BackgroundPurified from the supernatant of Bacillus subtilis QK02 culture broth, Subtilisin QK-2 is a type of effective thrombolytic reagent that has great exploitable potential. However, the unbearable flavor that occurs with fermentation and the complicated methods that are required to obtain pure products limit the application of this enzyme. Lactic acid bacteria (LAB)-based delivery vehicles are promising as cheap and safe options for medicinal compounds. The secretory expression and surface display using LAB may popularize Subtilisin QK-2 more easily and conveniently with minimal adverse effects.ResultsSubtilisin QK-2 was expressed successfully in two forms using lactic acid bacteria. For the secretory expression in Lactococcus lactis, Subtilisin QK-2 was efficiently secreted into the culture using the promoter PnisA and signal peptide SPUsp. The expression levels were not different in L. lactis NZ9000 and NZ3900 without the effect of different selection markers. However, leaky expression was only detected in L. lactis NZ3900. The biological activity of this secreted Subtilisin QK-2 was enhanced by modulating the pH of medium to slightly alkaline during induction and by codon optimization of either the entire gene sequence (qk′) or only the propeptide gene sequence (qkpro′). For surface display onto gram-positive enhancer matrix (GEM) particles, n LysM repeats from the C-terminal region of the major autolysin AcmA of L. lactis were fused to either the C-terminus (n = 1, 3, 5) or the N-terminus (n = 1) of the Subtilisin QK-2. These fusion proteins were secreted into the culture medium, and the QK-3LysM was able to bind to the surface of various LAB GEM particles without a loss of fibrinolytic activity. Furthermore, the binding capacity significantly increased with a higher concentration of QK-3LysM. Compared to the free-form Subtilisin QK-2, the QK-3LysM displayed on the surface of GEM particles was more stable in the simulated gastric juice.ConclusionsCombined with the safety and popularity of LAB, Subtilisin QK-2 may be easily applied worldwide to prevent and control thrombosis diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0478-7) contains supplementary material, which is available to authorized users.
Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that is linked to the development of various malignancies. There is an urgent need for effective vaccines against EBV. EBV envelope glycoprotein gp350 is an attractive candidate for a prophylactic vaccine. This study was undertaken to produce the truncated (codons 1-443) gp350 protein (gp350(1-443)) in Pichia pastoris and evaluate its immunogenicity. The gp350(1-443) protein was expressed as a secretory protein with an N-terminal His-tag in P. pastoris and purified through Ni-NTA chromatography. Immunization with the recombinant gp350(1-443) could elicit high levels of gp350(1-443)-specific antibodies in mice. Moreover, gp350(1-443)-immunized mice developed strong lymphoproliferative and Th1/Th2 cytokine responses. Furthermore, the recombinant gp350(1-443) could stimulate CD4(+) and CD8(+) T cell responses in vaccinated mice. Collectively, these findings demonstrated that the yeast-expressed gp350(1-443) retained strong immunogenicity. This study will provide a useful source for developing EBV subunit vaccine candidates.
Objective To establish a novel HBV specific immunoadsorbent for the removing of HBV particles. Methods The anti-HBsAg monoclonal antibody was immobilized on sepharose beads to produce a sepharose anti-HBs column. Then the immunoadsorbent was evaluated and characterized by scanning electron microscopy. In addition, time-dependent effects of the eradication capacity of anti-HBsAg functionalized sepharose beads against HBV were investigated. Results Proposed immunoadsorbents exhibited a favorable biocompatibility as well as specificity. With the optimized recycle time, the decontamination performance of HBV particles and quantity of HBsAg were assessed either by real-time quantitative PCR or ELISA, which showed that the immunoadsorbent could remove approximately 90% of the HBV and 90% of the HBsAg from human plasma samples. Conclusions All these results indicated that the novel immunoadsorbent could effectively remove HBV particles and likely serve as a novel therapy option or at least supplementary for the treatment regimen of HBV.
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