产单核细胞李斯特菌减毒突变株的构建及其免疫生物学特性的研究

发布时间：2018-12-23 19:20

【摘要】： 产单核细胞李斯特菌(Listeria monocytogenes,LM)是人兽共患李斯特菌病的病原,主要通过食用被LM污染的食品感染,能引发人的脑膜炎、发热性败血性胃肠炎、孕妇流产等,感染后发病死亡率约为25%。欧美国家曾多次暴发流行,LM对食品的污染与危害,已引起世界各国的普遍关注和高度重视,其中研制预防李斯特菌病的减毒活疫苗是重要的课题之一。另一方面,鉴于LM是胞内寄生菌,减毒LM是非常有前景的疫苗载体之一。为此,本研究通过同源重组的方式对血清型为1/2a的野生型菌株基因组中相邻的两毒力基因actA和plcB进行缺失,获得减毒重组菌株,并以减毒株开展了以原核和真核表达的不同方式运送模式蛋白GFP的研究。为了对减毒株的免疫保护力进行评价,以及从不同水平上证实基因的缺失,对毒力基因hly和actA在大肠杆菌中进行了原核表达并对ActA蛋白进行了单抗的研制。减毒突变株的获得不仅对李斯特菌病的预防具有重要作用,而且为构建预防人类和动物疫病的疫苗载体奠定了基础。此外,对于LM阐明毒力因子的致病机理与免疫保护作用提供了条件。 1.产单核细胞李斯特菌actA基因和hly基因的原核表达及ActA单克隆抗体的研制利用PCR技术从血清型1/2a的LM4株中扩增出actA基因,经克隆筛选和测序鉴定后,构建成该基因的原核表达载体pGEX-6P-1-actA及pET-actA,转入终宿主菌E.coli后,IPTG诱导目的蛋白的表达。SDS-PAGE电泳结果表明,actA基因在两种载体中均获得表达,融合蛋白的大小分别约为120KD和97KD。
[Abstract]:Listeria monocytogenes (Listeria monocytogenes,LM) is the pathogen of Listeria monocytogenes. It can cause meningitis, febrile septic gastroenteritis, miscarriage of pregnant women and so on by eating food contaminated with LM. The death rate after infection was about 25%. There have been many outbreaks in Europe and America, and the contamination and harm of food by LM has attracted worldwide attention. Among them, the development of live attenuated vaccine against Listeria monocytogenes is one of the most important topics. On the other hand, since LM is an intracellular parasite, attenuated LM is one of the promising vaccine vectors. In this study, two virulence genes, actA and plcB, were deleted from the genome of wild-type strains with serotype of 1 / 2a by homologous recombination, and the attenuated recombinant strains were obtained. The study on the transport of mode protein GFP by prokaryotic and eukaryotic expression was carried out with attenuated strain. In order to evaluate the immune protection of the attenuated strain and to confirm the deletion of the gene at different levels, the virulence genes hly and actA were expressed in E. coli and the monoclonal antibody against ActA protein was developed. The acquisition of attenuated mutants not only plays an important role in the prevention of Listeria, but also lays a foundation for the construction of vaccine vectors for the prevention of human and animal diseases. In addition, it provides conditions for LM to elucidate the pathogenicity of virulence factors and immune protection. 1. Prokaryotic expression of actA gene and hly gene of Listeria monocytogenes and preparation of ActA monoclonal antibody actA gene was amplified from 1 / 2 a LM4 strain of serotype by PCR technique. The expression of the target protein was induced by IPTG after the construction of prokaryotic expression vectors pGEX-6P-1-actA and pET-actA, into E.coli. The results of SDS-PAGE electrophoresis showed that actA gene was expressed in both vectors. The size of the fusion protein was about 120KD and 97kD, respectively.
【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R378

【相似文献】