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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
纸质出版日期:2015,
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王 毅, 肖异珠, 杨致邦, 等. 金黄色葡萄球菌重组表皮剥脱毒素A的原核表达[J]. 重庆医科大学学报, 2015,40(3):436-440.
Wang Yi, Xiao Yizhu, Yang Zhibang, et al. Prokaryotic expression of exfoliative toxin A from Staphylococcus aureus[J]. Journal of Chongqing Medical University, 2015,40(3):436-440.
目的:通过基因工程技术获取重组表皮剥脱毒素A(recombine exfoliative toxin A,rETA),为深入研究表皮剥脱毒素的致病机制提供实验材料。方法:根据GenBank公布的ETA基因序列设计引物,从金黄色葡萄球菌(Staphylococcus aureus,S.aureus)基因组中扩增ETA基因,插入原核表达载体pQE30,构建重组质粒pQE30-ETA。将pQE30-ETA转化表达菌E.coli M15,优化诱导表达条件,SDS-PAGE分析重组蛋白的表达,Western blot鉴定。重组蛋白经Ni2+-NTA树脂纯化后,注入BALB/C小鼠皮下,观察皮肤病理改变,鉴定重组蛋白的生物学活性。结果:重组质粒pQE30-ETA序列分析显示插入的基因片段全长927 bp,与基因文库中的ETA序列完全吻合。SDS-PAGE分析显示表达产物相对分子量约为27 kD,在IPTG终浓度0.1 mmol/L,诱导时间4 h,可得到最大表达量的可溶性蛋白,约为20%。Western blot显示与S.aureus产生的ETA蛋白在同一位置,重组蛋白经Ni2+-NTA树脂纯化后蛋白纯度达90%以上,注射重组蛋白的小鼠表皮内出现水疱和裂隙。结论:成功构建了重组质粒pQE30-ETA,表达了具有生物学活性的重组S.aureus表皮剥脱毒素A。
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.
金黄色葡萄球菌葡萄球菌性烫伤样皮肤综合征表皮剥脱毒素A原核表达
Staphylococcus aureusstaphylococcal scalded skin syndromeexfoliative toxins Aprokaryotic expression
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