Prokaryotic Soluable Expression Of Canine Parvovirus VP2 Gene And Development Of Monoclonal Antibodies Against VP2 Protein

Canine Parvovirus(CPV) is a small nonenvelop virus.It possesses single-stranded DNA that codes for the two capsid protein(VP1,VP2) and two nonstructural protein(NS1,NS2). The whole length of Genome is 5323nt, and VP2 is 1755nt. As the major antigen epitope and the major structure protein, VP2 produces neutralization against antibody. As the important optional antigen, VP2 produces protection against CPV which can induce the severe hemorrhagic enteritis and myocarditis. CPV can cause the disease in every season, and it happens with rapid speed, short process, powerful epidemic and the high death rate. So it is important to diagnosis immediately the infective canine.The reseach expressed the VP2 fragment in prokaryotic cell. According to published sequence of the CPV, three pair of primers were designed, and the relative gene fragment was amplified by polymerase chain reaction (PCR). According to the conventional method, the product of PCR was subcloned into the downstream of GST gene in expression plasmid pGEX-4T-2. The recombinant vector was transformed into E. coli BL21, GST fusion protein was expressed at the induction of IPTG. The fusion protein was 38.3KDa and 41KDa and have good antigenicity which confirmed by SDS-PAGE, indirect ELISA and Western blotting. The fusion protein was purified by metal-ligand affinity chromatography, which should be used as diagnostic reagent of CPV disease.In order to prepare the monoclonal antibodies (McAb) against VP2 protein of CPV, BALB/c mice were immunized with the purified CPV and its spleen cells were used to prepare the hybridoma, used the purified recombinant VP2 fusion protein as screening antigen. After subcloned, Eight hybridoma cell lines secreting McAb against CPV-VP2 have been established. They were respectively named 3D9C4,4F11D5,5F10F5,4F9B7,4G9G11,5F11C11. Eight hybridoma cell lines secreting McAb against CPV-VP2 have been established. They were respectively named 3D9C4,4F11D5,5F10F5,4F9B7,4G9G11,5F11C11. The neutralization test indicated that these four McAbs 5F10F5,4F9B7,4G9G11,5F11C11 can produces the neutralization effect against antibody, and the results of indirect ELISA,Western Blot and indirect immunofluorescence experiment indicated that these four McAbs were specific for VP2 and didn't have the mutual reaction with other canine viruses, so they can be used for specific detection of CPV.After the direct ELISA, indicated that among these four McAbs, 5F10F5,4G9G11 and 5F11C11 against the different epitope, 4F9B7 and 5F11C11 against the same epitope.Next, all these achievement can be used for specific detection of CPV, remedy of the CPV desease, reseach of the VP2 protein function, and establishment of the gold immune-chromatographic assay kit for rapid dectection of CPV and antibody.