Prokaryotic expression of exfoliative toxin A from Staphylococcus aureus
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Prokaryotic expression of exfoliative toxin A from Staphylococcus aureus
Journal of Chongqing Medical UniversityVol. 40, Issue 3, Pages: 436-440(2015)
作者机构:
1. 重庆医科大学基础医学院免疫学教研室,重庆,400016
2. 重庆医科大学附属儿童医院皮肤科,重庆,400014
3. 重庆医科大学基础医学院病原生物学教研室,重庆,400016
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Published:2015,
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Wang Yi, Xiao Yizhu, Yang Zhibang, et al. Prokaryotic expression of exfoliative toxin A from Staphylococcus aureus. [J]. Journal of Chongqing Medical University 40(3):436-440(2015)
DOI:
Wang Yi, Xiao Yizhu, Yang Zhibang, et al. Prokaryotic expression of exfoliative toxin A from Staphylococcus aureus. [J]. Journal of Chongqing Medical University 40(3):436-440(2015)DOI:
Prokaryotic expression of exfoliative toxin A from Staphylococcus aureus
Objective:To obtain the recombine exfoliative toxin A(rETA) by the technique of gene engineering and to provide materi-als to study the pathogenic mechanism of the exfoliative toxin(ET). Methods:The primers were designed according to the gene se-quence of ETA gene announced by GeneBank. The ETA gene was amplified from the genome of S.aureus and was inserted into the prokaryotic expression vector pQE30. The recombinant plasmid pQE30-ETA was built and was translated into E.coli M15. After the expression conditions being optimized,the recombinant plasmid pQE30-ETA was expressed. The expression product was analyzed by SDS-PAGE and confirmed by Western blot. After being purified with Ni2+-NTA resin,the purified protein was injected into the sub-cutaeous of BALB/C mice and pathological modifications of BALB/C mice were observed to identify the biologic activity of the rETA. Results:The gene sequence analysis of the recombinant plasmid pQE30-ETA showed that the inserted gene fragment was 927 bp and was corresponded with the ETA gene sequence in GenBank database. The analysis of SDS-PAGE showed that the relative molecular mass of expression was 27 kD and the recombinant solubility protein reached the highest expression(20%) when IPTG being at a fi-nal concentration of 0.1 mmol/L and the induction time of 4 h. Western blot showed that the recombinant protein was at the same lo-cation with the ETA protein produced by S.aureus. The purity of the recombinant protein was over 90% after being purified with Ni2+-NTA resin. The splitting and blister were appeared in the epidermis of mouse injected with recombinant protein. Conclusion:The construction of the recombinant plasmid pQE30-ETA and the expression of rETA with biologic activity are successful.