人布鲁氏菌病标准化PCR诊断方法及BP26基因标记疫苗研究

发布时间：2018-06-01 16:43

本文选题：人布鲁氏菌病 + PCR　；参考：《吉林大学》2009年博士论文

【摘要】： 布鲁氏菌病为动物源性疾病,是由细胞内寄生的布鲁氏菌引起的传染-变态反应性人兽共患传染病,在世界范围内广泛流行,对畜牧养殖业的发展和人类健康造成严重威胁,已成为重要的公共卫生问题。布鲁氏菌病的诊断方面存在两个主要的问题:一是快速准确诊断,二是现有减毒活疫苗的鉴别诊断。 PCR检测技术避免操作活菌,并能检测到标本中较低丰度的病原,适合布鲁氏菌这种高危险性病原的检测。用PCR方法对布鲁氏菌病早期、复发期以及伴随多种并发症的患者及药物治疗后患者进行诊断,具有较好的应用前景,我国目前尚无应用PCR检测方法在临床诊断上跟踪随访的研究报道。本研究针对布鲁氏菌的保守基因bp26设计了属的引物,针对插入性序列IS711设计了牛、羊、猪种引物,建立了单重和多重PCR检测方法,在临床上对免疫学诊断为患者、可疑患者和非患者人群进行检测,并结合流行病学个案数据库资料拟定了进一步跟踪随访检测的对象,为建立人布鲁氏菌病标准化PCR诊断方法提供研究依据。目前应用血清学方法不能区分布鲁氏菌减毒疫苗株的反应与自然感染,发展基因标记疫苗可解决这一问题。M5是我国广泛应用的减毒活疫苗株,本研究应用基因同源重组方法,用卡那抗性基因替换部分bp26基因,构建了bp26突变株,并根据布鲁氏菌的保守基因建立了能够区分bp26突变株与疫苗株的PCR鉴别方法。bp26突变株在胞内外的生存能力显示,bp26突变株在小鼠体内的免疫应答和免疫保护效果均低于M5疫苗株,表明bp26基因在M5疫苗株的免疫保护作用中发挥了作用。考虑到bp26基因在免疫原性和保护性方面的重要性,我们对BP26蛋白进行了表达及纯化,并致力于开发有效的布鲁氏菌亚单位疫苗和作为人布鲁氏菌病血清学的特异性诊断抗原用于临床诊断研究。
[Abstract]:Brucellosis, an animal borne disease, is an infectious and allergic zoonotic disease caused by brucella, which is widely prevalent in the world and poses a serious threat to the development of animal husbandry and human health. Has become an important public health problem. There are two main problems in the diagnosis of brucellosis: one is rapid and accurate diagnosis, the other is the differential diagnosis of the existing attenuated live vaccine. The PCR technique avoids the operation of live bacteria and can detect the pathogens with low abundance in the specimens, which is suitable for the detection of brucellosis, which is a high risk pathogen. PCR was used to diagnose brucellosis in early stage, relapse stage, complicated with many kinds of complications and after drug treatment. It has a good prospect of application. At present, there is no research report on the application of PCR in clinical diagnosis and follow-up. In this study, we designed primers for the conserved gene bp26 of brucella, designed primers for bovine, sheep and pig breeds for inserted sequence IS711, and established single and multiple PCR detection methods. Suspicious patients and non-patients were tested, and the data of epidemiological case database were used to determine the objects for further follow-up detection, which provided the basis for the establishment of standardized PCR diagnosis method for human brucellosis. At present, serological methods can not distinguish the response of attenuated brucellosis vaccine strain from natural infection. The development of gene marker vaccine can solve this problem. M5 is a widely used live attenuated vaccine strain in China. The bp26 mutant was constructed by replacing part of the bp26 gene with the kana-resistant gene. According to the conserved genes of Brucella, a method of PCR identification was established to distinguish bp26 mutant from vaccine strain. The viability of bp26 mutant in vitro and in vitro showed that the immune response and protective effect of Bp26 mutant in mice was lower than that of M5 vaccine strain. The results suggest that bp26 gene plays an important role in the immune protection of M 5 vaccine strain. Considering the importance of bp26 gene in immunogenicity and protection, we expressed and purified BP26 protein, We also focus on the development of effective brucellosis subunit vaccine and clinical diagnosis as a specific diagnostic antigen for human brucellosis.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R516.7;R392

【引证文献】