Extraction and purification of the H9N2 virus nucleoprotein: A simple and practical method

Background: Avian Influenza disease annually entails a significant economic loss to the poultry industry around the world.Influenza virus is a polymorphic virus of the orthomyxoviridae family (single-stranded RNA genome), and nucleoprotein (NP) is the structural and internal protein of the virus.The aim of the work was to purify nucleoprotein for further investigations with a simple, low-cost, fast and practical method.Methods: In this study, H9N2 influenza virus was isolated in specific pathogen-free embryonated chicken eggs by allantoically inoculating 103 to 105 egg-infective doses (EID50) for 9 to 11 days, purified by 10% (W/V) polyethylene glycol (PEG) 6000 with a sucrose gradient of 60% to 30%.The influenza virus proteins were collected and prepared as fractions by preparative electrophoresis.Finally, the purified NP was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot procedures.Results: The protein analysis with SDS-PAGE and silver nitrate staining indicated that the desired samples contained purified nucleoprotein and lacked other viral proteins.The results of the investigation of lyophilized fractions containing nucleoprotein on the SDS-PAGE revealed the absence of viral RNA in nucleoprotein and its high purity.Conclusion: According to this study, purified nucleoprotein can be used to produce nucleoprotein vaccines, as well as to study structural, molecular and diagnostic and therapeutic materials.