The Prokaryotic Expression Of Listeria Monocytogenes Hemolysin And Preparation Of Monoclonal Antibody To Hemlysin

Listeriosis was caused by Listeria monocytogenes (LMO) of human and animals. The disease is an important common food-borne zoonoses, which is an acute infectious disease with high fatality rate. LMO is a serious threat to the human food safety. Therefore, it is very important to establish a fast and accurate method that can detects and diagnoses pathogenic bacteria. The main detection of LMO in foods are immunological detection methods and molecular biology detection method. Many domestic and foreign scholars have done extensive researches on the application of monoclonal antibodies in the immunological detection and have confirmed this the view.The hemolysin (Listeriolysin O, LLO) protein encoded by the hlyA gene is the main pathogenic factor that plays a very important role in the process of Listeria monocytogenes infect host cells. Hemolysin is a secreted protein so that the content is very low in bacteria cultures and can not meet the actual needs. This research using the hemolysin protein as the object, cloned the LMO virulence gene hlyA, after identificated the sequences, the target gene was inserted into expression vector pET-30(+), and the recombinant plasmid pET-30a-hlyA was successly constructed that was transformed into E.Coli to empress the LLO after PCR and restriction enzyme identification. Under the optimized expression conditions (IPTG concentration is0.8mol/L, the temperature is34℃, the induction time is3h, we got a large amounts of soluble recombinant protein, which then was purified by using Ni-NTA chelating affinity chromatography. When imidazole concentration was50mmol/L and100mmol/L, the elution can get the best results according to the spectrophotometer determination and SDS-PAGE analysis. The BALB/c mice was immunized by the purified protein and getting positive serum, then the indirect ELISA method was initial established with the ment of confirming the immunogenicity of target protein. The spleen cells of immune mice were fused with bone marrow tumor cells SP2/0using polyethylene glycol (PEG), ELISA method based on LLO was applied to screen for positive clones. Senven hybridomas cell lines was obtained by limiting dilution after subcloning three times, which can stably secrect McAbs anti-LLO. Rapid qualitative kits was used to determinate their subclasses, the results show that2strains were IgG2b and5strains were IgGl; Detecting mcAbs specificity with various strains that were kept in the laboratory as coating antigen, and the results were that2strings hybridoma cell supernatant could interact weakly with Escherichia coli. Detecting the mcAbs antigen recognition site by superposition experiment. The hybridoma cells were cultured3months, then detecing the titer of cells’ supernatant periodically and analysis the stability of McAbs. The results were that7strains of McAbs remain stable basically.