The Immunogenicity Of Cyprinid Herpesvirus 2 ORF25B And Preliminary Screening Of Its Receptor Protein On Host Cells

The hemopoietic necrosis disease of gibel carp infected by Cyprinid herpesvirus 2(CyHV-2)has caused enormous economic losses to the farming industry of this fish.It has a serious threat to the healthy development of crucian carp breeding industry because of a wide range of prevalence,rapid spread and high mortality.At present,the research on the mechanism and control measures of the disease has been paid closely attention.In this paper,1)the coding gene and its 3’ non-coding region gene of 20 isolates CyHV-2 ORF25B collected from gibel carp in Jiangsu and Hubei Province have been sequenced and compared;2)The bioinformatics software of TMHMM Server v.2.0,SignaIP 5.0 and DNA Star 7.0(Protean)were used to analyze CyHV-2 ORF25B gene,and truncated the antigen epitopes rich region of CyHV-2 ORF25B encoding protein,and then expressed it in pET32a(+)system,and prepared the polyclonal antibody anti-CyHV-2 ORF25B.The immunogenicity of CyHV-2 ORF25B protein was testified by immunoprotection assay;3)Using baculovirus expression vector system expressed the truncated gene of CyHV-2 ORF25B.And the titer of the virus was determined after transfection the plasmid of Bacmid-JD25B into SF9 cells.Using SDS-PAGE and Western blot assay,the protein expression of recombinant baculovirus was researched,and finally its immunogenicity of the recombinant baculovirus was studied by immunoprotection experiments;4)The receptor protein of CyHV-2 ORF25B interacting with gibel carp brain(GiCB)was screened by yeast two hybrid system.The research contents details are as follows:1.Gene sequence comparison of the coding gene and its 3’ non-coding region gene of 20 isolates CyHV-2 ORF25B collected from Jiangsu Province and Hubei ProvinceThe gene sequences of 20 strains CyHV-2 ORF25B coding region from Crucian carp farms in Jiangsu Province and Hubei Province were highly similar to those of SY-C1 and YZ-01 strains,with only a little gene deletion by gene sequence comparison.And their similarity of amino acid sequences was 95.4-100%.The comparison of 3’non-coding region gene sequences of ORF25B,the similarity of the three isolates was 99.1-100%with ST-J1 strain and 67.6-71.3%with the others.The sequence similarity of the remaining 17 isolates with SY-C1,YZ-01,1301 and SY strains ranged from 97.2%to 100%.Based on the comparative analysis of CyHV-2 ORF25B coding gene and its 3’ non-coding region gene,SY-C1 strain and YZ-01 strain were the main pathogenic strains caused CyHV-2 in Jiangsu and Hubei Province.2.Immunogenicity induced by prokaryotic expression of CyHV-2 ORF25B proteinThe epitope rich region of CyHV-2 ORF25B was selected by bioinformatics software,the recombinant protein pET-JDORF25B expressed by prokaryotic expression system was about 53 kDa,which result was expected.The recombinant protein pET-JDORF25B was purified and imm unized rabbits for several times.And the polyclonal antibody anti-CyHV-2 ORF25B was obtained.The results of Western blot testified that the antibody could specifically identify the protein of pET-JDORF25B.The indirect immunofluorescence assay indicated that GiCB cells inoculated with CyHV-2 could be specifically combined with the polyclonal antibody.It is demonstrated that the relative survival rate of Carassius auratus immunized with purified recombinant protein pET-JDORF25B was 38%by the challenge test.3.Expression and characterization of CyHV-2 ORF25 trucated protein in Baculovirus expression vector systemThe CyHV-2 extracted from the positive tissues of crucian carp was amplified by the specific primers.Then cloned it into the baculovirus expression vector pFastBacl and transformed the plasmid into E.coli DH10Bac,and constructed the recombinant shuttle plasmid Bacmid-JD25B.Then Bacmid-JD25B was transferred into SF9 cells with the help of liposome CellfectionTM Ⅱ.And the P1 generation recombinant baculovirus with high titer was obtained.After the virus was inoculated up to P3 generation,the TCID50 of the virus was 10-7.04/0.1 ml.After expanded culture of the virus,the protein molecular weight was used to analyze by SDS-PAGE is 36 kDa,and Ni column was used for affinity purification to obtain a single band,which indicated that the purification is successful.The polyclonal antibody pET-JDORF25B was incubated with Bacmid-JD25B expressed by baculovirus expression vector system was tested by Western blot.At last,the results showed that the target protein was exactly 36 kDa,which indicated that the recombinant protein was successfully expressed in SF9 cells and it could be specifically attached to the polyclonal antibody.After immunizing crucian carp with purified Bacmid-JD25B recombinant protein,the relative survival rate of CyHV-2 was 54%,the results showed that Bacmid-JD25B recombinant protein expressed by baculovirus expression vector system had good immunogenicity,and it was higher than the relative survival rate(38%)of pET-JDORF25B recombinant protein expressed by prokaryotic expression system.4.polymorphism detection of yeast cDNA Library of GiCB cellThe yeast two hybrid GiCB cDNA library preserved by laboratory was recovered,and its titer is about 1.92 × 106 CFU/ml.Single colony was randomly selected and extracted yeast plasmid from it to amplify by PCR.As it revealed that the inserted fragment sizes were staggered up and down,and most of them were more than 1.0 KB,which demonstrated that the library had good polymorphism and could be used in the next two hybrid experiment.5.Construction of a bait vector and preliminary screening of host proteins interacting with Cyprinid herpesvirus 2 ORF25BUsing PCR technology to amplify the CyHV-2 extracted from the positive tissues of crucian carp.and the amplified product was inserted into bait vector pGBKT7.Then it was successfully constructed the bait recombinant plasmid pGBKT7-tORF25B and transformed it into yeast Y2H Gold.It was suggested that the bait strain pGBKT7-tORF25B/Y2H Gold was obtained.Using the auxotroph medium,the transcriptional self-activation and toxicity of the bait plasmid were tested,and the bait plasmid was shown to have no toxic effect on yeast Y2H Gold and no self-activation phenomenon.The bait strain pGBKT7-tORF25B/Y2H Gold was hybridized with GiCB cDNA library used by yeast two hybrid technique.There were four candidate interacted host proteins with tORF25B gene were preliminarily obtained.In summary,this study had sequenced and compared the coding gene and its 3’non-coding region gene of 20 isolates CyHV-2 collected from Jiangsu Province and Hubei Province,and it was certified that CyHV-2 ORF25B gene had satisfactory immunogenicity.Furthermore,The GiCB receptor protein that CyHV-2 ORF25B interacting with were preliminarily obtained.The results of this paper have laid an important theoretical support for further study on the protein function of CyHV-2 and the mechanism of this virus invasion into host cells,and also the innovations of novel drugs and subunit vaccines against CyHV-2.