Preparation of reference protein for infectious hematopoietic necrosis virus (IHNV)

To avoid the biosecurity risk of live virus used as a reference material, the glycoprotein gene of infectious hematopoietic necrosis virus (IHNV) was expressed in Pichia expression system to prepare reference protein,then it can be used as reference material in immunological assay. The IHNV-uk glycoprotein gene (IHNVG) was amplified by RT-PCR with the specific primers designed according to the GenBank IHNV gene sequence,then the IHNV-G was cloned to pPICZαA and transformed into GS115 to make gene fusion system of the target gene and yeast. The fusion gene was induced by 1% methanol to get soluble glycoprotein, the molecular weight of protein is than 70ku in SDS-PAGE analysis.and reactinogenicity of expressed protein was determined with goat polyclonal antibodies against IHNV by Western Blot analysis. The reactinogenicity of 0.1531 mg expressed protein was comparable to 0.3125TCID₅₀ IHNV in ELISA analysis, and the expressed protein can exist stably for two months at -20 ℃. So it was proved that the expressed protein can replace IHNV as reference material. The study provides a foundation for the development of virus ELISA test kit.