肠出血性大肠埃希菌O157∶H7 espF基因缺陷突变株的构建及其特性初步研究

Construction and characterization of enterohemorrhagic Escherichia coli O157∶H7 espF deletion mutant

目的为研究肠出血性大肠埃希菌O157∶H7效应分子EspF功能,构建EHECO157∶H7espF基因缺陷突变株,并对其生物学特性进行初步研究。方法采用OL-PCR和自杀性质粒pCVD442介导的同源重组方法构建中间缺失162bp的espF基因缺陷突变株,并比较突变株与野生株对结肠癌细胞Lovo的黏附性。结果 PCR及序列分析证实,缺陷突变株的espF基因缺失了162bp碱基,野生株对Lovo细胞的黏附率明显高于突变株(P<0.001)。结论成功构建EHECO157∶H7espF基因缺陷突变株,突变株黏附Lovo细胞能力减弱,表明EspF促进细菌的粘附,为进一步研究其在EHECO157∶H7的A/E损伤中的作用奠定基础。

The purpose of this study was to construct espF deletion mutant of EHEC O157∶H7 and investigate its properties.espF deletion fragment（△espF）was amplified by two pairs of primers designed according to the sequence of espF and its upper and down streams.The △espF fragment was cloned into suicide plasmid pCVD442 and reconstructed suicide plasmid pCVD442-△espF by conjugating into EHEC O157∶H7 from E.coli SM10λpir.espF deletion mutant was screened with resistance sign,sucrose and PCR.Then the adhesion activity of mutant was compared with the wide type.The sequencing result showed that 162bp of espF was deleted.The adhesion rate of the wide type to LoVo cells was higher than that to the mutant（P〈0.001）.It＇s concluded that espF deletion mutant of EHEC O157∶H7 was constructed successfully,terming as EHEC O157∶H7（△espF）.The adhesion activity of the mutant was attenuated.espF could promotes EHEC＇s adhesion to LoVo cells.