The Purification Of A Recombinant Listeriolysin O Expressed In Escherichia Coli And Preliminary Detection Of Listeria Monocytogenes By Blocking ELISA

Listeria monocytogenes (LMO) is a Gram-positive, foodborne, human and animal pathogen. This bacterium causes an infection, named listeriosis, which especially affects immunocompromised patients, new-borns, and pregnant women and is characterized by a variety of severe syndromes, such as encephalitis, meningoenpephalitis, septicemia, and abortion. Owing to its ability to survive in various environmental conditions, such as low pH, high NaCl concentrations, and very low temperatures, LMO is present both in raw and processed foods; moreover, being a facultative anaerobe its growth is not significantly affected by vacuum packaging. Following the ingestion of contaminated food, bacteria cross the intestinal barrier, reach the liver and the spleen, and then disseminate to the brain and placenta. LMO is a facultative intracellular micro-organism capable of internalizing into macrophages, as well as into cells devoid of phagocytic activity, by inducing its own phagocytosis. After phagocytosis, the pathogen escapes from the bacterium-containing vacuole by lysis of the membrane and reaches the host cytosol in which it multiplies.LMO can be found widely in nature. After a number of recent foodborn outbreaks, it has been demonstrated that this bacterium poses a significant health risk and has attracted the attention of the food safety professionals and public health officers.This thesis introduces the general characteristics of this bacterium on preface, including its physiological and biochemical traits, virulent factors, infection routes, symptoms, therapy, and epidemiology. This thesis is also especially emphasized on the introduction and comparison of the differences, the advantages, as well as the disadvantages between the traditional and rapid diagnosis methods.Listeriolysin O (LLO) is the virulence factor that allows the LMO escape from the primary phagocytic vacuole. LLO could be isolated and purified from LMO culture medium, Unfortunately this method requires high culture volumes, is time-consuming and provides very low yields. As consequence, studies on the peculiar LLO moleculal mechanism of action and protein employment in pharmaceutical application, are very difficult. A way out to the problem has been found optimum expressing the protein in Escherichia coli.In the thesis.The recombinant expression plasmid pGEX-6P-hly was transformed into Escherichia coli cell and induced by IPTG First of all the induction conditions were optimized inorder to obtain the expression of the largest amount of recombinant protein in a soluble and active form. In particular, the influence of length and temperature of induction , the bacterial density and the IPTG concentration were examined. The recombinant proteins can be observed by SDS-PAGE and analysed by BandScan. Experimental results indicated that under shake incubator, LB medium and inducing 2.5hours with37"C, hly expression quantity was the most when ultima concentration of IPTG was 0.6mmol/L. Western-blot analysis sho 'ed that recombinant fusion protein could react with a specific anti-LMO0586 serum.This provided dependable experimental accordance for industry production of Listeriolysin O when IPTG was inducer.In the Escherichia coli. induced cells,the toxin was expressed in the cytoplasmic compartment. Therefore, to purify the recombinant LLO it was necessary to lyse the induced bacteria and to recover the proteins in soluble form. In this experiment, the optimum conditions to acquire the cytoplasmic compartment was found. As a result, the high-level expressed recombinant LLO was purified to homogeneity by an adsorption chromatography.In the experiment, we found LMO cultured by BHI medium could express native LLO optimally. We can even detect it by SDS-PAGE. This discovery was very important to our study. So, after die purification of recombinant LLO, w<) immunized the rabbits with the recombinant LLO to acquire anti-recombinant LLO antibody which we use it as a diagnostic antibody, we established a preliminary method by ELISA to detect the LMO in the laboratory. In the experiment, the optimal concentration of antigen and the reference serum was decided. The sensitivity and specificity was very well in this method. It was successful to establish the method of Blocking ELISA for determination of LLO secreted by native LMO in this study.