Preliminary investigation of human serum albumin-Vβ inhibition on toxic shock syndrome induced by staphylococcus enterotoxin B in vitro and in vivo
Section snippets
Instruction
SEB is produced by Staphylococcus aureus and has been implicated in many human diseases, such as food poisoning and toxic shock syndrome (TSS) (Torres et al., 2001). Moreover, SEB may be a potential biological warfare agent because it is simple and inexpensive to produce, stable in aerosol form, and easy to disperse (Henghold, 2004). Accordingly, SEB is listed as a category B priority agent by the Centers for Disease Control and Prevention (CDC). As a type of superantigen, SEB stimulates robust
HSA-Vβ preparation
HSA and Vβ were fused by overlap polymerase chain reaction (PCR). The linker between HSA and Vβ was GGGGSGGGGSGGGGS. The fusion protein was expressed in GS115 cells cultured in BMGY medium at 30 °C with shaking at 250 rpm. Every 24 h, 0.5% methanol was added to the culture. The supernatant was precipitated with 1 M (NH4)2SO4 and then purified using Phenyl Sepharose HP and Source 30Q (GE Healthcare Life Science, Pittsburgh, PA, USA). The purity was evaluated by sodium dodecyl sulfate
HSA-Vβ was expressed in yeast and exhibited high affinity for SEB
In order to be expressed stably in yeast, the gene sequence of Vβ was optimized and fused with HSA. For optimization, yeast biased codons were chosen to improve the stability of mRNA. Moreover, by fusion with HSA, HSA-Vβ was expressed in soluble form in GS115 yeast cells. The production yield reached about 25%, and after purification, the fusion protein was more than 95% pure (Fig. 1). Affinity testing using BLItz showed that the KD for the interaction between HSA-Vβ and SEB was 7.272 × 10−9 M (
Discussion
TSS is characterized by a hyperinflammatory response due to microbial infection. The superantigen SEB can induce a so-called “cytokine storm”, which may cause TSS. As no vaccine is available for SEB, here we constructed this fusion protein HSA-Vβ which had therapeutic effects on SEB-induced TSS both in vitro and in vivo, highlights its potential for treating SEB intoxication.
Bioinformatics analysis has shown that the secondary structure of Vβ is mainly random coils and sheets, indicating that
Conclusion
In this study, we constructed the fusion protein HSA-Vβ. In vitro and in vivo evaluations showed that HSA-Vβ could effectively inhibit cytokine production and protect mice from lethal challenge by SEB. By fusing Vβ with HSA, the preparation of Vβ was simplified, and production yields were improved, solving key problems for future use of this target.
Conflict of interest
The authors declare no conflicts of interest.
Ethical statement
All of the experiments in the paper were conducted under the supervision of the Institutional Review Board of the Academy of Military Medical Sciences (No. IACUC of AMMS-2014-033).
Acknowledgments
This work was supported by grants from the National 863 Foundation of China (No. 2012AA022001-04), Chinese National Instrumentation Program (No. 2012YQ180117), and the National Nature Science Foundation of China (No. 81441062, 81401642).
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These authors contributed equally to this manuscript.