The Expression of CPV-2 VP2 Gene in SF9 and Development of Indirect Elisa for Serum Antibodys
|School||Nanjing Agricultural College|
|Course||Preventive Veterinary Medicine|
|Keywords||canine parvovirus(CPV) VP2 gene expression Sf9 indirect ELISA|
Canine parvovirus (CPV) belongs to the family parvovirus. It is one of the major pathogen 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 is a significant mean of prevention of this disease. Therefore, it is important to establish an accurate, specific and sensitive diagnostic method for early diagnosis and isolation and treatment of canine distemper dogs. The researches showed that the outer capsid glycoprotein VP2 of CPV particls is major protective antigen and excellent diagnosis antigen, and gaining VP2 gene and its expressing product is key to the preparation of the new type vaccine and specific diagnosis antigen.In this study, a pair of primers were designed to amplify VP2 gene by PCR according to the published sequence of CPV VP2 gene with primer5.0 software.The product of PCR named VP2 is approximate 1.755kb in length. The VP2 gene was cloned into the vector pMD-19-T, and the recombinant was named pMD-19-T-VP2. Identifications of restriction enzyme digestion, PCR and sequencing indicated that the VP2 gene has been cloned successfully. The pMD-19-T-VP2 was analyzed in comparison with the other CPV strain VP2 genes. The Gene sequence comparison indicated that VP2 shared 98.97%、98.75% and 98.69% identities with CPV-2a CPV-2b and CPV-2c respectively. The deduced amino acid sequences were 98.12%、97.60% and 97.60% correspondingly.The VP2 was inserted in EcoRI and XhoⅠmultiple cloning sites of the vector pFastBacTMTA, and the recombinant transfer vector pFastBacTMTA-VP2 was obtained. The recombinant shuttle vector Bacmid-VP2 was obtained by blue white screeing when the recombinant transfer vector pFastBacTMTA-VP2 was transformed into DH10Bac. The recombinant baculovirus rBac-VP2 was obtained when the recombinant shuttle vector Bacmid-VP2 was transfected into insect cells Sf9 using the liposome. The analysis results with IFA、SDS-PAGE and Western-blotting showed that VP2 gene was expressed in Sf9.Recombinant fusion protein was used as detecting antigen in indirect enzyme linked immunosorbent assay(ELISA) to detect serum antibody against CPV. The optional ELISA reactional system were confermed. The coating buffer was PBS (pH7.4). The blocking buffer was 5% CS/PBS. The optional serum dillution buffer was 0.05% CS/PBS. The washing buffer was 0.5M NaCl/0.5% Tween-20/PBS. The optimal incubation time for the primary antibody was 1.0h, and the chromogenic condition was incubated at 37℃for 15min. The development of the indirect ELISA provided a technological tool for CPV diagnose and serological detection of CPV.