Cloning,Expression And Identification Of Immunogenicity ORF5 Truncation Genes Of Cyprinid Herpesvirus 2

Carp herpes virus disease is the only viral diseases that be found to infection allogynogenetic crucian carp at present.the pathogen of the disease is the carp herpesvirus type 2(CyHV-2).CyHV-2 named goldfish hematopoietic organ necrosis virus(GFHNV)and herpes hematopoietic organ necrosis virus(HVHN V),mainly infected goldfish.common carp and its variants.CyHV-2 was found in Japan for the first time,which led mass mortality of the goldfish.caused great damage to the Japanese goldfish breeding industry.With the increasing of international trade,currently the disease has spread widely in various areas of the world,which has drawn a great attention of various countries.This research mainly for CyHV-2 ORF5 truncation gene(registration number:AFJ20457.1)bioinformatics analysis,and carries on the gene cloning,construct recombinant prokaryotic expression plasmid.in vitro induced expression of the recombinant plasmid and protein purification,animal immune experiment,testing its immunogenicity.in order to provide references for further research of CyHV-2.1、Bioinformatics analysis of CyHV-2 ORF5 truncation geneOne pair of primers were designed and synthesized according to the ORF5 truncation gene of CyHV-2 nucleic acid sequence(AFJ20457.1)published in the GenBank.The ORF5 truncated gene of CyHV-2 was amplified by PCR from viral DNA.then cloning and sequence.The result showed the full length sequence of ORF5 truncated gene was 357bp.which encoded 119 amino acids consisiting of 9 strongly basic amino acids(K.R).16 strong acidic amino acid(D,E).42 of the hydrophobic amino acids(A,I,L,F,W,V)and 38 polar amino acid(N,C,Q,S,T,Y),The relative molecular mass was 13059.70u,having no signal peptide and transmembrane region.having 4 epitopes.The biggest hydrophobic index of the recombinant protein was 1.578,the smallest hydrophobic index was-2.500,the hydrophilic amino acids were even distribution and the number of hydrophilic amino acid was greater than hydrophobic amino acids.2、Construction of the prokaryotic expression vector and Optimizing the recombinant protein expressionThe expression vector pET32a-ORF5 was constructed,after PCR amplification and double enzyme identification.transformed into E.coli BL21 cells.By the detection of SDS-PAGE,the recombinant protein was 33 kD.agreed with expected size,and formed an insoluble inclusion body,and the expression optimal condition was 0.4 mM IPTG at 37℃for 4 h.3、Immunogenicity study of recombinant proteinRenaturating and purificating the protein,and determinating the concentration was 0.534 mg/mL.The protein was used to immune mices to preparation of recombinant protein from serum.The Western blot showed that recombinant protein had good specificity.Allogynogenetic crucian carp were divided into four groups and were injected physiological saline,10 μg protein.25 μg protein and 50 μg protein respectively.The blood was collected on 0 day.7 days.14 days.21 days and 28 days.By ELISA method to analysis the immune response in carp,the result showed that CyHV-2 specific antibody can be detected in the blood of carp,the different antibody levels can be detected by injecting different dose proteins,but significant difference was found in antibody levels compared with controls(p<0.05).Poison attack on crucian carp protective test showed that injection of saline group protection for 0.injection of 10 μg protein group of protection for 20%,injection of 25 μg protein group protection for 30%,injection of 50 μg protein group protection ratio of 35%.In conclusion,ORF5 truncation membrane proteins immunogenic,has certain protective effect for the allogynogenetic crucian carp,but low recovery rates.