The Construction And Immunobiological Properties Of An Attenuated Strain Of Listeria Monocytogenes

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...