Eukaryotic Expression Of Canine Parvovirus HL-1 VP2 Gene And Development Of An Indirect Elisafor The Detection Of Viral Antibodies With The Expression Produce

Canine parvovirus(CPV)belongs to the family parvovirus.It is one of the major pathogens that cause bleeding enteritis and myocarditis in dogs.CPV-2 only infected dogs and was highly contagious.Its infectious ratio is 20%-100%,and its mortality in dogs less 12 weeks is 10%-50%.Canine parvovirus enteritis's clinical symptom is severely sickness, diarrhea and excreting rotten dejecta.Accurate diagnosis and effective vaccination are main means for the prevention of canine parvovirus antigen.Research showed that the outer capsid glycoprotein VP2 of CPV particls is major protective antigen and excellent diagnosis antigen. So gaining VP2 gene and its expressing product is key to the preparation of the new type vaccine and specific diagnosis antigen. In this study, canine parvovirus strain HL-1 was propagated on F81 cell.Apair of primers was designed to amplify VP2 gene by PCR according to the published sequence of CPV's VP2 gene with primer5.0 software. The product of PCR named VP2 is approximate 1.8kb in length.The VP2 gene was cloned into the vector pMD-18-T and the recombinant was named pMD-18-T-VP2.Identifications of restriction enzyme,PCR and sequencing indicated that the VP2 gene has been cloned successfully. pMD-18-T-VP2 was analyzed in comparison with the other CPV strain VP2 genes.Gene sequence comparison indicated that HL-1 VP2 shared 98.97%,98.75% and 98.69% identities with CPV-d(CPV-2),CPV-15(CPV-2a) and CPV-39(CPV-2b) respectively.The deduced amino acid sequences were 98.12% ,97.60% and 97.60% correspondingly.The results affirmed that VP2 gene of CPV strain HL-1 was not conservative. VP2 was inserted in Hindâ…¢and Salâ… multiple cloning sites of the vector pMel Bac C by restriction enzyme. pMel Bac C-VP2 was identified and analyzed by PCR on the basis of the genetic sites of pMel Bac C,which was identified as VP2 gene of CPV.Purified pMel Bac C-VP2 and Bac-N-Blue DNA were cotransinfected insect cell Sf9 and harvested Recombinant baculovirus 3d later.Identification with PCR showed VP2 gene has been recombinated correctly with baculovirus DNA .Recombined purificated baculovirus was gained through blue plaque purification for three times. The analysis results with SDS-PAGE and Western-blotting showed that HL-1 VP2 gene was expressed in baculovirus. Recombinant fusion protein was used as detecting antigen in indirect enzyme linked immunosorbent assay (ELISA) to detect anti-CPV serum antibody.The optional ELISA reactional system were decided.The coating buffer was PBS (pH7.4).The blocking buffer was 0.5%BSA/PBS.The optional serum dillution buffer was 4%PEG6000/PBS.The washingbuffer was 0.5M NaCl/0.5%Tween-20/PBS. The optimal incubation time for the firstsera was 1 to 1 .5h . Development of an Indirect ELISA provides a technology for CPV's diagnose and serological detection.