牙龈卟啉单胞菌重组菌毛蛋白对人外周血单个核细胞中TNF-α和IL-6的影响

发布时间：2018-03-08 00:26

本文选题：P.gingivalis　切入点：Fim　出处：《锦州医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的探讨牙龈卟啉单胞菌菌毛(Porphyromonas gingivalis Fimbriae,P.gingivalis Fim A)融合蛋白对人外周血单个核细胞(Peripheral Blood Mononuclear Cells,PBMCs)中TNF-α和IL-6表达的影响。方法1.收集对数期P.gingivalis菌液,提取细菌DNA,通过PCR克隆Fim A基因,构建可原核表达质粒PGEX-6P-Fim A,转化至大肠杆菌DH5α感受态细胞中,经过不同时间、温度及不同浓度IPTG诱导,选取最佳条件诱导Fim A融合蛋白表达,通过谷胱甘肽巯基转移酶亲和层析法,纯化Fim A融合蛋白。考马斯亮蓝染色和Western blot检测蛋白的准确性。2.于锦州医科大学附属第二医院随机选择3例牙周健康志愿者,采集静脉血提取PBMCs。设计不同的实验组:a.分别设置不同的Fim A融合蛋白浓度(0μg/ml、1μg/ml、2μg/ml、3μg/ml、4μg/ml和5μg/ml)刺激PBMCs 24 h;b.选取3μg/ml Fim A融合蛋白刺激PBMCs 0 h、8 h、12 h、16 h、20 h和24 h。收集各组细胞上清,应用ELISA法检测TNF-α和IL-6的表达水平。结果1.经考马斯亮蓝染色和Western blot检测,成功纯化出P.gingivalis Fim A融合蛋白。2.不同浓度的Fim A融合蛋白刺激PBMCs 24 h对细胞因子的影响:TNF-α中1μg/ml组与对照组相比,差异无统计学意义(P=0.362);其余各组间相比,差异有统计学意义(P0.05)。IL-6中各组间相比,差异有统计学意义(P0.05)。3.不同时间点3μg/ml Fim A融合蛋白刺激PBMCs对细胞因子的影响:TNF-α和IL-6各时间组相比,差异有统计学意义(P0.05)。结论P.gingivalis Fim A融合蛋白可以使PBMCs中TNF-α和IL-6致炎细胞因子上调,为后续制备多克隆抗体和牙周病亚单位疫苗的研制提供了部分实验基础。
[Abstract]:Objective to investigate the effect of Porphyromonas gingivalis Fimbriaeae P.gingivalis Fim A fusion protein on the expression of TNF- 伪 and IL-6 in peripheral Blood Mononuclear cells PBMCs.Methods 1.The bacterial DNA was extracted from P. gingivalis in logarithmic phase and the Fim A gene was cloned by PCR. The prokaryotic expression plasmid PGEX-6P-Fim A was constructed and transformed into Escherichia coli DH5 伪 competent cells. The expression of Fim A fusion protein was induced by different time, temperature and concentration of IPTG. Fim A fusion protein was purified by glutathione mercaptotransferase affinity chromatography. Coomassie brilliant blue staining and Western blot were used to detect the protein. 2. Three healthy volunteers were randomly selected from the second affiliated Hospital of Jinzhou Medical University. Blood was collected from vein blood to extract PBMCs. Different experimental groups were designed: a. Different concentrations of Fim A fusion protein were set at 0 渭 g / ml 1 渭 g / ml / ml 2 渭 g / ml / ml 3 渭 g / ml / ml and 5 渭 g / ml respectively) stimulated PBMCs 24 h / ml, 3 渭 g / ml Fim A fusion protein was selected to stimulate PBMCs 0 h / 8 h / h / 12 h / h / 16 h / h and 24 h / h. Cell supernatants were collected for 20 h and 24 h for PBMCs 0 h / 8 h ~ 12 h ~ (-1) h ~ (-1) / h ~ (-1) and 5 渭 g / ml ~ (-1) 渭 g / ml / ml respectively. Results 1. Coomassie brilliant blue staining and Western blot were used to detect the expression of TNF- 伪 and IL-6. P.gingivalis Fim A fusion protein .2.The effect of different concentrations of Fim A fusion protein on cytokines stimulated by PBMCs for 24 h in 1 渭 g / ml group of 1 渭 g / ml of 1 渭 g / ml of Fim A fusion protein was not significantly different from that in control group, but there was no significant difference among other groups. There was significant difference between the three groups in P0.05U. IL-6. The difference was statistically significant. The effect of PBMCs stimulated by 3 渭 g / ml Fim A fusion protein on cytokines at different time points: TNF- 伪 was compared with that of IL-6 at different time points. Conclusion P.gingivalis Fim A fusion protein can up-regulate TNF- 伪 and IL-6 inflammatory cytokines in PBMCs, which provides some experimental basis for the subsequent preparation of polyclonal antibody and periodontal disease subunit vaccine.
【学位授予单位】：锦州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.4

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