【作者】 王平；

【导师】 张文慧；

【作者基本信息】 吉林农业大学 ， 微生物学， 2018， 硕士

【摘要】 由结核分枝杆菌感染引起的结核病(Tuberculosis,TB)仍然是对人类健康的重大威胁。结核病是全世界的第九大死因,在传染性疾病中排名第一,超过艾滋病。卡介苗(Bacillus Calmette-Guerin Vaccine,BCG)是唯一世界公认的免疫接种方式,但近些年研究表明,BCG的相对保护率仅为0%-80%,不能对机体进行有效的保护。同时,现有结核病的检测方法不能满足临床上对特异性和灵敏度的需求。因此,特异性更好的有效疫苗和快速检测试剂盒仍然是防治结核病的两项重要任务。BCG与结核基因组存在着差异区(Region of Difference,RDs),BCG在长期传代过程中丢失了结核分枝杆菌和牛分枝杆菌RD1-RD16的一些基因包含129个开放阅读框,其中一些基因可能与毒力有关,并且可能与BCG疫苗的保护率低有关系。近年来,关于结核分枝杆菌的研究主要集中在RD1区,其它15个差异区的研究不全面,因此本研究拟对RD1-RD16区的潜在T、B细胞抗原表位进行预测与分析,筛选优势抗原表位肽,建立应用优势抗原表位肽诊断结核病的ELISA方法,并对优势抗原表位肽对TLR4/MyD88信号通路的影响进行初步研究。1.使用生物信息学方法对RD1-RD16区129种蛋白质进行了系统的分析,通过对蛋白质的二级结构、亲疏水性及糖基化位点等分析,全面解析了结核分枝杆菌RD区蛋白的特性,并针对蛋白的同源性以及空间结构等信息对候选的B细胞抗原表位和T细胞抗原表位进行了筛选,最终得到优势B细胞抗原表位117条;优势T细胞抗原表位18条。2.根据表位筛选结果,分别合成1条T细胞表位肽和1条B细胞表位肽。B细胞表位肽序列:P1:LKTQIDQVESTAGSLQ GG KWDATATELNNALQNL GG TVCGFRSRLYA;T细胞表位肽序列:P2:SLDLLDPIY GG KTDAATLAQ GG MTEQQWNFA GG ASDNNAGDY。3.应用B细胞表位合成肽P1作为包被抗原,通过间接ELISA方法检测结核病人血清中特异性抗体,建立了以抗体为检测靶标的结核病诊断方法。确定了P1-ELISA最佳包被浓度为5μg/mL、血清的最佳稀释倍数为1:400,酶标二抗的最佳工作浓度为1:5000,底物最佳作用时间为15min。敏感性实验确定血清最高稀释倍数为1:2000,特异性强。4.T细胞表位合成肽P2与佐剂乳化免疫C57小鼠,将60只C57小鼠平均分为三组,每组20只,A组:CFA+P2 1:1混合,B组:IFA+P2 1:1混合,C组:PBS组。免疫一周后取脾,利用荧光定量技术检测TLR4/MyD88信号通路相关因子的mRNA表达水平。结果显示:A组MyD88、TRAF6、JUN、IL-6、IL-12、TNF-a表达量与PBS对照组有明显差异(P<0.05);FOS、IL-8表达量与PBS对照组有显著差异(P<0.01);MAP2K4表达量与PBS对照组有极显著差异(P<0.001),B组中,TLR4、TRAF6、MAP2K4、FOS、IL-6表达量与PBS对照组有明显差异(P<0.05);MyD88、JUN、IL-12表达量与PBS对照组有显著差异(P<0.01)。试验结果表明,合成肽P1可以作为诊断结核病的候选抗原;合成肽P2能够调节TLR4/MyD88信号通路相关因子的mRNA表达水平。更多还原

【Abstract】 Tuberculosis(TB)caused by Mycobacterium tuberculosis infection remains a major threat to human health.Tuberculosis is the ninth leading cause of death in the world,ranking first among infectious diseases and surpassing AIDS.Bacillus Calmette-Guerin Vaccine(BCG)is the only world-accepted immunization method.However,studies in recent years have shown that the relative protection rate of BCG is only 0%-80% and cannot effectively protect the body.At the same time,the existing detection methods for tuberculosis cannot meet the clinical needs for specificity and sensitivity.Therefore,effective vaccines and rapid test kits with better specificity remain two important tasks in the fight against tuberculosis.There is a Region of Difference(RDs)between BCG and tuberculosis genomes.BCG lost some genes of Mycobacterium tuberculosis and Mycobacterium bovis RD1-RD16 during long-term passaging.There are 129 open reading frames,some of which may be The virulence is related and may be related to the low protection rate of the BCG vaccine.In recent years,studies on Mycobacterium tuberculosis have been mainly concentrated in the RD1 region,and the study of 15 other differential regions is not comprehensive.Therefore,the potential T and B cell epitopes of RD1-RD16 region are to be predicted and analyzed in this study.The predominant epitope peptides were used to establish an ELISA method for diagnosis of tuberculosis using dominant epitope peptides,and preliminary studies were conducted on the influence of dominant epitope peptides on TLR4/MyD88 signaling pathways.1.A systematic analysis of 129 proteins in the RD1-RD16 region was performed using bioinformatics methods.A comprehensive analysis of the RD region protein of Mycobacterium tuberculosis was performed by analyzing the secondary structure,hydrophobicity,and glycosylation sites of the protein.And screened the candidate B-cell epitopes and T-cell epitopes against the homology and spatial structure of the proteins,and finally obtained 117 predominant B-cell epitopes;dominant T-cell epitopes.18 articles.2.According to the result of epitope screening,one T-cell epitope peptide and one B-cell epitope peptide were synthesized.B cell epitope peptide sequence: P1: LKTQIDQVESTAGSLQ GG KWDATATELNNALQNL GG TVCGFRSRLYA;T cell epitope peptide sequence: P2: SLDLLDPIY GG KTDAATLAQ GG MTEQQWNFA GG ASDNNAGDY.3.Using the B-cell epitope synthesis peptide P1 as the coating antigen,indirect ELISA was used to detect the specific antibodies in the serum of tuberculosis patients,and a method for the diagnosis of tuberculosis using antibodies as the target was established.It was determined that the optimal coating concentration of P1-ELISA was 5 μg/mL,the optimal dilution of serum was 1:400,the optimal working concentration of enzyme-conjugated secondary antibody was 1:5000,and the optimal working time of substrate was 15 minutes.Sensitivity experiments determined that the highest dilution of serum was 1:2000 with strong specificity.4.T-cell epitope synthesis peptide P2 and adjuvant immunization C57 mice were immunized,and 60 C57 mice were equally divided into three groups with 20 in each group.Group A: CFA+P2 1:1 mixture,Group B: IFA +P2 1:1 mix,group C: PBS group.One week after the immunization,the spleens were taken and the mRNA expression levels of TLR4/MyD88 signaling pathway related factors were detected by fluorescence quantitative technique.The results showed that the expression levels of MyD88,TRAF6,JUN,IL-6,IL-12 and TNF-a in group A were significantly different from those in PBS control group(P<0.05);the expression levels of FOS and IL-8 were significantly different from those in PBS control group.Difference(P<0.01);MAP2K4 expression was significantly different from PBS control group(P<0.001).In group B,the expression of TLR4,TRAF6,MAP2K4,FOS,IL-6 was significantly different from PBS control group(P <0.05);MyD88,JUN,IL-12 expression levels and PBS control group were significantly different(P <0.01).The experimental results show that the synthetic peptide P1 can be used as a candidate antigen for the diagnosis of tuberculosis;the synthetic peptide P2 can regulate the mRNA expression level of TLR4/MyD88 signaling pathway related factors.更多还原