Anti-HCV envelope prevalence in blood donors from Baden-Württemberg

Cardoso, M. da Silva; Sturm, Daniela; Koerner, K.; Dengler, T.; Kerowgan, M.; Kubanek, B

doi:10.1007/s002770050271

Cited by 5 publications

(1 citation statement)

References 6 publications

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“…In several studies HCV core antigen based ELISA has been used as a screening method for the identification of HCV infection in human sera [29-31]. No screening method has been developed so far in Pakistan based on HCV types and isolates exist in this region and the sensitivity and specificity of the commercially available methods may be low for HCV strains common in Pakistan.…”

Section: Discussionmentioning

confidence: 99%

Expression of core antigen of HCV genotype 3a and its evaluation as screening agent for HCV infection in Pakistan

Yousaf

Idrees

Saleem

et al. 2011

Virol J

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BackgroundPakistan 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.MethodsAfter 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.ResultsThe 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.ConclusionIn 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.

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Section: Discussionmentioning

confidence: 99%

Expression of core antigen of HCV genotype 3a and its evaluation as screening agent for HCV infection in Pakistan

Yousaf

Idrees

Saleem

et al. 2011

Virol J

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Isolation and characterization of human monoclonal antibodies against hepatitis C virus envelope glycoproteins

et al. 1998

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The isolation and characterization of human monoclonal antibodies (humAbs) against the hepatitis C Virus (HCV) glycoproteins E1 and E2 are described. B-cells from blood donors with anti-HCV were transformed with Epstein-Barr virus. The supernatants of the resulting lymphoblastoid clones were screened by ELISA with an extract of cells infected with a recombinant vaccinia virus RMPA95 expressing the envelope proteins E1 and E2 of an HCV genotype 1a virus (H strain). Positive clones were fused to the heteromyeloma cell line K6H6/B5. Fifteen heterohybridoma cell lines have been established. The specificity of the isolated humAbs was determined both by ELISA and Western blot assays. Several recombinant extracts expressing either the E1 or E2 protein or truncated forms were used in an attempt to map the epitopes on the viral glycoproteins. Some of the humAbs were used successfully for immunofluorescence investigation of transfected cells. Seven specific anti-E2 humAbs, which react with the envelope protein 2 of genotype 1a and 1b isolates, were characterized.

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et al. 2002

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High-level expression of hepatitis C virus (HCV) E2 protein fragments encompassing its C-terminal hydrophobic region (downstream of aa 661) in Escherichia coli has been proven difficult. The extreme hydrophobicity of this region has been suspected to be detrimental to the host. In this work, we found that the C-terminal region (downstream of aa 565) of E2 ectodomain interfered with full-length expression of E2 fragments in E. coli. Nonetheless, when the central region (aa 484-622) of E2 was deleted, full-length protein was efficiently produced. C-terminal region aa 567-700 could not be efficiently expressed individually or as mouse dihydrofolate reductase (DHFR) fusion protein. However, a mutant that emerged in the cloning process was able to express full-length DHFR fusion protein. Sequencing analysis reveals the mutation to be a short frame-shift around the fusion junction, altering aa 568-571 of E2. C-terminal region of E2 ectodomain (aa 567-730) carrying this mutation was successfully expressed as hexa-histidine-tagged protein to a high level. The protein was highly insoluble and was purified under denaturing conditions. The purified protein displayed HCV E2-specific antigenicity in Western blot and specific rabbit antiserum was raised against it. These results demonstrate that hydrophobicity of the C-terminal region of E2 ectodomain is not harmful to E. coli host and has no dominative adverse effect on its bacterial expression. Other nucleotide and/or amino acid sequence properties seem to play a more important role. This finding opens up new possibilities for the development of novel bacterially-derived E2 proteins for research and clinical applications.

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Anti-HCV envelope prevalence in blood donors from Baden-Württemberg

Cited by 5 publications

References 6 publications

Expression of core antigen of HCV genotype 3a and its evaluation as screening agent for HCV infection in Pakistan

Expression of core antigen of HCV genotype 3a and its evaluation as screening agent for HCV infection in Pakistan

Isolation and characterization of human monoclonal antibodies against hepatitis C virus envelope glycoproteins

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