Optimization of the expression of hepatitis B virus e gene in Pichia pastoris and immunological characterization of the product.
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.
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ABSTRACT: Interleukin (IL)-25 (also known as IL-17E) is a distinct member of the IL-17 cytokine family which induces IL-4, IL-5, and IL-13 expression and promotes pathogenic T helper (Th)-2 cell responses in various organs. IL-25 has been shown to have crucial role between innate and adaptive immunity and also a key component of the protection of gastrointestinal helminthes. In this study, to produce bioactive recombinant human IL-25 (rhIL-25), the cDNA of mature IL-25 was performed codon optimization based on methylotropic yeast Pichia pastoris codon bias and cloned into the expression vector pPICZαA. The recombinant vector was transformed into P. pichia strain X-33 and selected by zeocin resistance. Benchtop fermentation and simple purification strategy were established to purify the rhIL-25 with about 17 kDa molecular mass. Functional analysis showed that purified rhIL-25 specifically bond to receptor IL-17BR and induce G-CSF production in vitro. Further annexin V-FITC/PI staining assay indicated that rhIL-25 induced apoptosis in two breast cancer cells, MDA-MB-231 and HBL-100. This study provides a new strategy for the large-scale production of bioactive IL-25 for biological and therapeutic applications.Applied Microbiology and Biotechnology 10/2013; · 3.81 Impact Factor
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ABSTRACT: Coxsackievirus and adenovirus receptors (CARs) are the common cellular receptors which mediate coxsackievirus or adenovirus infection. Receptor trap therapy, which uses soluble viral receptors to block the attachment and internalization of virus, has been developed for the inhibition of virus infection. In this study, we have constructed a pPIC3.5K/CAR-Fc expression plasmid for the economical and scale-up production of CAR-Fc fusion protein in Pichia pastoris. The coding sequence of the fusion protein was optimized according to the host codon usage bias. The amount of the CAR-Fc protein to total cell protein was up to 10% by 1% methanol induction for 96h and the purity was up to 96% after protein purification. Next, the virus pull-down assay demonstrated the binding activity of the CAR-Fc to coxsackievirus. The analyses of MTT assay, immunofluorescence staining and quantitative real-time PCR after virus neutralization assay revealed that CAR-Fc could significantly block coxsackievirus B3 infection in vitro. In coxsackievirus B3 infected mouse models, CAR-Fc treatment reduced mortality, myocardial edema, viral loads and inflammation, suggesting the significant virus blocking effect in vivo. Our results indicated that the Pichia pastoris expression system could be used to produce large quantities of bioactive CAR-Fc for further clinical purpose.Journal of Biotechnology 01/2013; · 3.18 Impact Factor
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ABSTRACT: Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus associated with the development of both lymphoid and epithelial tumors. EBNA1 is the only viral protein expressed in all EBV-associated malignancies and plays important roles in EBV latency. Thus, EBNA1 is thought to be a promising antigen for immunotherapy of all EBV-associated malignancies. This study was undertaken to produce recombinant EBNA1 protein in Pichia pastoris and evaluate its immunogenicity. The truncated EBNA1 (E1ΔGA, codons 390-641) was expressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast P. pastoris and purified by Ni-NTA affinity chromatography. The purified proteins were then used as antigens to immunize BALB/c mice for production of polyclonal antibodies. Western blot analysis showed that the polyclonal antibodies specifically recognized the EBNA1 protein in B95-8 cell lysates. The recombinant E1ΔGA also induced strong lymphoproliferative and Th1 cytokine responses in mice. Furthermore, mice immunized with E1ΔGA developed CD4(+) and CD8(+) T cell responses. These findings showed that the yeast-expressed E1ΔGA retained good immunogenicity and might be a promising vaccine candidate against EBV-associated malignancies.Applied Microbiology and Biotechnology 05/2013; · 3.81 Impact Factor