)變化與葡萄球菌腸毒素致炎癥的關(guān)系。分別以豬T、B、PBMC、PAM細(xì)胞總RNA為模板,RT-PCR擴(kuò)增6種豬免疫相關(guān)表面分子CD28、CD25(T細(xì)胞),CD86(B細(xì)胞),IL17A(PBMC),MHC I、MHC II(PAM細(xì)胞),克隆至克隆載體pUC T simple獲得6種豬免疫相關(guān)基因。測(cè)序結(jié)果顯示,6種分子與GenBank/NCBI已登陸序列同源性高于95%。編碼蛋白序列一級(jí)結(jié)構(gòu)預(yù)測(cè)表明,除IL17A為胞外蛋白外,其余5種分子均存在跨膜域;CD28、CD86、MHC I、MHC II均含一個(gè)Ig結(jié)構(gòu)域,CD25包含兩個(gè)重復(fù)的補(bǔ)體控制蛋白結(jié)構(gòu)域。對(duì)6種分子糖基化位點(diǎn)、抗原表位以及磷酸化位點(diǎn)預(yù)測(cè),應(yīng)用于后期6種分子信號(hào)通路的研究。通過(guò)生物信息學(xué)分析軟件對(duì)6種豬免疫相關(guān)表面分子蛋白特性進(jìn)行分析,分別以6種克隆的分子質(zhì)粒為模板,構(gòu)建了缺失信號(hào)肽或跨膜域的重組表達(dá)質(zhì)粒pET28a-CD28、pET28a-CD86、pET28a-CD25、pET28a-IL17A、pET28a-MHC I、pET28a-MHC II。構(gòu)建的pET28a-X系列重組菌經(jīng)誘導(dǎo)表達(dá)、包涵體蛋白純化,獲得高純度的重組蛋白CD28、CD86、CD25、IL17A、MHC I、MHC II。對(duì)6種重組蛋白亞細(xì)胞分布和三級(jí)結(jié)構(gòu)進(jìn)行預(yù)測(cè),同源模建了幾種葡萄球菌腸毒素與豬MHC II分子互作的分子細(xì)節(jié),分析其對(duì)不同腸毒素超抗原活性的影響。設(shè)計(jì)了豬腸道上皮細(xì)胞上的趨化因子及其相應(yīng)受體CXCL2、CXCL8-CXCR2;MCP-1-CCR2;CCL25-CCR9、CCL28-CCR10、CXCL10-CXCR3,以及6種豬免疫相關(guān)表面分子的特異性檢測(cè)引物,運(yùn)用相對(duì)熒光定量PCR的方法,檢測(cè)了SEs體內(nèi)外刺激后各類分子轉(zhuǎn)錄水平變化。SEs體外刺激豬IEC、PBMC、PAM細(xì)胞后,趨化因子及其受體均在48 h或72 h時(shí)表達(dá)量上調(diào),表明SEs刺激可引發(fā)細(xì)胞表面趨化因子分泌,募集表達(dá)其相應(yīng)受體的免疫細(xì)胞,引發(fā)炎性反應(yīng);而體內(nèi)刺激變化趨勢(shì)不定,特別是SEs體內(nèi)刺激后,PBMCs和PAM細(xì)胞表面相應(yīng)趨化因子及其受體表達(dá)受抑制;SEO體內(nèi)刺激后,CD28、CD25、IL17A、MHC II轉(zhuǎn)錄水平顯著提高,其他腸毒素作用不明顯,可能與機(jī)體內(nèi)免疫系統(tǒng)多元、多向、時(shí)空調(diào)控的復(fù)雜性有關(guān)。
[Abstract]:Staphylocucal enterotoxins (SES) is a kind of soluble extracellular toxin produced by Staphylococcus, which has good heat resistance and high stability. Staphylococcal enterotoxin (staphylococcal enterotoxin) exerts its toxic effect through two mechanisms of potential gastrointestinal toxin and superantigen and induces inflammatory reaction. However, the molecular details of its inflammatory mechanism are still unclear. Several immune-related surface molecules were cloned and expressed, their molecular characteristics and structures were analyzed, and the effects of several recombinant enterotoxins on the expression of immune molecules in immune cells were studied. The relationship between the expression of these molecules and the inflammation induced by staphylococcal enterotoxin was evaluated. Six porcine immune-associated surface molecules CD28, CD25 and T cells were amplified by RT-PCR using the total RNA of porcine TBX PBMC pam cells as template, and cloned into six kinds of porcine immune-associated surface molecule CD28 / CD25 / T cells. Six kinds of porcine immune-related genes were obtained by pUC T simple. The sequence analysis showed that the homology between the six molecules and the landing sequence of GenBank/NCBI was higher than that of 95%. With the exception of IL17A as extracellular protein, all the other five molecules have transmembrane domain CD28, CD86, MHC, MHCII and one Ig domain. CD25 contains two repeated complement control protein domains. The glycosylation sites, antigenic epitopes and phosphorylation sites of six molecules are predicted. The characteristics of immune-related surface proteins of six kinds of pigs were analyzed by bioinformatics analysis software, and six cloned molecular plasmids were used as templates. The recombinant expression plasmid pET28a-CD28a-CD86pET28a-CD86pET28a-CD25pET28a-MHC-pET28a-MHC-pET28a-MHC IHCI was constructed. The recombinant strain of pET28a-X was induced to express, purified, and purified the inclusion body protein, and the recombinant plasmid pET28a-CD28pET28a-MHC-pET28a-MHC-pET28a-MHC-pET28a-MHC-pET28a-MHCIIIwas constructed. A high purity recombinant protein CD28, CD86, CD25, IL17, IHC and MHCII. was obtained. The molecular details of interaction between several staphylococcal enterotoxins and porcine MHC II molecules were constructed by homologous modeling. The chemokine and its receptor CXCL8-CXCR2 on porcine intestinal epithelial cells were designed, and the primers for specific detection of CXCL10-CXCR3, CCL25-CCR9, CCL28-CCR10, CXCL10-CXCR3, and six porcine immune-related surface molecules were designed. The expression of chemokines and their receptors were up-regulated at 48 or 72 h after stimulation of SEs in vitro and in vivo. The results showed that SEs stimulation could induce the secretion of chemokines on the cell surface. Recruitment of immune cells expressing the corresponding receptors triggers inflammatory response, while the tendency of changes in stimuli in the body is uncertain. In particular, the expression of chemokines and their receptors on PBMCs and PAM cells stimulated in vivo by SEs was significantly increased after stimulation in vivo, and the transcription level of CD28, CD25, CD25, IL-17AHC-MHCII was significantly increased, and other enterotoxins were not obvious, which may be multiple and multidirectional with the immune system in vivo. The complexity of space-time regulation is related.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.28

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