Production And Use Of Monoclonal Antibodies For The Detection Of Taura Syndrome Virus

Taura syndrome(TS) of prawn is caused by infection with Taura syndrome virus(TSV),which can cause significant disease outbreaks and mortalities of the principal host species(Penaeus vannamei and P. stylirostris).Three major polypeptides VP1,VP2and VP3(55,40and24kD) and one minor polypeptides VP0(58kD) constitude its proteinic capsid.The gene VP1have the major antigenic epitope.In this research, take the laboratory strains which have been building in VP1gene’s conservied region.Through the recovery and expand training, using IPTG to induce gene expression. A large number of inducible expression and purification of target protein. The Balb/c mice were immunized with the purified protein.Through three times immunization to producing monoclonal antibody, and make the serum get up to1:10-4, then use the protein to immuned another time three days before experiment. Under the aseptic condition, using the spleen cell of mice to hybridize the SP2/0cell to get the hybridoma.Detecting the antibody titer by indirect enzyme-linked immunoadsorb assay (ELISA). Through three times subclone, finding the cell which can steadily secretes antibody. Expanding culture the cell, then to inoculate the cell to the belly of the Balb/c mice to get the ascites. Every mice inoculates cell about106,after seveen to ten days,we can get the ascites.The McAb titer is about1:10-6and have Specificity through Western-BlotThe monoclone antibody was used to build a test strip.Two diameter of colloidal gold (20nm and30nm) were used respectively to label the monoclonal antibodies to find the best experiment condition. The assay was constructed in the form of sandwich by using the anti-VP1cr recombinant monoclonal antibodies.The monoclone antibody labeled with20nm colloidal gold was made a conjugated pad.Mouse anti-VPlcr recombinant polyclonal antibody was immobilized in a defined detection zone on a porous nitrocellulose membrane. Negative control were tested successfully ande the color is red. Using this test strip to detect the VP1recombinant protein. The control line and the test line is obviously red.