title: Cell culture extraction and purification of rabies virus nucleoprotein.
abstract: BACKGROUND
  Rabies is a major zoonotic viral disease and is detected using the World Health Organization standard diagnostic techniques. Rabies detection is preferably done using the fluorescent antibody technique (FAT) that provides reliable diagnosis with almost 100% accuracy for all variant strains, if a proper conjugate is used. Rabies virus nucleoprotein (NP) is the most important protein used in production of a specific diagnostic conjugate.
  
  
  OBJECTIVE
  The aim of this study was to extract the cell-associated rabies virus NP from infected Baby Hamster Kidney cell clone (BSR) with rabies virus (Pasteur vaccine strain/PV) and purify for a future project to produce an anti-NP conjugate.
  
  
  METHODS
  Pasteur vaccine strain (PV) as the standard rabies vaccine strain with a focus-forming dose (FFD) of 105 was inoculated in to the BSR cell culture at a concentration of 10(6) cells per milliliter. Infected cells were harvested 72 hours after infection and the rabies NP was extracted from these cells by low-speed centrifugation and purification by ultracentrifugation in cesium chloride (CsCl) gradient. For analysis, the purified NP was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
  
  
  RESULTS
  The volume of the lysate was 15 mL and it became 2.5 mL after purification, with a concentration of 3.25 mg/mL. The corresponding band to the cell lysate protein on the SDS-PAGE had a molecular weight of 50 KDa, similar to the molecular weight of NP in rabies virus.
  
  
  CONCLUSIONS
  The rabies virus NP could be extracted and purified in an appropriate amount from infected cell culture. The results of SDS-PAGE analysis showed that the intact rabies virus NP had been purified properly and thus could be used for further steps to produce the specific diagnostic rabies conjugate.
authors.affiliation: Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, IR Iran.
authors.forename: Mahshid
authors.initials: M
authors.lastname: Dastkhosh
authors.affiliation: Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Pooneh
authors.initials: P
authors.lastname: Rahimi
authors.affiliation: Department of Microbiology, Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, IR Iran.
authors.forename: Setareh
authors.initials: S
authors.lastname: Haghighat
authors.affiliation: Influenza Research Laboratory, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Peyvand
authors.initials: P
authors.lastname: Biglari
authors.affiliation: WHO Collaborating Center for Reference and Research on Rabies, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Nader
authors.initials: N
authors.lastname: Howaizi
authors.affiliation: Department of Biochemistry, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Reza
authors.initials: R
authors.lastname: Saghiri
authors.affiliation: Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, IR Iran.
authors.forename: Akram
authors.initials: A
authors.lastname: Roohandeh
keywords: Diagnosis; Fluorescent Antibody Technique; Isolation and Purification; Nucleoproteins; Rabies Virus