title: Immunoreactivity assessment of hepatitis C virus NS3 protease and NS5A proteins expressed in TOPO cloning system.
abstract: BACKGROUND
  Hepatitis C virus (HCV) is a major cause of acute and chronic liver disease. Numerous screening assays based on the detection of immunoresponses to HCV structural and nonstructural proteins have been designed. Various studies have demonstrated genotype-specific differences in anti-HCV antibody responses to different HCV proteins.
  
  
  METHODS
  Full-length NS3 protease and N-terminally truncated NS5A were expressed using pET TOPO 102/D system. Antigenicity of the purified recombinant proteins was assessed by immunoblotting and indirect enzyme-linked immunosorbent assay (ELISA). Furthermore, anti-HCV antibody responses to the recombinant proteins were evaluated in three prevalent genotypes in Iran.
  
  
  RESULTS
  We were able to express and purify NS5A and NS3 protease using TOPO cloning system. The HCV NS3 protease and NS5A produced in BL21 Star (DE3) was immunoreactive. Our results demonstrate that NS3 protease and NS5A have good immunoreactivity, but they are not sufficient for detecting all HCV-positive sera. No significant genotype-specific differences were detected in immunoresponses to the recombinant proteins.
  
  
  CONCLUSIONS
  In conclusion, we successfully isolated, expressed, and purified substantial amount of HCV NS3 protease and N-terminally truncated NS5A, and used them as capturing antigens in a screening ELISA assay with high sensitivity, reproducibility, and specificity. Accordingly, it is well confirmed that TOPO cloning system can be used as a dynamic system in order to express higher amount of immunoreactive viral proteins.
authors.affiliation: Department of Medical Biotechnology, Faculty of Allied Medical Sciences, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Mahsa
authors.initials: M
authors.lastname: Alaee
authors.affiliation: Department of Medical Biotechnology, Faculty of Allied Medical Sciences, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Peyman
authors.initials: P
authors.lastname: Rajabi
authors.affiliation: Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.
authors.forename: Zohreh
authors.initials: Z
authors.lastname: Sharifi
authors.affiliation: Department of Medical Biotechnology, Faculty of Allied Medical Sciences, Tehran University of Medical Sciences, Tehran, Iran. Electronic address: mfarajol@tums.ac.ir.
authors.forename: Mohammad Morad
authors.initials: MM
authors.lastname: Farajollahi
mesh_terms: D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology; D003001:Cloning, Molecular; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics*; D005838:Genotype; D016174:Hepacivirus / Q000145:classification / Q000235:genetics / Q000276:immunology; D006526:Hepatitis C / Q000276:immunology / Q000382:microbiology; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000914:Antibodies, Viral; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase