Cloning And Expression Of Canine Parvovirus VP2 Gene On Diagnostic Application

Canine Parvovirus disease is an acute disease of canine caused by canine parvovirus (CPV). CPV can be found in dogs of different ages and species all over the world. The incidence rate and death rate caused by this disease are particularly high in weaned puppies. With the development of canine industry, the prevention and control of canine disease become more and more important. Therefore, it is especially important to establish positive and effective preventive measures for obtaining prompt and accurate diagnostic information.In this study, through design and synthesis of specific primers, which can be used to duplicate VP2 genes. The VP2 gene is expressed in prokaryotic system. The highly purity of VP2 recombinant protein harvested by purification. The specificity, sensitivity and highly detection rate of indirect ELISA was established using VP2 recombinant protein, meanwhile the specific CPV McAbs were obtained by this method.A pair of specific primers is synthesized according to the CPV VP2 sequence published by Genbank. The fragment is amplified by PCR. Then the amplified fragments are cloned into pMD18-T vector. The positive plamid were directionally subcloned into the expression vector pET-30a, after sequencing . They were transformed into component bacteria Rosetta. The recombinant protein was expressed with the yield accounting for 29% of total bacterial proteins. Western blot analysis showed that the recombinant protein had a molecular weight of approximate 67ku, and had special antigenicity. The target protein was harvested with a concentration of 0.7mg/ml by ProBond^TMPurification Systerm.The optimization condition and the determination standard is determined by using VP2 recombinant protein as coated antigen. The reaction between the antigen and the standard positive serum can only be blocked by CPV and there is not any cross reaction with the positive sera of Canine distemper, Canine hepatitis, canine Bordetella bronchiseptica and canine parainfluenza. Compared with the imported CPV ELISA kit, the 92 serum samples detection result indicated the coincidence rate is 97.8%. After four times of duplicate test of 48 serum samples, the coefficient of variability of reproducibility testing is below 16%. Therefore, the indirect ELISA for detection is successful established. Carries on the examination using this method in some domestic areas of serum sample, finally we discovered in 227 serum samples 164 of which is positive, the positive rate is 72.25%.Conventional hybridoma techniques were used to fuse BALB/c mice myeloma cell line SP2/0 with B lymphocytes of spleen of BALB/c mice which were immunostimulated with the purified CPV-Z. Three hybridoma cell lines were obtained and named E4F11F7, E3B9C4 , F10G10A10 respectively. They stably produce antibodies against recombinant VP2 protein were picked out and identified by indirect ELISA. The subtypes of monoclonal antibodies(McAbs) belong to IgG2a and IgM, all the light chains were Kappa. Western blot analysis showed that the McAbs specifically recognized certain antigenic epitope of VP2 protein of CPV, but not reacted with other reference viruses. The McAbs against CPV VP2 recombinant protein with high activity and specificity had been established successfully.