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目的利用CRISPR/Cas9基因编辑技术抑制肠出血性大肠埃希菌(EHEC)O157∶H7StxⅡ基因表达,并评价其对细菌生长的影响及细胞毒性。方法针对EHEC O157∶H7StxⅡ基因设计引物,构建CRISPR/Cas9表达质粒pdCas9-StxⅡ并转化EHEC O157∶H7感受态细胞,采用RT-PCR及胶体金法检测StxⅡ基因表达情况,绘制菌株生长曲线,将菌株培养上清液接种Vero细胞观察细胞病变情况。结果测序分析显示pdCas9-StxⅡ表达质粒被成功构建;转化EHEC O157∶H7(00G097)感受态细胞后,StxⅡ基因mRNA、蛋白表达均受抑制,EHEC O157∶H7生长曲线未受影响(P值均>0.05)。pdCas9-StxⅡ-00G097菌株培养上清液对细胞的毒性效应(CPE为30%)显著低于对照菌株00G097和pdCas9-00G097(CPE均>80%)。结论成功构建的pdCas9-StxⅡ表达质粒能特异抑制EHEC O157∶H7StxⅡ基因表达、降低细胞毒性,为进一步研究志贺毒素StxⅡ在EHEC O157∶H7致病机理和基因工程减毒活菌苗奠定了基础。
OBJECTIVE: To inhibit the expression of O157: H7StxⅡ gene of enterohemorrhagic Escherichia coli (EHEC) by using CRISPR / Cas9 gene editing technology and evaluate its effect on bacterial growth and cytotoxicity. Methods The primers of EHEC O157:H7StxⅡgene were designed. The CRISPR / Cas9 expression plasmid pdCas9-StxⅡ was constructed and transformed into EHEC O157:H7 competent cells. The expression of StxⅡ gene was detected by RT-PCR and colloidal gold method. The growth curve of the strain was drawn. The supernatant was seeded into Vero cells to observe the cytopathic effect. Results Sequencing analysis showed that pdCas9-StxII expression plasmid was constructed successfully. After transfection of EHEC O157: H7 (00G097) competent cells, the expression of StxⅡ mRNA and protein were inhibited. The growth curve of EHEC O157: H7 was not affected (P> 0.05). The cytotoxic effect of pdCas9-StxII-00G097 culture supernatant (CPE 30%) was significantly lower than the control strains 00G097 and pdCas9-00G097 (CPE> 80%). Conclusion The constructed pdCas9-StxII expression plasmid can specifically inhibit the expression of EHEC O157: H7StxⅡ gene and reduce the cytotoxicity, which lays a foundation for further study on the pathogenesis of Shiga toxin Stx Ⅱ and the live-attenuated vaccine of EHEC O157:H7.