title: Expression of core antigen of HCV genotype 3a and its evaluation as screening agent for HCV infection in Pakistan.
abstract: BACKGROUND
  Pakistan is facing a threat from hepatitis C infection which is increasing at an alarming rate throughout the country. More specific and sensitive screening assays are needed to timely and correctly diagnose this infection.
  
  
  METHODS
  After RNA extraction from specimen (HCV-3a), cDNA was synthesized that was used to amplify full length core gene of HCV 3a. After verification through PCR, DNA sequencing and BLAST, a properly oriented positive recombinant plasmid for core gene was digested with proper restriction enzymes to release the target gene which was then inserted downstream of GST encoding DNA in the same open reading frame at proper restriction sites in multiple cloning site of pGEX4t2 expression vector. Recombinant expression vector for each gene was transformed in E. coli BL21 (DE3) and induced with IPTG for recombinant fusion protein production that was then purified through affinity chromatography. Western blot and Enzyme Linked Immunosorbant Assay (ELISA) were used to detect immuno-reactivity of the recombinant protein.
  
  
  RESULTS
  The HCV core antigen produced in prokaryotic expression system was reactive and used to develop a screening assay. After validating the positivity (100%) and negativity (100%) of in-house anti-HCV screening assay through a standardized panel of 200 HCV positive and 200 HCV negative sera, a group of 120 serum specimens of suspected HCV infection were subjected to comparative analysis of our method with commercially available assay. The comparison confirmed that our method is more specific than the commercially available assays for HCV strains circulating in this specific geographical region of the world and could thus be used for HCV screening in Pakistan.
  
  
  CONCLUSIONS
  In this study, we devised a screening assay after successful PCR amplification, isolation, sequencing, expression and purification of core antigen of HCV genotype 3a. Our developed screening assay is more sensitive, specific and reproducible than the commercially available screening assays in Pakistan.
authors.affiliation: Division of Molecular Virology & Molecular Diagnostics, National Centre of Excellence in Molecular Biology, University of the Punjab, Pakistan. mzubairyousaf@gmail.com
authors.forename: Muhammad Z
authors.initials: MZ
authors.lastname: Yousaf
authors.forename: Muhammad
authors.initials: M
authors.lastname: Idrees
authors.forename: Zafar
authors.initials: Z
authors.lastname: Saleem
authors.forename: Irshad U
authors.initials: IU
authors.lastname: Rehman
authors.forename: Muhammad
authors.initials: M
authors.lastname: Ali
mesh_terms: D000956:Antigens, Viral / Q000235:genetics; D019411:Clinical Laboratory Techniques / Q000379:methods*; D016174:Hepacivirus / Q000145:classification / Q000235:genetics / Q000378:metabolism; D006526:Hepatitis C / Q000175:diagnosis*; D018937:Hepatitis C Antibodies / Q000097:blood*; D006801:Humans; D008403:Mass Screening / Q000379:methods*; D010154:Pakistan; D011994:Recombinant Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D014758:Viral Core Proteins / Q000235:genetics
chemical_list: D000956:Antigens, Viral; D018937:Hepatitis C Antibodies; D011994:Recombinant Proteins; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus