Hosseini Seyedeh Saeedeh, Taheri Kayvan, Hosseini Seyedeh Marzieh, Gholami Mehrdad, Kouhsari Ebrahim, Edalati Elahe, Madani Rasoul, Mohammadzadeh Rokhsare, Zahedi Bialvaei Abed, Sholeh Mohammad, Golchin Far Fariba
Department of Botany, Biotechnology and Bioinformatics, Payame Noor University, Tehran, Iran.
Department of Biology, Microbiology, Azad University, Damghan, Iran.
Med J Islam Repub Iran. 2018 Dec 21;32:128. doi: 10.14196/mjiri.32.128. eCollection 2018.
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. 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. 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. 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.
禽流感疾病每年给全球家禽业带来巨大的经济损失。流感病毒是正粘病毒科的一种多形病毒（单链RNA基因组），核蛋白（NP）是该病毒的结构蛋白和内部蛋白。这项工作的目的是用一种简单、低成本、快速且实用的方法纯化核蛋白，以便进一步研究。在本研究中，通过在无特定病原体的鸡胚中尿囊接种103至105个鸡胚感染剂量（EID50），持续9至11天，分离出H9N2流感病毒，然后用10%（W/V）聚乙二醇（PEG）6000在60%至30%的蔗糖梯度中进行纯化。收集流感病毒蛋白，并通过制备性电泳制备成组分。最后，对纯化的NP进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质印迹分析。SDS-PAGE和硝酸银染色的蛋白质分析表明，所需样品含有纯化的核蛋白，且不含其他病毒蛋白。对含有核蛋白的冻干组分进行SDS-PAGE分析的结果显示，核蛋白中不存在病毒RNA且纯度很高。根据这项研究，纯化的核蛋白可用于生产核蛋白疫苗，以及研究结构、分子、诊断和治疗材料。
