基于牛病毒性腹泻病毒核心蛋白C间接ELISA抗体检测方法的建立OA

To establish an indirect ELISA detection method based on the core protein C of bovine viral diarrhea virus(BVDV),this study amplified the BVDV C gene by PCR,cloned it into the prokaryotic expression vector pET-32a,and constructed the recombinant plasmid pET-32a-C.After IPTG-induced expression and purification,the recombinant protein was used to immunize BABL/c mice to prepare polyclonal antibodies.Using the protein C as the coating antigen,an indirect ELISA detection method was established.The results showed that the pET-32a-C recombinant plasmid was successfully constructed,and the recombinant protein C,with a molecular weight of approximately 30 kDa,could be expressed in soluble form.Optimal soluble expres-sion was achieved under induction with 0.8 mmol/L IPTG at 30 ℃ for 6 hours.The serum antibody titer from im-munized BABL/c mice reached 1∶128 000 and could specifically recognize protein C.The optimal conditions for the ELISA method were as follows:coating with 0.5 μg/mL antigen at 37 ℃ for 2 h,blocking with 10 g/L BSA at 37 ℃ for 3 h,incubation with 1∶16 000 diluted serum for 2 h,secondary antibody incubation for 40 min,and light-protected color development for 10 min.The critical value for distinguishing positive and negative re-sults was set at 0.224,demonstrating good specificity and repeatability.When applied to detect sera from non-vaccinated cattle suspected of BVDV infection,the positive concordance rate with RT-qPCR nucleic acid detection reached 94.73％.These results indicated that the indirect ELISA antibody detection method based on protein C exhibits strong specificity,high sensitivity,and excellent repeatability,providing a founda-tion for the clinical diagnosis of BVDV and the development of protein C-based indirect ELISA detection kits.