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Publications (2)4.53 Total impact

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    ABSTRACT: Escherichia coli-derived hepatitis B e antigen (HBeAg) is widely used for serological tests of hepatitis B virus (HBV). Because it exhibits cross-reactivity with HBcAb in human sera, current antibody to HBeAg (HBeAb) immunoassays are based on competitive inhibition enzyme-linked immunosorbent assay (ELISA) rather than sandwich ELISA, which interfere with the specificity and sensitivity of HBeAb detection. Pichia pastoris has advantages of eukaryotic cells, while having the capacity of high-level secretion of foreign proteins. To explore the diagnostic suitability of recombinant HBeAg (rHBeAg), we expressed the wild type HBV e gene (wt-e-gene) and the synthetic HBV e gene (syn-e-gene; native HBV e gene modified based on synonymous codon usage bias) in P. pastoris. The recombinant antigen was secreted into the medium. The expression level of rHBeAg was enhanced by optimizing HBV e gene. The yield of syn-e-gene product was approximately five-fold greater than wt-e-gene. The protein represented 66% of the total supernatant protein, and was simply purified to 90%. P. pastoris-derived HBeAg showed high HBe antigenicity, while lacking any HBc antigenicity and cross-reactivity between all proteins derived from the culture of P. pastoris and normal human sera. P. pastoris-derived HBeAg has higher specificity and sensitivity for detection HBeAb in the diagnostic assay than the commercial HBeAb ELISA kits.
    Journal of Biotechnology 08/2008; 138(1-2):1-8. · 3.18 Impact Factor
  • Bo Hu, Guoqiang Hong, Zhaoxia Li, Jue Xu, Zhenyu Zhu, Lin Li
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    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.
    Biotechnology and Applied Biochemistry 06/2007; 47(Pt 1):59-69. · 1.35 Impact Factor