DNA Immunization Porcine Transmissible Gastroenteritis S Gene Monoclonal Antibody Prepation And Characterization Analysis

Porcine transmissible gastroenteritis (TGE) caused by transmissiblegastroenteritis virus (TGEV) is a highly contagious disease characterizedby vomiting, diarrhoea and dehydration. The mortality rate of TGE inseronegativesuckling piglets may reach100%. TGE prevalence causesenormous economic losses to swine-breeding unitsIn this study, we used an eukaryotic plasmid containing antigenicsites A, B, C, and D (termed S gene) with PVAX vetor to immunizeBALB/c mice. we established that an indirect ELISA detection methodwhich packaged ELISA plate with TGEV,5%skimmed milk as blockingagent, primary antibody and second antibody Incubated time both are60min.Then,the antibody levels produced is detected by indirect ELISA method after immunization42days. Cell fusion was performed accordingto the traditional method.. It was carried out to screen hybridoma cell lineusing supernatant of TGEV propagation as for the first kind of antigen byin direet ELISA assay, using the limiting dilution method,we finallyobtained two hybridoma cell line which named1H4and4E9after threecloned.The antibodies of3hybridoma cell lines were belong to IgMsubgroup.In indircct immunofiuorescence assays, ST cell monolayers wasinfected with TGEV probed by the antibodies or supematant of SP2/0cellculture respectively.After staining with FITC labeled secondary antibodies,strong light green fluorescent could be observed on membrane ofcells.However,no fluoreseence was detected with the addition ofsupematant of SP2/0cell culture. The1H4monocloned antibody werecapable of reacting with the TGEV S protein which was supernatant ofTGEV propagation analysed by Western blot. In indirect ELISA assay,the result of specific recognition characterswas proved that used the supernatants of TGEV,PEDV,PRRSV and IBVpropagatlonas antigens and monoclonal antibodies that they could not reactwith PEDV,PRV or PrV. It was confirmed that these specific antibodiescould be used for TGEV diagnosis.Then,we used the Plaque techniqueproved that the1H4monoclonal antibody had the activity of neutralizingand could reduce cell infection by TGEV in a dose-dependent manner.Dot-ELISA was performed to determine the character of TGEV,weused the different dilutions and puried TGEV as antigen which was reactedwith monoclonal antibodies.The dark mark showed that two monoclonalantibodies could reacted with TGEV, but the supernatant of SP2/0cellculture could not recognize TGEV.TGEV and monoclonal antibodies were used for basic detectionreagent as indirect competitive ELISA method to detect the virus,according to the criteria for the yin and yang which the inhibition rate is greater than10.77%of the sample was positive.This detection method fordetecting TGEV,PRV and PEDV had no cross-reaction, and the compliancerate reached to94.11%which compared with the PCR method.The resultof indirect ELISA confirmed with the best stability by repeatedly test.Indirect competitive ELISA not only to establish e a more simple, rapid,sensitive and widely used TGEV detection methods for field applisction,but provided a basis for the practical production.