title: Expression of Hepatitis C Virus Core and E2 antigenic recombinant proteins and their use for development of diagnostic assays.
abstract: BACKGROUND
  Early diagnosis of HCV infection is based on detection of antibodies against HCV proteins using recombinant viral antigens. The present study was designed to select, clone and express the antigenic regions of Core and E2 genes from local HCV-3a genotype and to utilize the antigenic recombinant proteins (Core & E2) to develop highly sensitive, specific and economical diagnostic assays for detection of HCV infection.
  
  
  METHODS
  The antigenic sites were determined within Core and E2 genes and were then cloned in pET-28a expression vector. The right orientation of the desired inserted fragments of Core and E2 were confirmed via sequencing prior to expression and were then transformed in BL21 (DE3) pLysS strains of E. coli and induced with 0.5mM Isopropyl-b-D-thiogalactopyranoside (IPTG) for the production of antigenic recombinant proteins. The produced truncated antigens were then purified by Nickel affinity chromatography and were confirmed by western blotting, immunoblotting and enzyme-linked immunosorbent assay (ELISA).
  
  
  RESULTS
  The expressed Core and E2 recombinant antigens were used to develop immunoblotting assay for the detection of anti-HCV antibodies in sera. With immunoblotting, a total of 93-HCV infected sera and 35-HCV negative individuals were tested for the presence of anti-HCV antibodies to the Core and E2 antigens. Recombinant antigen showed 100% reactivity against HCV infected sera, with no cross reactivity against HCV-negative sera. The immunoblot assay mixture of recombinant antigens (Core+E2) showed a strong reaction intensity in the test area (TA) as compared to the individual truncated Core and E2 recombinant antigens. In the in-house ELISA assay, mixed Core and E2 recombinant antigens showed 100% reactivity against a standardized panel of 150-HCV-positive sera and non reactivity against a standardized panel of 150 HCV-negative sera while also being non reactive to sera positive for other viral infections. The antigenic recombinant antigens also were tested for the 30 sera of known genotypes. The antigens did not detect antibodies to genotype-3a, but detected antibodies to all genotypes and did not discriminate them genotype wise. A panel of 175 of HCV-suspected serum samples was subjected to comparative analysis with our in-house ELISA assay and with commercial HCV screening assays. After subjecting the results to the formulas for determining the quality parameters, immunoblot assay had 100% sensitivity and specificity, while the ELISA assay had 100% sensitivity and 98.8% specificity as compared to commercially available assays.
  
  
  CONCLUSIONS
  This study indicates that a mixture of Core and E2 antigens are potentially valuable antigens and there is the possibility of developing serological assays for monitoring HCV infection.
authors: Department of Biotechnology University of Malakand Chakdara, Dir (lower) Khyber Pakhtunkhwa, Pakistan.
authors: Amjad
authors: A
authors: Ali
authors: Department of Botany University of Malakand Chakdara Dir (lower), Khyber Pakhtunkhwa, Pakistan.
authors: Muhammad
authors: M
authors: Nisar
authors: National Centre of Excellence in Molecular Biology, 87-West Canal Bank Road Thokar Niaz Baig, Lahore-53700, University of the Punjab, Lahore, Pakistan. Electronic address: Idrees.khan96@yahoo.com.
authors: Muhammad
authors: M
authors: Idrees
authors: Centre of Applied Molecular Biology, 87-West Canal Bank Road Thokar Niaz Baig, Lahore, Ministry of Science & Technology Govt. of Punjab, Pakistan.
authors: Shazia
authors: S
authors: Rafique
authors: Centre of Applied Molecular Biology, 87-West Canal Bank Road Thokar Niaz Baig, Lahore, Ministry of Science & Technology Govt. of Punjab, Pakistan.
authors: Muhammad
authors: M
authors: Iqbal
mesh_terms: D003429:Cross Reactions / Q000276:immunology; D042241:Early Diagnosis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006526:Hepatitis C / Q000175:diagnosis*; D018937:Hepatitis C Antibodies / Q000276:immunology; D018936:Hepatitis C Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D012680:Sensitivity and Specificity; D014758:Viral Core Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology
chemical_list: D018937:Hepatitis C Antibodies; D018936:Hepatitis C Antigens; D011994:Recombinant Proteins; D014758:Viral Core Proteins; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus
keywords: Core; E2; ELISA; HCV; Immunoblotting; antigens; genotype-3a; mixture