The Immunogenicity Of Cyprinid Herpesvirus ? ORF25 Protein And The Screening Of Its Receptor On Host Cells

Hematopoietic necrosis of crucian carp?Carassius auratus?is a newly emerged infectious viral disease caused by Cyprinid herpesvirus 2?CyHV-2?,which is characterized by wide-range spreading,fast transmission,high mortality rate,etc.,representing a major threat to the healthy aquaculture of crucian carp in China.Curently,the researches on the pathogenic mechanism and control methods of this disease have become a great concern.In this study,1)the partial gene sequences of 13 isolates CyHV-2 collected from different areas have been sequenced and compared,including ORF25,ORF25B,ORF25D,ORF55 and ORF72 genes;2)the epitopes,hydrophobic and hydrophilic sites were analyzed,the antigen-rich region of CyHV-2 ORF25 gene was truncated and expressed in prokaryotic expression system,and the polyclonal antibody against the expressed protein was generated.Using the indirect immunological fluorescent assay and the immune protection test,the immunogenicity of the expressed CyHV-2 ORF25 protein was invetigated;3)in Pichia pastoris,the CyHV-2 ORF25truncated gene was expressed and the immunogenicity of the expressed protein was studied by the inhibition of virus replication,the colloidal gold immunoelectron microscopy,the serum neutralization and the immune protection test;4)the receptors on gibel carp brain?GiCB?cells that the CyHV-2 ORF25 encoding protein interacted with were screened by yeast two-hybrid system.The research details are as follows:1.Gene sequence comparison of 13 isolates CyHV-2 collected from different areasThe genes of CyHV-2 collected from different areas,including ORF25,ORF25B,ORF 25D,ORF55 and ORF72,were sequenced and compaired,the results demonstrated that the genes of ORF25 and ORF72 were conserved,showing a high similarity with the same genes in the reference isolate SY-C1 of China?GenBank Access No.KM200722.1?and in the reference isolate ST-J1 of Japan?GenBank Access No.NC₀19495.1?.The ORF55 of 13 isolates CyHV-2 had same sequence compared with the reference isolate CyHV-2 SY-C1 of China.However,the ORF55 gene had a 9 base pairs deletion in 13isolates CyHV-2,compared with the gene sequence of the reference isolate CyHV-2ST-J1.In addition,the sequences of ORF25B and ORF25D of 13 isolates CyHV-2showed difference compared with the reference isolate of CyHV-2 SY-C1 of China.2.Immune response induced by the truncated CyHV-2 ORF25 protein expressed in prokaryotic expression system.Using bioinformatics techniques,the epitope,hydrophobic and hydrophilic sites were comprehensively analyzed.After that,the antigen-rich region of CyHV-2 ORF25was trucately cloned and expressed in prokaryotic expression system.The trucated recombinant protein,tORF25,had a molecular weight 28 kDa,which was the same as the predicted size.Ployclonal antibody was raised in Japanese white rabbit by immunizing the animal with purified tORF25 protein and the Western blot assay showed that this polyclonal antibody can recongnize the tORF25 protein.The indirect fluorescent assay demonstrated that this polyclonal antibody can combine with the GiCB cells infected with CyHV-2.The challenge test showed that the Relative Percentage Survival?RPS?of gibel carp immunized with the tORF25 protein reached 48%.3.Expression and characterization of CyHV-2 ORF25 trucated protein in Pichia pastorisThe CyHV-2 ORF25 truncated gene was amplified by PCR and then was cloned into yeast expression plasmid pPICZ?B,named pPICZ?B-tORF25.The recombinant plasmid was transformed into yeast strain KM71 for expression and screening.The expression of recombinant y-tORF25 truncated protein was purified and incubated with CyHV-2infected GiCB cells,and the y-tORF25 protein could specifically attach to the surface of CyHV-2 infected cells confirmed by colloidal gold immunoelectron microscopy.After incubation of the y-tORF25 protein and GiCB cells,then the GiCB cells were infected with CyHV-2.The titer of virus was TCID₅₀0 10^4.1/mL,which was significantly lower than that of the control group.4.Immune response of y-tORF25 protein in gibel carpHealthy gibel carps were immunized with 20?g of y-tORF25 protein by injection into the back muscle of the fish,and the control group received the same volume of PBS.Interleukin 11?IL-11?expression in the spleens of immunized fish peaked at day 4 and the complement component C3?C3?gene expression were significantly up-regulated at day 7 post-immunization.Specific antibodies were measured in the immunized groups and the titer reached 327.When the immunized fish were challenged with CyHV-2 by intraperitoneal injection,the Relative Percentage Survival?RPS?of the immunized fish was 75%.These results suggested that y-tORF25 had a good immunogenicity.5.Construction of yeast cDNA Library of GiCB cellThe yeast two hybrid cDNA library of GiCB cells was constructed and transfected into E coli DH5?,the library titer was 1.93×10⁶ CFU/mL.The positive colonies confirmed by PCR demonstrated that most of the inserted fragment sizes were about 1.5kb.Transforming yeast Y187 with the cDNA libtray extracted from E coli,the transfomation efficiency was 1.03×10⁶ CFU/?g and the titer of yeast cDNA library was about 1.24×10⁶CFU/mL.These results indicated that the yeast cDNA library of GiCB cells could be used to the interaction study between CyHV-2 and GiCB cells.6.Construction and identification of bait vector of CyHV-2 ORF25 encoding protein for yeast two-hybridThe specific primer pairs were designed according to the CyHV-2 ORF25 sequences and the gene was amplified by PCR.The full-length CyHV-2 ORF25 gene was cloned into yeast expression vector pGBKT7 to construct the bait vector pGBKT7-ORF25 and then the yeast Y2H Gold was transfected with plasmid pGBKT7-ORF25 to generate pGBKT7-ORF25/Y2H Gold.Self-activation and toxicity analysis showed that the pGBKT7-ORF25/Y2H Gold had white colonies on SD/-Trp and SD/-Trp/X-?-Gal plates,but none colony appeared on SD/-Leu and SD/-Trp/AbA+/X-?-Gel plates.These results indicated that the fusion protein expressed by recombinant pGBKT7-ORF25/Y2H Gold could not self-activate the report gene and was non-toxic to Y2H Gold.7.The initial screening of GiCB cell receptor of CyHV-2 ORF25 encoding proteinThe recombinant strain pGBKT7-ORF25/Y2H Gold was fused with the yeast two hybrid cDNA Library of GiCB cells.One positive strain was obtained by the highly efficient screening media and its ORF was correct with a length of 618 bp.Through homology sequences analysis in GenBank database,it was found that the sequence encoding protein of positive strain was highly homologous to the protein kinase C1receptor?Receptor of protein kinase C1,RACK1?.In summary,this study had comparatively analyzed the partial gene sequences of CyHV-2 isolated from different areas,and proved that CyHV-2 ORF25 encoding protein had good immunogenicity.Moreover,the GiCB cell receptor that CyHV-2 ORF25encoding protein interacted with had been initially screened.These results may lay a foundation for the further studies on CyHV-2 infection mechanism and the innovations of specific targeted drugs and therapeutic vaccines against CyHV-2.