金黄色葡萄球菌重组表皮剥脱毒素A的原核表达

Prokaryotic expression of exfoliative toxin A from Staphylococcus aureus

摘要:

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。

Abstract:

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.