Expression of VCA (viral capsid antigen) and EBNA1 (Epstein-Barr-virus-encoded nuclear antigen 1) genes of Epstein-Barr virus in Pichia pastoris and application of the products in a screening test for patients with nasopharyngeal carcinoma.
ABSTRACT EBV (Epstein-Barr virus) serological tests have been used for many years as accessory diagnostic predictors of NPC (nasopharyngeal carcinoma). To date, IF (indirect immunofluorescence) assays still serve as the 'gold standard' for EBV serodiagnosis. However, IF assays are time-consuming, unsuitable for automatic handling and difficult to standardize. This makes their application in mass screening of populations inconvenient. Some of the technical difficulties associated with IF have been overcome by the development of specific ELISAs, but, at present, high sensitivity and specificity cannot be achieved simultaneously by using recombinant protein-based ELISAs, as the diagnostic value of different fragments of EBV in NPC is different. In an attempt to determine a suitable recombinant EBV protein for diagnostic purposes, fragments of EBV VCA (viral capsid antigen) and EBNA1 (Epstein-Barr-virus-encoded nuclear antigen 1) genes were expressed in the methylotrophic yeast Pichia pastoris, and a novel ELISA was established using P. pastoris-expressed VCA-BALF4 [aa (amino acids) 287-623; the BALF4 gene encodes the EBV glycoprotein gp125], EBNA1 (aa 390-641) and VCA-BFRF3 (the gene BFRF3 encodes a viral structural capsid protein or tegument protein VCA p18) proteins. Serum samples were collected from patients with NPC and healthy controls and were tested using this ELISA. The sensitivity of VCA-BFRF3, VCA-BALF4 and EBNA1 tests in the NPC sera were 65.0 (195/300), 76.3 (229/300) and 81.4% (244/300) respectively, whereas the specificity of normal individuals were 92 (460/500), 96 (480/500) and 95.8% (479/500). The optimum combination is VCA-BALF4 plus EBNA1, which identified 90.3% (271/300) of the NPC patients and had a specificity of 92.8% (464/500) for normal individuals. The results obtained from the evaluation of three antibodies to EBV as markers for detecting NPC suggests that a combination of EBNA1 (aa 390-641) and VCA-BALF4 (aa 287-623) assays would give better results in screening for NPC.
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ABSTRACT: The study reports heterologous expression in Pichia pastoris of active neuraminidase derived from avian influenza virus A/Viet Nam/DT-036/2005(H5N1). A gene encoding the neuraminidase N1 head domain (residues 63-449) was fused directly in-frame with the Saccharomyces cerevisiae alpha-factor secretion signal in pPICZ(A vector. Recombinant N1 neuraminidase was expressed in P. pastoris as a 72kDa secreted, soluble protein. Glycopeptidase F treatment generated a 45kDa product, indicating that the secreted recombinant N1 neuraminidase is an N-linked glycoprotein. Kinetic studies and inhibition tests with oseltamivir carboxylate demonstrated that the recombinant N1 neuraminidase has similar K(m) and K(i) values to those of the viral N1 neuraminidase. This yeast-based heterologous expression system provided functionally active recombinant N1 neuraminidase that should be useful in anti-influenza drug screening, and also as a potential protein-based vaccine.Journal of Virological Methods 12/2008; 156(1-2):44-51. · 1.90 Impact Factor
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ABSTRACT: Current single Epstein-Barr virus (EBV) markers fail to reach 100% sensitivity for serodiagnosis of acute and malignant diseases associated with EBV infection. Previous study had identified immunodominant epitopes of VCA-p40 and VCA-p18, and indicated that these two VCA antigens may have diagnostic value for EBV-related diseases. A recombinant protein of the full-length BdRF1 fused to the immunodominant domain of BFRF3 as 6-his tagged protein in Escherichia coli was developed. The recombinant protein was extracted in 8M urea solution and purified by metal-affinity chromatography yielding a 55 kDa product (VCA-p40+18). VCA-p40+18 blot-strips examined for IgM reactivity in infectious mononucleosis samples yielded 100% sensitivity and specificity, with improved reactivity compared with IgM/VCA-p18-ELISAs. A recent study described a synthetic peptide-based IgA/[EBNA1+VCA-p18]-ELISA (IgA/EBV-ELISA), with a sensitivity of 90% for diagnosing nasopharyngeal carcinoma. Immunoblot analysis of biopsy-confirmed nasopharyngeal carcinoma cases with low or negative IgA/EBV-ELISA showed 100% IgG reactivity to VCA-p40 and VCA-p18 proteins. Evaluation of VCA-p40+18 as an additional marker for screening and diagnosis of nasopharyngeal carcinoma was carried out. The data showed positive IgA/VCA-p40+18 reactivity by ELISA for 63.6% (14 of 22) nasopharyngeal carcinoma samples that were missed by peptide-based IgA/EBV-ELISA, suggested VCA-p40+18 as an improved marker for nasopharyngeal carcinoma serodiagnosis. The VCA-p40+18 may be combined with an EBNA1 synthetic peptide as an antigen mixture in one or separate IgA ELISA for improved nasopharyngeal carcinoma serodiagnosis.Journal of virological methods 10/2010; 169(1):79-86. · 2.13 Impact Factor
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ABSTRACT: Serological tests for Epstein-Barr virus (EBV) have been used for many years as diagnostic predictors of nasopharyngeal carcinoma. It has been shown previously that the conventional immunofluorescence assay has a limited diagnostic value, especially in young patients from North African area. In the search for more reliable immunoglobulin (Ig) G or IgA antibody markers for the diagnosis of nasopharyngeal carcinoma, immunoblot analysis was performed using a full spectrum of EBV proteins. Sera were collected from 108 patients with nasopharyngeal carcinoma and three control groups composed of 18 patients with lymphoma, 18 other patients with autoimmune diseases and 55 healthy EBV carriers. It was observed that the IgA Epstein-Barr nuclear antigen 1 (EBNA1), IgA early antigen (EA)-p138 and IgG EA-p138 antibodies represent the most specific anti-EBV responses in either young or older patients with nasopharyngeal carcinoma which yield higher positive rates compared to the three control groups. Since the IgA EBNA1 response showed the highest sensitivity value for the detection of nasopharyngeal carcinoma, a novel enzyme-linked immunosorbent assay (ELISA) was established using a GST-EBNA1 protein expressed in bacteria, containing the P-threonine EBNA1 subtype cloned from DNA EBV sequence of C15 xenograft cells. Detection rates were 85.7% and 94.9% in young and older patients with nasopharyngeal carcinoma respectively, while only 3.6%, 11.1%, and 16.6% in healthy EBV carriers, patients with lymphoma and patients with autoimmune diseases, respectively. Thus, IgA EBNA1 ELISA may be useful for early diagnosis and mass screening of nasopharyngeal carcinoma in Tunisia even in young patients.Journal of Medical Virology 07/2009; 81(8):1412-21. · 2.37 Impact Factor