Preparation And Identifition Of Encephalitis Virus EDⅢ Protein Monoclonal Antibodies

Japanese encephalitis is an acute infectious disease caused by Japanese encephalitis virus which attacks the central nervous system disease. There are about more than two million JE patients in our country every year, and 20%-30% patients are dead. Epidemic encephalitis B belongs to the natural focal disease and a variety of animals can be infected among horse, donkey and monkey, casing clinical symptom obviously, whose mortality rates are relatively high. Swine infected with encephalitis B virus is more common, whose mortality rate is relatively low, and most of them don’t appear clinical symptom. Pregnant sows infected with Japanese encephalitis can be manifested as high fever, abortion, stillbirth and mummy. The boar can be manifested as orchitis and other animals appear slient infection.Epidemic encephalitis B virus belongs to the flaviviridae family, genus flavivirus, and virus particles are 30-40nm in diameter. Structural protein of Japanese encephalitis virus are C, PrM/M and E. E protein is the major structural protein of virus body, playing an important role in immune and pathogenic. The monomer structure of E protein contains three domains, domain Ⅲ (EDⅢ) which is domain immunoglobμlin similar with fold is the main binding site of a receptor with high immunogenicity. It is valuable for this region in diagnostic analysis of virus. In addition, monoclonal antibody has the advantages of good specificity, high purity, high potency, low cost and can be produced in large quantities. Monoclonal antibody also has very large development value and function research in the laboratory testing, protein purification and functional study, diagnosis and treatment of disease and identification of the epitopes. This article induces the expression of EDIII protein with glycerol bacteria containing pET-28a-EDⅢ plasmid preserved by laboratory. BALB/c mouse is applied by subcutaneous injection, preparing two hybridoma cell lines secreting monoclonal antibodies against EDⅢ protein of encephalitis B virus. Antigenic potency, stability and secretion of the monoclonal antibodies are identified and the neutralizing activity is identified in cells and rat brain.1. Preparation and identification of Japanese encephalitis virus of EDIII protein monoclonal antibodyThis article induces the expression of EDⅢ protein with glycerol bacteria containing pET-28a-EDⅢ plasmid preserved by laboratory, mixing with Freund’s adjuvant emμlsified equivalent after purification, and then BALB/c mouse was applied by subcutaneous injection. The monoclonal antibodies were prepared by fusing mouse myloma cells with spleen cells from BALB/c mice immuned with purified EDⅢ protein. Two hybridoma cell lines secreting monoclonal antibodies against EDⅢ protein were screened by indirect enzyme-linked immunosorbent assay and named as 1C3、1C4. Western-blot and indirect immunofluorescence assy resμlts showed that the obtained monoclonal antibodies reacted specifically with epidemic encephalitis B virus. It could be concluded that the specific monoclonal antibodies against EDⅢ protein of epidemic encephalitis B virus were developed and they may be useful in the development of detection assays and provide basis for mapping the epitopes of EDⅢ protein.2. Study on the effect of ED Ⅲ protein monoclonal antibodies to the virus neutralizationIn order to identify the neutralizing activity of the two hybridoma cell lines secreting monoclonal antibodies against EDⅢ protein which had been obtained in laboratory,1C3 monoclonal antibodies had been tested by plaque reduction cross neutralization and was found having a certain neutralizing neutralizing activity. The neutralizing activity of 1C3 monoclonal antibody was researched in the brain tissue of suckling mouse ulteriorly, and the brain tissue of suckling mouse was anatomized and dyed by hematoxylin-eosin, then 1C3 monoclonal antibody had a certain ability of neutralizing the virus, but neutralizing activity was weak.