关键词: 犬细小病毒; VP2基因; 表达; 抗体; 竞争ELISA

Abstract: This study focused on the preparation of canine parvovirus VP2 antibodies, and establishing a competitive ELISA detection method, so as to provide technical support for detection of the effect of CPV vaccine and serological investigations of CPV infection. A pair of primers adding EcoR Ⅰ and Xho Ⅰ restriction site were designed according the gene sequence of canine parvovirus VP2. Full-length VP2 gene was obtained by PCR amplification, then cloned into pET28a(＋) vector to construct a prokaryotic expression vector pET28a-VP2, and transformed into E.coli host strain Rosetta. Finally, the VP2 recombinant protein was expressed and purified. Rabbits were immunized with purified VP2 recombinant protein, then VP2 polyclonal antibody was prepared, and labeled with horseradish peroxide enzyme, and detected by indirect ELISA method, finally, established a competitive ELISA method initially. The result of SDS-PAGE showed that the VP2 recombinant protein expressed was correct, and the molecular weight size was 67 ku. The result of Western blotting showed that this protein could be recognized by CPV antiserum, demonstrated that it was specific. Indirect ELISA results showed that best packages concentration of the VP2 recombinant protein was 5 μg/mL, and coated at 4 ℃ overnight, the best dilution of enzyme labeled antibody was 1∶3200, the optimal solution for block was the 10% bovine serum, 1∶5000 dilution of the goat anti-rabbit IgG-HRP was the best work concentration, coloration time was 25 min. The results of competitive ELISA test showed that the method was stability. In conclusion, this study provides a technological means for determination the effect of CPV vaccine and serological surveys.

Key words: CPV; VP2 gene; expression; antibody; competive ELISA