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.