结核分枝杆菌PPE蛋白家族免疫原性初探

发布时间：2018-08-14 13:36

【摘要】：结核病(Tuberculosis, TB)是一种由结核分枝杆菌(Mycobactium tuberculosis，简称结核杆菌)感染引起的人畜共患慢性传染病，仍然是当今威胁人类健康的最主要疾病之一。结核病的传统诊断方法易受多种因素的干扰而产生较高的假阳性或假阴性结果，不利于结核病的早期确诊与治疗，因此，发展新颖有效、灵敏度高和特异性强的诊断技术非常必要。尽管不少结核杆菌抗原已经被鉴定，但是其中大多数不适合应用于鉴别诊断卡介苗（Bacillus Calmette-Guerin，BCG）免疫接种的健康人群与真正的结核病患者。结核杆菌基因组约10%序列用于编码PE（theproteins which contain Pro-Glu (PE) motif）和PPE（the proteins which containPro-Pro-Glu (PPE) motif）蛋白家族，且该家族蛋白为结核杆菌所特有。目前已有一些关于PPE蛋白家族成员已被成功鉴定并加以应用，但较为系统地分析PPE蛋白免疫原性的报道尚不多见。本研究应用系统进化分析法选取10个PPE蛋白家族成员作为研究对象，PCR扩增PPE蛋白编码基因后克隆至pET32a或pET41a质粒中构建重组PPE蛋白表达载体，转化大肠杆菌BL21（DE3）并筛选阳性表达菌株。重组表达的PPE融合蛋白用金属螯合镍离子柱亲和层析分离纯化，纯化的重组PPE融合蛋白作为目标抗原分别包被96孔酶标板，建立标准的ELISA为基础的血清学检查方法，用于结核病的早期诊断。60例临床确诊的结核病人和30例健康人的血清被用作ELISA检测，所得OD值经SPSS17.0软件分析制作ROC曲线，通过ROC曲线来计算ELISA灵敏度和特异性，并进一步评估各个PPE抗原用作早期血清学诊断的价值。本研究在大肠杆菌中克隆、表达了10个选择的PPE抗原，然而PPE68未能纯化成功。以纯化的PPE蛋白作为包被抗原建立了ELISA血清学诊断方法，结果发现所有以PPE蛋白作为抗原的ELISA检测灵敏度均比Ag85A（antigen85A）高。Ag85A抗原的ROC曲线下面积仅0.573，而所有PPE蛋白ROC曲线下面积均大于0.679，说明PPE蛋白作为抗原的血清学诊断的准确性更高。这是因为Ag85A不仅存在于结核杆菌中，，也存在于BCG等其他分枝杆菌中，易引起交叉反应，因而特异性不高。在选择的五类PPE抗原中，第一类PPE蛋白（PPE68）未能成功纯化；第二类PPE蛋白（PPE37）检测的特异性最高（99.3%），仅次于结核杆菌特异性抗原ESAT-6（early secretory antigenic target-6），但其检测灵敏度（65%）和准确性（ROC曲线下面积0.738）均比ESAT-6高，且国内外未见有报道，提示其可作为一种新型的候选抗原应用于结核病的早期血清学诊断研究；第三类PPE蛋白（PPE36、PPE41、PPE57、 PPE58、PPE59、PPE69）的检测灵敏度（65%~80%）和准确度（ROC曲线下面积0.761~0.805）均要高于PPE37，然而其特异性略低（80%~93.3%），与相关文献报道的结果一致；第四类PPE蛋白（PPE17）的检测灵敏度（63.3%）、特异性（83.3%）和准确性（ROC曲线下面积0.679）均不高；第五类PPE蛋白（PPE64）的检测灵敏度（63.3%）、特异性（90%）和准确性（ROC曲线下面积0.757）与PPE37相近，提示其也可以作为新型血清学诊断的候选抗原之一。本研究成功克隆、表达并纯化了9个PPE抗原，应用ELISA方法初步验证了PPE抗原的良好免疫原性，第二类PPE抗原PPE37和第五类PPE抗原PPE64的检测灵敏度、特异性和准确性都要明显优于ESAT-6，且目前国内外尚未有相关研究报道，提示这两种PPE蛋白可作为血清学诊断的候选抗原。我们还将进一步应用这些纯化的PPE抗原刺激结核病人的外周血单核细胞并分析其引起的细胞免疫反应，综合评价PPE抗原的免疫原性并探讨其在结核病诊断或疫苗研究中的意义。
[Abstract]:Tuberculosis (TB) is a chronic zoonotic infectious disease caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis) infection. It is still one of the most important diseases threatening human health. Traditional diagnostic methods of tuberculosis are susceptible to interference from many factors and produce high false positive or false negative results. As a result, it is not conducive to the early diagnosis and treatment of tuberculosis. Therefore, it is necessary to develop novel, effective, sensitive and specific diagnostic techniques. True tuberculosis patients. About 10% of the genome of Mycobacterium tuberculosis is used to encode PE (the proteins which contain Pro-Glu (PE) motif) and the PPE (the proteins which contain Pro-Pro-Glu (PPE) motif) protein family, and the family proteins are unique to Mycobacterium tuberculosis. However, a systematic analysis of the immunogenicity of PPE protein is rare.
In this study, 10 members of PPE protein family were selected as the research object by phylogenetic analysis. The PPE protein coding gene was amplified by PCR and cloned into pET32a or pET41a plasmid to construct recombinant PPE protein expression vector. The recombinant PPE fusion protein was transformed into E. coli BL21 (DE3) and the positive expression strain was screened. The purified recombinant PPE fusion protein was separated and purified by affinity chromatography and coated with 96-well enzyme plate as the target antigen. A standard ELISA-based serological method was established for the early diagnosis of tuberculosis. The sera of 60 clinically confirmed tuberculosis patients and 30 healthy persons were used for ELISA detection. The OD values were analyzed by SPSS17.0 software. ROC curves were made to calculate the sensitivity and specificity of ELISA, and to further evaluate the value of each PPE antigen for early serological diagnosis.
In this study, 10 selected PPE antigens were cloned and expressed in E. coli. However, PPE68 was not purified successfully. ELISA was established by using purified PPE protein as coating antigen. The results showed that the sensitivity of ELISA using PPE protein as antigen was higher than that of Ag85A. The area under ROC curve of Ag85A antigen was only small. The area under the ROC curve of all PPE proteins was greater than 0.679, indicating that the serological diagnostic accuracy of PPE proteins as antigens was higher. This is because Ag85A not only exists in Mycobacterium tuberculosis, but also exists in other Mycobacterium such as BCG. It is easy to cause cross-reaction and therefore has low specificity. White (PPE68) was not purified successfully; the specificity of PPE37 was the highest (99.3%) after ESAT-6 (early secretory antigenic target-6), but its sensitivity (65%) and accuracy (ROC area 0.738) were higher than that of ESAT-6, and it was not reported at home and abroad, suggesting that PPE37 could be used as a specific antigen of Mycobacterium tuberculosis. The sensitivity (65%-80%) and accuracy (ROC curve area 0.761-0.805) of the third class of PPE proteins (PPE36, PPE41, PPE57, PPE58, PPE59, PPE69) were higher than those of PPE37, but their specificity was slightly lower (80%-93.3%) and were consistent with the results reported in the relevant literature. The detection sensitivity (63.3%) of PPE-like protein (PPE17), specificity (83.3%) and accuracy (0.679 under ROC curve) were not high; the detection sensitivity (63.3%) of PPE-like protein (PPE64), specificity (90%) and accuracy (0.757 under ROC curve) were similar to PPE-37, suggesting that PPE-like protein could also be used as a candidate antigen for new serological diagnosis.
Nine PPE antigens were successfully cloned, expressed and purified in this study. The immunogenicity of PPE antigens was preliminarily verified by ELISA. The sensitivity, specificity and accuracy of PPE37 and PPE64 of the second and fifth PPE antigens were significantly better than those of ESAT-6, and there were no reports on these two kinds of PPE eggs at home and abroad. We will further apply these purified PPE antigens to stimulate peripheral blood mononuclear cells of tuberculosis patients and analyze their cellular immune responses, evaluate the immunogenicity of PPE antigens and explore their significance in tuberculosis diagnosis or vaccine research.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

【相似文献】