葡萄球菌肠毒素致炎作用与猪免疫相关分子关系的研究

发布时间：2018-02-10 08:17

本文关键词： 葡萄球菌肠毒素免疫相关表面分子重组蛋白肠道上皮细胞趋化因子及其受体相对荧光定量PCR　出处：《天津大学》2015年硕士论文　论文类型：学位论文

【摘要】：葡萄球菌肠毒素(Staphyloccucal enterotoxins,SEs)是由葡萄球菌产生的一类耐热性好、稳定度高的可溶性胞外毒蛋白,可引发人类多种免疫疾病。葡萄球菌肠毒素通过潜在的胃肠毒素及超抗原两种机制发挥其毒性作用,诱导炎性反应,但其致炎机制的分子细节尚不清楚。论文克隆表达了几种免疫相关表面分子,并分析了其分子特性及结构,研究了几种重组新型肠毒素对免疫细胞表达相关免疫分子的影响,评价了这些分子表达变化与葡萄球菌肠毒素致炎症的关系。分别以猪T、B、PBMC、PAM细胞总RNA为模板,RT-PCR扩增6种猪免疫相关表面分子CD28、CD25(T细胞),CD86(B细胞),IL17A(PBMC),MHC I、MHC II(PAM细胞),克隆至克隆载体pUC T simple获得6种猪免疫相关基因。测序结果显示,6种分子与GenBank/NCBI已登陆序列同源性高于95%。编码蛋白序列一级结构预测表明,除IL17A为胞外蛋白外,其余5种分子均存在跨膜域;CD28、CD86、MHC I、MHC II均含一个Ig结构域,CD25包含两个重复的补体控制蛋白结构域。对6种分子糖基化位点、抗原表位以及磷酸化位点预测,应用于后期6种分子信号通路的研究。通过生物信息学分析软件对6种猪免疫相关表面分子蛋白特性进行分析,分别以6种克隆的分子质粒为模板,构建了缺失信号肽或跨膜域的重组表达质粒pET28a-CD28、pET28a-CD86、pET28a-CD25、pET28a-IL17A、pET28a-MHC I、pET28a-MHC II。构建的pET28a-X系列重组菌经诱导表达、包涵体蛋白纯化,获得高纯度的重组蛋白CD28、CD86、CD25、IL17A、MHC I、MHC II。对6种重组蛋白亚细胞分布和三级结构进行预测,同源模建了几种葡萄球菌肠毒素与猪MHC II分子互作的分子细节,分析其对不同肠毒素超抗原活性的影响。设计了猪肠道上皮细胞上的趋化因子及其相应受体CXCL2、CXCL8-CXCR2;MCP-1-CCR2;CCL25-CCR9、CCL28-CCR10、CXCL10-CXCR3,以及6种猪免疫相关表面分子的特异性检测引物,运用相对荧光定量PCR的方法,检测了SEs体内外刺激后各类分子转录水平变化。SEs体外刺激猪IEC、PBMC、PAM细胞后,趋化因子及其受体均在48 h或72 h时表达量上调,表明SEs刺激可引发细胞表面趋化因子分泌,募集表达其相应受体的免疫细胞,引发炎性反应;而体内刺激变化趋势不定,特别是SEs体内刺激后,PBMCs和PAM细胞表面相应趋化因子及其受体表达受抑制;SEO体内刺激后,CD28、CD25、IL17A、MHC II转录水平显著提高,其他肠毒素作用不明显,可能与机体内免疫系统多元、多向、时空调控的复杂性有关。
[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.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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