| Infectious hematopoietic necrosis is the most important rhabdoviral disease of salmon and trout caused by infectious hematopoietic necrosis virus (IHNV). Outbreaks of IHN result in losses approaching 100%, depending on the species and size of the fish, the virus strain and environmental conditions. So far the IHNV has spread into many countries of America, Europe, Australia and Asia. Since the 1990s, the main producing areas of rainbow trout in China were threatened by IHN outbreaks resulted inasharp decline in rainbow trout production. Recently, IHN becomes the main problem which hinderes the healthy and sustainable development of China's cold-water fish farming industry. Therefore, diagnosis and treatment of the IHN cause the attention of the mankind.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.This research used one-step RT-PCR method to amplify the truncated glycoprotein (375 bp) from genome RNA extract of IHNV-Snl203 isolated (GenBank accession no. KC660147). The truncated glycoprotein was inserted into plasmid pET-27b to construct recombinant plasmid pET-27b-short G. The plasmid was transformed into E. coli (Rosetta) and expressed. The transformed E. coli was cultured for 4 h by inducing with 1 mM IPTG. SDS-PAGE showed that target protein, with suspect molecular weight of 13 kDa, was highly expressed as inclusion body. After dissolution of inclusion bodies in buffer containing 8 M Urea and 2 M Urea, high purity protein was achieved. The purified protein was used for rabbit polyclonal antibody preparation. The rabbit polyclonal antibody was analyzed in ELISA assay and immunofluorescence test. The titers of the rabbit polyclonal antisera with recombinant short G and IHNV-Sn isolate were 1:80 000 and 1:40 000, respectively. This result showed that the rabbit polyclonal antibody could recognize both the short G and natural glycoprotein of IHNV isolate, which indicated that the expressed short G could efficiently fold and had good immunogenicity approximately similar with surface glycoprotein of IHNV. The result of immunofluorescence test indicated that the rabbit polyclonal antibody can specifically recognize the IHNV-Sn1203 isolate without cross-reaction with VHSV.Collectively, the expressed truncated IHNV glycoprotein possesses the satisfied immunogenicity and immunoreactivity, and polyclonal antibodies against the truncated glycoprotein cannot cross-react with VHSV in immunological detection methods. It enhanced the specificity of the IHNV immunodetection method and made the IHNV immunodetection method better. This study paves a material basis and theoretical reference for the detection of the current IHNV in China. It also provides theory guidance for the development of subunit vaccine of IHNV. |
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