Truncationand Expression Of IHNV Glycoprotein And Detection Of Immunogenicity

Infectious Hematopoietic Necrosis(IHN)is an acute viral infectious disease which is commonly occurs in salmon.The disease is caused by infectious hematopoietic necrosis virus(IHNV),and often cause 50%-100%mortality of larvae and fry.The surface protein of IHNV is a glycoprotein referred as G protein.G protein plays a critical role in processes of recognition and binding of the virus to the cell receptor,and it is also related with virulence of IHNV.Additionally,G protein,containing the protective epitopes,is the major antigen of the virus,can induce specific neutralizing antibodies.Therefore,preparation of G protein and its antibody is very important for the IHNV diagnosis,G protein functional research and vaccine design.Genotype of IHNV isolates in China is different from that of IHNV isolates from Europe and America.Different genotype may results in different immunogenicity of IHNV glycoprotein.Therefore,it is necessary to establish immunological method for identification of Chinese IHNV isolates.Previous studies indicated that there was cross-reactivity between antibodies against IHNV full-length glycoprotein with viral haemorrhagic septicemia virus(VHSV).To improve the specificity of the immunological method,IHNV glycoprotein was truncated based on bioinformatics analysis and expressed in E.coli.Purified truncated G was used to prepare polyclonal antibodies.This study aimed to establish an immunological detection method with high specificity for Chinese IHNV isolates.In order to study the immunogenicity of the G protein,this paper analyzed transmembrane domain,hydrophobicity and antigenic determinants of G protein using relati software,and after which the truncation express of G protein was made.Polyacrylamide gel electrophoresis(PAGE)showed that the truncated glycoprotein Gs size was 40kDa.After purified,the protein was analysised by high performance liquidChromatography(HPLC)system,and the purity of Gs protein reached 90%.The rabbit antiserum was also made by the purified Gs protein,and the antisera against glycoprotein prepared in this study could react specifically with both natural glycoprotein of the IHNV-Sn and the recombinant truncated glycoprotein in ELISA test,and the titer against the natural glycoprotein was 1:20 000,the recombinant glycoprotein was 1:80000.IFA result showed that the antisera could recognize the glycoprotein on the surface of IHNV-Sn and IHNV reference strain.These results indicated that the antigenicity of truncated glycoprotein was as good as that of natural glycoprotein of IHNV.This study lays a foundation for the immunofluorescence assay and DNA vaccine study of IHNV.