PREPARATION OF POLYCLONAL ANTIBODIES TO CYHV-2 ORF66 TRUNCATED PROTEIN AND SCREENING OF INTERCALATING PEPTIDES

Cyprinid herpesvirus 2 (CyHV-2) causes herpesviral haematopoietic necrosis, which is a serious threat to the health of the crucian carp farming industry. ORF66 is an immunogenic capsid protein of CyHV-2. To investigate the biological function of cyprinid herpesvirus 2 during infection, a prokaryotic expression plasmid pET28a-tORF66 with ORF66 truncated gene was constructed based on a region with abundant antigenic table, transformed into BL21 receptor cells and then induced to express the protein using IPTG (Isopropyl-beta-D-thiogalactopyranoside) at 16℃. The lysed recombinant protein was obtained by urea lysis dialysis and then immunized in 6-week-old mice to prepare a murine anti-tORF66 polyclonal antibody. The solubilised recombinant proteins were screened for intercalating peptides by phage display techniques. The Western Blot assay showed that the antibody was able to recognize CyHV-2 in infected GICF cells with high potency and good specificity. The results of the phage elution showed by bioinformatics analysis that a peptide with the highest frequency of occurrence, N′-LHLHQNRMSLSR-C′, was obtained. The polypeptide has high homology with three genes in the goldfish genome, including the leukotriene B₄ receptor 1 (BLT1) gene, which has six consecutive amino acid repeats with the polypeptide, so it is inferred that the polypeptide may interact with the rORF66 recombinant protein. This will provide a new basis for an in-depth investigation of the biological function of ORF66 during CyHV-2 virus infection, the development of new anti-CyHV-2 virus drugs and the search for potential drug targets.