【作者】 殷月兰；

【导师】 焦新安；

【作者基本信息】 扬州大学 ， 预防兽医学， 2006， 博士

【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。更多还原

【Abstract】 Listeria monocytogenes (LM) is an important food-borne intracellular pathogen which can cause animal and human serious listeriosis such as meningitis, pyrogenic and septic gastroenteritis, fetal death, premature births, sepsis, encephalitis, with the mortality about 25%. Several outbreaks have taken place in the Occident. Contamination to food by LM and its serious consequence have gained considerable attention in the worldwide. Generation of an attenuated live vaccine to prevent listeriosis had become an important task. On the other hand, attenuated LM is a promising vaccine vector for its intracellular parasitism. In this study, an attenuated recombinant strain was obtained by deleting actA and plcB virulence genes in the wild type strain serotype l/2a; and based on the attenuated LM, using prokaryotic and eukaryotic expression methods to deliver model protein GFP were studied. In order to evaluate the protective efficacy of the attenuated strain, and to verify the deletion of genes in different levels, virulence genes of hly and actA were expressed in the E.coli and specific monoclonal antibodies were prepared.The successful construction of the mutant has a very important effect on preventing listeriosis, and establishes a solid foundation to construct vaccine vector to prevent human and animal diseases. Furthermore, it provides the possibilities to elucidate the mechanisms of pathogenesis by LM virulence factors and immune response induced.1. Prokaryotic expression of actA and hly genes and preparation of ActA monoclonal antibodiesThe actA gene was amplified from LM4 strain of Listeria monocytogenes serotype l/2a by PCR and inserted into prokaryotic expression vector pGEX-6P-l and pET respectively. The fusion protein expressed in both vectors had molecular weight approximately 120KD and 97KD. The expressed ActA protein was purified and used as antigen to prepare specific monoclonal antibodies. Four hybridoma cell lines secreting antibody against ActA protein were established, the ELISA titer of更多还原