Prokaryotic Expression Of Major Antigenic Domains And Development Of Monoclonal Antibodies For GB And GCof Duck Enteritis Virus

Duck virus enteritis (DVE), also known as duck plague (DP), is caused by duck virus enteritis (DEV). DVE is an acute, septicaemic contagious viral disease with high fever which can infect a variety of Anseriformes birds, such as ducks, geese and swan. DVE, characterized by widely distribution, rapidly spread, high morbidity and mortality, is one of the most serious diseases for waterfowl industry. DEV belongs to alpha herpesvirus families. Glycoprotein B and C plays an important role in receptor recognition and adsorption to host cells during DEV infection. In this study, we cloned the main antigenic domain encoding sequences distributed in DEV gB and gC gene, performed prokaryotic expression on them, and obtained the monoclonal antibodies against glycoprotein B and glycoprotein C. These laid the foundation for studying biological characteristics of DEV further and setting up a rapid, accurate and specific method for DEV detection.We designed five primer pairs to amplify the main antigenic domain encoding sequences distributed in standard virulent strain DPV-F37gB and gC gene by PCR. All purified gene fragment were subcloned into prokaryotic expressing vector pGEX-6P1. After sequencing success, the recombinant plasmid, named pGEX-6P1-gB1、pGEX-6P1-gB2、pGEX-6P1-gB3、 pGEX-6P1-gC1and pGEX-6P1-gC2, were transformed into E.coli BL21(DE3) and expressed under the induction of IPTG. The size of expressed proteins approximately were35KD,45KD,37KD,38KD and40KD respectively. Western-blot result showed that all recombinant proteins had specific reaction with polyclonal rabbit antiserum against DEV. These results suggested that the fusion proteins have similar antigenicity with nature proteins encoded by DEV.Six-week-old BALB/c mice were subcutaneously injected with purified DEV. An intraperitoneal injection of the same dose of DEV was given4days before splenocytes were collected and fused to SP2/0myeloma cells. The ELISA plates were coated with purified gB and gC protein and hybridoma culture supernatants were screened by indirect ELISA method, then selected the positive to subclone and obtained7hybridoma cell lines secreting monoclonal antibody anti-gB or anti-gC antigen. They were named6F7、3G11、4B5、1A12H、4D4and10A6. Subclass of monoclonal antibodies6F7,4D4and10A6IgG1,4B5and1A12were IgG2b,3G11and2H9were IgM. Western-blot approved that10A6had specific reaction with gBl,1A12reacted with gB3, the others reacted with gC2. Indirect immunofluorescence was positive when monoclonal antibodies were specific against the DF1affected DEV.