Isolation And Identification Of Infectious Hematopoietic Necrosis Virus (IHNV) And Antiserum Preparation Of IHNV Glycoprotein

In this study, a strain of virus was isolated from diseased rainbow trout in a Jilin fishing ground, No. IHNV-1008. Both reverse transcription polymerase chain reaction (RT-PCR) and the results of electron microscope showed the isolated virus was infectious hematopoietic necrosis virus (Infectious hematopoietic necrosis virus, IHNV). Further studying physicochemical properties and biological characteristics of the virus. At the same time, the establishment of IHNV prokaryotic expression system, expression of IHNV protein as immunogen, immuned mouse for antiserum preparation, and related applications. The results were as follows:The RT-PCR amplified IHNV specific bands in the isolated virus, further observation on the infected cells with electron microscopic and a large number of virus particles appearing typical bullet-shape particles with the size of (70～90) nm×(150～170) nm were observed in the cytoplasm. After the purification, the study of characteristics showed that the virus can proliferated in5cell lines including EPC and FHM. The CPE performance was cell rounding,shrinking and detaching. Growth curve experiments of virus showed the maximum infection CPE can be observed after5days and virus tite reached about106.6TCIDso/100μL. The experimental results show that the proliferation temperature of virus temperature was10℃～25℃and the optimum was15℃. The virus was sensitive to heat, the fat solvent and acid, insensitive to the alkali. SDS-PAGE electrophoresis showed the virus protein bands were same as the identical documents.The1527bp length gene of IHNV glycoprotein was cloned by RT-PCR from cell culture, and was subcloned into pCWori plasmid. Then the recombinant pCWori-G was transformed into DH5α. The recombinant protein was got by fermentation. The57kDa target protein was expressed successfully induced by1mmol/L IPTG after5hours by SDS-PAGE analysis, which was mainly in the form of inclusion bodies. The soluble glycoprotein has been got after purification and renaturation processing. Western-blot analysis indicated that the recombinant protein can be identified specifically by the goat anti-IHNV serum. Using the recombinant protein to immunize mice to produce glycoprotein antiserum. The prepared antiserum reacted specifically with IHNV by indirect ELISA test. In this study, the IHNV glycoprotein was expressed by prokaryotic expression system and polyclonal antiserum was got by immunized mice. This may set up a foundation for the immunological detection methods of IHNV.