Establishment Of Monoclonal Antibody Mediated Detecting Method To AEV And Preliminary Research Of The Structural Protein VP3

Avian encephalomyelitis is an infectious disease of poultry which is characterized by neurological signs such as ataxia, paralysis and tremors of the neck and head. Which can affect young chickens , pheasants, quail, and turkeys. The disease was of great economic importance to the poultry industry prior to the widespread use of vaccines. Now avian encephalomyelitis almost can be found in every countries with poultry industry. The disease has been reported in some provinces of China since 1980.The disease can be transmitted laterally via the faeces of infected birds or vertically via the eggs to chicks. Consequently contamination of vaccine is likely if eggs used for vaccine production are derived from flocks infected with avian encephalomyelitis virus(AEV). Two methods are recommended for the detection of AEV in vaccines. Both methods are cumbersome, time-consuming and expensive. They involve inoculating vaccines into five- or six-day-old embryos, allowing them to hatch and looking for signs attributable to AEV. Alternatively one-day-old chicks are inoculated intracerebrally and observed for clinical signs. Now, several new techniques such as indirect immunofluorescent assay (IFA) and indirect enzyme-linked immunosorbent assay (ELISA) have been developed to detect AEV. In these methods adsorbed antibodies against AEV were used to reduce nonspecific reaction. Monoclonal antibodies are specific to antigens and considered a reliable method for diagnosis of pathogens.In order to establish a specific detecting method to AEV, monoclonal antibody against AEV was prepared with AEV purified by cesium-chloride equilibrium centrifugation propagated using SPF eggs via the yolk sac inoculation. Then monoclonal antibody mediated methods such as indirect fluorescent assay, indirect ELISA and Dot-ELISA were preliminarily established for detecting AEV.In the family Picornaviridae, the structural protein VP3 is considered relating tothe persistent infection of virus, T cell immunity and the reaction with virus receptors. Although AEV was classified to enterovirus, several researches indicated that it is closest to hapatovirus. There are some neutralization locations in the VP3 of hepatitis A virus and VP2 and VP3 of poliovirus indicates that the YP3 of AEV maybe in some degree function as protection protein resisting infection of virus. In this research, a pair of primers specific to VP3 gene were designed according to the published sequence of VP3 gene of AEV strain 1143 and VP3 gene was amplified by RT-PCR from the total RNA of semipurified AEV VR strain. Prokaryotic and eukaryotic vectors expressing VP3 were constructed and some researches have been done to VP3.