Prokaryotic Expression Of Avian Pasteurella Multocida Genome And Adsorption Of Infected Serum

Avian pasteurellosis, also known as fowl cholera, caused by Pasteurella multocida, is a contact, septic and infectious diseases. The diseases with a sudden and popular feature are created high morbidity and mortality. It causes huge economic losses in the poultry industry. Therefore it is listed as one of the focus control poultry diseases. Vaccination as an effective measure to control pasteurellosis, scholars has been attached great importance to the diseases prevention and control research over the years. Therefore the development of effective vaccines has become a hot topic.The antigen of avian pasteurellosis is avian Pasteurella multocida. The genes of avian pasteurellosis are important significance to understanding its pathogenesis and effective prevention of avian pasteurellosis. Pathogens in the host and not expressed in vitro expression of these genes function called " in vivo induced genes "(in vivo-induced gene). Numerous studies show that these unique genes expressed in vivo and in vitro is often not expressed in vivo are very important of survive and pathogenicity of pathogens. Vivo induced antigen technology (in vivo-induced antigen technology, IVIAT) is the use of a host infected with a pathogenic bacteria in the sera screened pathogen specific gene expression in vivo in a host. The positive colonies contain the genes which could be specially expressed in vivo and might be potential targets to novel vaccines, drugs and diagnostic methods. And by prokaryotic expression analysis, a new gene was found which paved the way for further in-depth the vaccine development.The first a genomic expression library of avian Pasteurella multocida CVCC474: The genome of avian Pasteurella multocida was extracted. It cuts with the restriction enzyme Sau3A I. The genomic of500-3000bp DNA fragment was recovered after digestion. Prokaryotic expression vector pET32a/b/c was digested by restriction endonuclease BamH I. The linear vectors were obtained and dephosphorylated with CIAP. After genomic fragments connected with prokaryotic expression vector pET32a/b/c. They were transformed into competent cells E. coli BL21(DE3). The genomic expression library of avian Pasteurella multocida CVCC474was constructed. The DNA expression library total capacities were l.5×104. Nine randomly picked clones were digestied experiments. They were consisted with expression vectors and gene fragments. It proved that genomic expression library of avian Pasteurella constructed successfully. The recombinant plasmid is extracted by alkaline lysis and induced by IPTG. It proved that protein wes expressed by recombinant plasmid.Serum adsorption and the screening of avian Pasteurella genomic expression library: The serum of avian Pasteurella chicks was collected. Chicken serum was adsorbed by cultured in vitro with E. coli BL21(DE3)(containing pET30a vector) and avian Pasteurella. In order to removing non-specific antibodies in serum.The titer of antibodies was detected by indirect ELISA assay. The results showed that antibody titers decreased in serum after the third adsorption. OD values for antibodies remained unchanged, the effect of absorption is good. Only the antibody was reacted with avian Pasteurella in vivo induction expression in serum. Then genomic expression library was situ immunochemistry, to search the possible clones. However, no positive clones, which is pending further study.