Serological Detection Methods Of Anti Koi Herpesvirus Antibody

Koi herpesvirus disease (KHVD), caused by Koi herpesvirus(KHV), is an infection disease of common carp (Cyprinus carpio) with contagious and acute viraemia. The disease has caused severe financial losses to fish breeders. Many diagnosis methods of KHV have been developed. Among these methods, the Enzyme-linked immunosorbent assay (ELISA) has the excellence of convenience, shortcut, inexpensive and suitably spread in grass roots. In this study, using the purified KHV as coating antigen. An indirect ELISA method was successfully developed for detecting anti-KHV antibody in the fish serum. The developed ELISA provided a rapid diagnostic method of KHV. At the same time, the envelope protein ORF59gene of KHV-347was amplified from the KHV genome by PCR and the combinant ORF59protein was expressed effectively in E. coli BL21. It would be useful for developing genetic engineering subunit vaccine. The results of the research are as follows:1The virus strain of KHV-347was grown in Koi fin cells (KF-1) and Carp epithelial tumor cells (EPC). Viruses from cell suspensions were purified by supercentrifuge. In this way the high purity antigen can be prepared. This study found that KHV is not easy to produce CPE on Koi fins cell line, but KHV can produce better CPE on cros-culture of EPC and KF cell lines.2The purified KHV-347was used as a coating antigen to establish an indirect ELISA for the detection of antibodies against KHV. After selecting the conditions with the optimal, the best coating concentration of KHV-347, confining solution, the serum dilution, Rabbit anti-koi IgM dilution and Goat anti-rabbit HRP-IgG dilution were0.5μg/mL,10%milk,1:800,1:4000and1:2000resperctively. The developed indirect-ELISA assay was high specificity, good sensitivity and easy manipulation, which provided an available technique for detection and serological survey of KHVD The anti-KHV antibody was detected in84 fish serum samples, and the results showed that the antibody positive ratio was2.38%.3The ORF59gene of virus strain KHV-347was amplified by PCR. The ORF59gene was cloned into pET-32a vector to construct recombinant expression vector, and then the vector was transformed into the host bacteria E. coli BL21. The20KD fusion protein was purified by affinity column with His-tag SDS-PAGE and Western blot showed that the fusion protein shows nice antigenicity and could be developed an. engineering subunits vaccine of KHV.