Purification of Nucleoprotein of H9N2 Avian Influenza Virus Strain by Electroelution

Abstract

Background: Avian influenza virus (AIV) belongs to the family of Orthomyxoviruses type A and causes avian influenza (AI) infectious disease. Currently, serological diagnostic techniques such as agar gel propagation (AGP), hemagglutination inhibition, and enzymelinked immunosorbent assay (ELISA) are considered as important tools for the antibodies detection against viral antigens. Due to antigenic variation in the surface of AIV glycoproteins (hemagglutinin and neuraminidase), these proteins cannot be used in serological tests. Development of assays to detect AI surface glycoproteins is problematic because a great variety of combinations of these subtypes are found in nature. The internal antigen determinants on the nucleoprotein (NP) are highly conserved within influenza viruses, making this protein more appropriate for a serological test. Objectives: In the experimental present study, an effectual method was expanded to purify NP of H9N2 AIV based on Electroelution method. Methods: AIV strain A/flash chicken/Iran/772/1998 (H9N2) was acquired from the Department of Avian Influenza Reference Laboratory, Razi Vaccine and Serum Research Institute, Iran, about 2 cc, in 2017. Nucleoprotein of AIV (H9N2) was purified by an efficient and simple modified method directly from native polyacrylamide gel electrophoresis (PAGE) according to the Electroelution method. The purified protein concentration was defined by the Lowry method, and the purified NP protein (60 KDa) was examined by Tricine-SDS-PAGE. Results: The protein concentration of the virus solution was 4.62 mg/mL by the Lowry method. The purified Nucleoprotein concentration was 0.296 mg/mL by Lowry method and the Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed only a 60-KDa protein band in the gel. Conclusions: The current technique was simple and rapid and made it possible to isolate NP from the H9N2 virus. The Nucleoprotein antigen is an appropriate candidate potential to detect antibodies against all subtypes of AIV and used as the main target antigen for the diagnosis of influenza virus due to its very high scale of sequence preserved among exist strains.

Electroelution of 31 kDa Immunogenic Protein Fraction from the Salivary Gland of Aedes aegypti and Aedes albopictus (Diptera: Culicidae)

“…The protein in the glass tube will be eluted out of the gel through the frit and will be collected on the membrane. The membrane in the electroeluter has pores of 12-15 kDa so that proteins with a molecular weight larger than the pores cannot exit the membrane [38]. The confirmation of 31 kDa is in Fig.…”

“…This tool has several advantages, a shorter time, avoids contaminants, does not require expensive reagents, and produces a good recovery rate of protein [21,38]. Protein recovery is affected by several factors such as the physicochemical properties of the protein, time and temperature, and the amount of purified protein [39].…”