Author/Authors :
Hosseini, Saeedeh Department of Botany - Biotechnology and Bioinformatics - Payame Noor University, Tehran, Iran , Taheri, Kayvan Department of Biology - Microbiology Azad University, Damghan, Iran , Hosseini, Marzieh Department of Biology - Microbiology Azad University, Karaj, Iran , Gholami, Mehrdad Department of Microbiology and Virology - Faculty of Medicine - Mazandaran University of Medical Sciences, Sari, Iran , Kouhsari, Ebrahim Department of Microbiology - School of Medicine - Iran University of Medical Sciences, Tehran, Iran , Edalati, Elahe Department of Botany - Biotechnology and Bioinformatics - Payame Noor University, Tehran, Iran , Madani, Rasoul Department of Botany - Biotechnology and Bioinformatics - Payame Noor University, Tehran, Iran , Mohammadzadeh, Rokhsare Department of Microbiology - School of Medicine - Iran University of Medical Sciences, Tehran, Iran , Zahedi Bialvaei, Abed Department of Microbiology - School of Medicine - Iran University of Medical Sciences, Tehran, Iran , Sholeh, Mohammad Department of Microbiology - School of Medicine - Iran University of Medical Sciences, Tehran, Iran , Golchin Far, Fariba Department of Botany - Biotechnology and Bioinformatics - Payame Noor University, Tehran, Iran
Abstract :
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.
Keywords :
Purification , Extraction , Nucleoprotein , H9N2 , Avian influenza virus
