几种新型重组葡萄球菌肠毒素生物功能的研究

发布时间：2018-04-25 18:21

本文选题：重组葡萄球菌肠毒素 + 细胞因子　；参考：《天津大学》2012年硕士论文

【摘要】：葡萄球菌肠毒素(Staphyloccucal enterotoxins,SEs)主要是由葡萄球菌产生的一类结构相关、毒力相似的可溶性胞外毒蛋白,可引发急性肠胃炎及其它免疫性疾病。已发现的肠毒素及肠毒素样基因型共20余种,但有关新型肠毒素的研究主要停留于基因型检测水平。本课题主要研究了几种新型重组肠毒素的毒性和超抗原活性,并初步探讨了蛋白分子结构和功能之间的关系。应用构建的pET28α-ses重组载体经诱导表达、镍鳌合层析纯化,获得高纯度且具有活性的重组蛋白SEA-HIS、SEO-HIS、SEG-HIS、SEK-HIS、SEU-HIS、和SEQ-HIS。将4周龄小鼠平均分成15组,每组7只,尾静脉注射20μg/g和5μg/kg的六种重组蛋白和天然SEA,以PBS做空白对照,72h后取胸腺、脾脏和肝脏。计算脏器指数并检测肝脏外源化合物代谢酶活性及IL-2、IL-4、IL-6、TNF-α、IFN-γ几种细胞因子的转录、表达水平。应用于体外细胞实验进一步评价肠毒素对T细胞增殖、凋亡和细胞因子转录、表达的影响。利用origin 7.5独立样本T检验法分析肠毒素处理组与PBS对照组差异的显著性。在20μg/g至毒剂量下,SEA-HIS、SEQ-HIS能下调体内Th1类细胞因子(IL-2、IFN-γ)和Th2类细胞因子(IL-4、IL-6)的转录水平,SEK-HIS、SEG-HIS和SEO-HIS能上调炎性因子IFN-γ。A、O、G、K、Q型肠毒素均能影响肝脏POD、SOD、GSH-PX、LDH的活性,上调TNF-α的转录水平,在50μg/mL剂量下诱导体外T淋巴细胞凋亡。在4μg/mL剂量下,四种新型重组肠毒素(K、Q、G、O型)与经典天然SEA相似,刺激体外T淋巴细胞增殖,具有超抗原活性,且超抗原活性强弱与T细胞TCR的Vβ亚型和MHCII分子有关。按促进T淋巴细胞增殖能力的强弱排序,肠毒素的超抗原活性由强至弱的顺序为:K、Q、A、G、O,U型肠毒素不表现超抗原活性,这与根据蛋白分子α3-β8环及Zn离子结合位点结合能力所预测的超抗原活性强弱顺序一致。在5μg/kg体内剂量和4μg/mL的体外剂量下, A、O、G、K、Q型肠毒素均能促进IL-2、IL-4、IL-6、TNF-α、IFN-γ的转录和表达。综上所述,重组葡萄球菌肠毒素可引起机体肝脏外源化合物代谢酶的变化,诱发炎症反应,具有一定的肝脏毒性; A、O、G、K、Q型肠毒素具有差异化影响机体细胞因子转录,淋巴细胞分化的能力,表现出不同的超抗原活性。新型葡萄球菌肠毒素毒性及其超抗原活性提出食品安全新问题。
[Abstract]:Staphylocucal enterotoxin (SES) is a kind of soluble extracellular toxin produced by Staphylococcus, which has similar virulence and can cause acute gastroenteritis and other immune diseases. There were more than 20 enterotoxin and enterotoxin like genotypes, but the study of new enterotoxins was mainly at the level of genotypic detection. The toxicity and superantigen activity of several new recombinant enterotoxins were studied, and the relationship between molecular structure and function of proteins was discussed. The recombinant protein SEA-HIS-SEO-HIS-SEG-HIS-SEK-HIS-SEU-HISS, and SEQ-HISs were obtained by using the recombinant pET28 伪 -ses recombinant vector, which was induced and purified by nickel chelation chromatography. The recombinant proteins SEA-HIS-SEO-HIS-SEG-HIS-SEK-HIS-HIS-SEU-HISS and SEQ-HISwere obtained. Four week-old mice were divided into 15 groups with 7 mice in each group. Six recombinant proteins (20 渭 g / g and 5 渭 g/kg) and natural sea were injected into caudal vein. Thymus, spleen and liver were taken after 72 hours of control with PBS. The organ index was calculated and the activity of exogenous metabolizing enzymes in liver and the transcription and expression level of IL-6 TNF- 伪 -IFN- 纬 cytokines were measured. The effects of enterotoxin on T cell proliferation, apoptosis and cytokine transcription and expression were further evaluated in vitro. The significance of the difference between enterotoxin treatment group and PBS control group was analyzed by origin 7.5 independent sample T test. At doses of 20 渭 g / g to 20 渭 g / g, SEA-HIS-SEQ-HIS could down-regulate the transcription levels of Th1 cytokines (IL-2tIFN- 纬) and Th2 cytokines (IL-4). SEK-HISG-HIS and SEO-HIS could up-regulate the activity of inflammatory factor IFN- 纬. AOGKKQ enterotoxin, and up-regulate the transcriptional level of TNF- 伪. In vitro T lymphocyte apoptosis was induced at the dose of 50 渭 g/mL. At the dose of 4 渭 g/mL, the four new recombinant enterotoxins, named KGN, are similar to the classical natural SEA, and stimulate the proliferation of T lymphocytes in vitro. The superantigen activity is related to the V 尾 subtype of T cell TCR and the MHCII molecule. According to the order of promoting T lymphocyte proliferation, the order of superantigen activity of enterotoxin from strong to weak was that the superantigen activity of Enterotoxin was not superantigen. This is consistent with the order of superantigen activity predicted by the binding ability of 伪 3- 尾 8 and Zn binding sites of protein molecules. Both in vivo and in vitro doses of 5 渭 g/kg and 4 渭 g/mL could promote the transcription and expression of TNF- 伪 -IFN- 纬. In conclusion, recombinant staphylococcal enterotoxin can induce the changes of exogenous metabolizing enzymes in the liver, induce inflammatory reaction, and have certain liver toxicity. The ability of lymphocyte differentiation showed different superantigen activity. The toxicity of new staphylococcal enterotoxin and its superantigen activity raise new food safety problems.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

【相似文献】