Prokaryotic Expression of Giant Panda-derived Canine Parvovirus VP2 Gene and Establishment of an Indirect ELISA Detection Method

Purpose To establish an indirect enzyme linked immunosorbent assay (ELISA) method for detecting canine parvovirus (CPV) antibodies in giant panda serum.

Methods The VP2 gene was amplified by PCR using CPV DNA derived from giant pandas as a template, then the VP2 gene was expressed using a prokaryotic expression system, and the purified protein was used as an ELISA coated antigen. Rabbit anti-giant panda IgG was prepared and purified, and labeled with horseradish peroxidase (HRP) as an indirect ELISA enzyme-labeled secondary antibody. The conditions such as the mass concentration of coated antigen and dilution of serum were determined by the checkerboard method; and the consistency rate between the established method and the test sample results of commercial kits was evaluated.

Results Prokaryotic expression successfully obtained about 70 ku of VP2 protein, which was used as an indirect ELISA coated antigen. The optimal mass concentration of coated antigen was 2.0 μg/mL, and the optimal dilution of serum was 1∶400. When using this method to detect serum samples OD₄₅₀≥0.258 was positive, and vice versa was negative. The positive rate of giant panda serum samples detected by the ELISA method established in this study was 60.0%, which was higher than the positive rate of commercial detection kits (52.5%), and the total coincidence rate reached 87.5%.

Conclusion This study establishs an indirect ELISA method based on the giant panda-derived CPV VP2 protein as the antigen, and homemade HRP-labeled rabbit anti-giant panda IgG as the enzyme-labeled secondary antibody for the first time. This method has strong specificity, high sensitivity and good repeatability, and provides technical support for the detection of CPV antibodies in giant panda serum.