【摘要】 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。更多还原

【Abstract】 Objective To inhibit StxⅡ gene expression in Enterohemorrhagic Escherichia coli(EHEC)O157∶H7 by CRISPR/Cas9 genome editing technology;to evaluate consequent impacts to bacteria growth and cytotoxicity.Methods Primers were designed based on EHEC O157∶H7StxⅡ gene.The pdCas9-StxⅡ expression plasmid targeting for StxⅡ gene of EHEC O157∶H7was constructed and transformed into EHEC O157∶H7competent cells.The expression of the StxⅡ gene was detected by RT-PCR and colloidal gold method.Growth curves were drawn,cell cytopathic effects(CPE)of Vero cell incubating with bacteria culture supernatants were observed.Results Sequencing analysis confirmed success construction of pdCas9-StxⅡ expression plasmid.Upon transformation of pdCas9-StxⅡ into EHEC O157∶H7(00G097)competent cells,both StxⅡ mRNA and protein expression were inhibited;while the growth curve was not effected significantly(all P>0.05).CPEs were significant decreased in pdCas9-StxⅡ-00G097incubation(around 30%)compared to 00G097/pdCas9-00G097incubation(all>80%).Conclusion EHEC O157∶H7StxⅡ gene can be inhibited effectively by pdCas9-StxⅡ expression plasmid constructed based on CRISPR/Cas9 technology,which resulted in lower cytotoxicity.The research lay a foundation for further study of Shiga toxin StxⅡ in EHEC O157∶H7pathogenic mechanism and construction of EHEC O157∶H7genetically engineered live attenuated vaccines.更多还原