【摘要】 目的构建致病性大肠杆菌(EPEC)espH删除株,探讨espH分子的转位、定位以及在A/E损伤中的作用。方法构建自杀质粒pcvd442-△H::kana,通过结合传递、等位交换,抗性筛选获得espH删除株,并对删除株进行PCR鉴定;构建带HA标签的espH表达载体pTrc99a-espH:HA,转化espH删除株,感染HeLa细胞,免疫荧光技术和Western blot检测espH的转位及定位;构建espH和绿色荧光蛋白融合表达质粒p-espH-EGFP,转染HeLa细胞,荧光显微镜观察融合蛋白的分布;用espH删除株感染HeLa、Caco-2和TC-7细胞,分别用荧光显微镜技术、上皮细胞电阻(TER)检测和扫描电镜(SEM)探讨espH在基垫形成、屏障功能障碍和微绒毛脱落损伤中的作用。结果酶切、测序及PCR鉴定表明自杀质粒pcvd442-△H::kana和espH删除株构建正确。espH依赖T3SS转位到宿主细胞,转位后,Western blot检测发现espH主要分布于细胞的可溶性成分,免疫荧光检测发现espH分布于细胞膜基垫形成处,espH与EGFP的融合蛋白分布于细胞膜。感染试验表明,espH删除株可以形成基垫,并造成Caco-2细胞屏障功能障碍和TC-7细胞微绒毛脱落损伤。结论已成功构建了EPECespH删除株。espH是EPEC的一个依赖T3SS转位的效应分子,转位后,蛋白分布于细胞膜基垫形成处,但未发现espH在EPEC基垫形成、屏障功能障碍和微绒毛脱落方面具有重要作用。更多还原

【Abstract】 Objective To construct an espH gene-deleted enteropathogenic E.coli(EPEC)mutant and investigate the translocation, location and the role of espH molecule in A / E lesion.Methods Suicide vector pcvd442-△H::kana was constructed, based on which espH gene-deleted EPEC mutant was obtained by conjugation, allelic exchange as well as kanamycin-and nalidixic acid-resistant screening, and identified by PCR.The espH expression vector pTrc99a-espH:HA was constructed and transformed to espH gene-deleted EPEC mutant, and the recombinant strain was infected to HeLa cells.The translocation and location of espH were determined by IFA and Western blot.The fusion expression vector p-espH-EGFP was constructed and transfected to HeLa cells, and the distribution of fusion protein was observed by fluorescent microscopy.HeLa, Caco-2 and TC-7 cells were infected with espH gene-deleted EPEC mutant respectively, and the role of espH in A / E lesion was investigated by fluorescent microscopy, transepithelial electrical resistance(TER)and scanning electrical microscopy(SEM).Results Restriction analysis, sequencing and PCR proved that both suicide vector pcvd442-△H::kana and espH gene-deleted EPEC mutant were constructed correctly.IFA proved that espH gene was translocated to host cells dependent on type Ⅲ secretion system(T3SS).After translocation, Western blot showed that espH was mainly distributed in the soluble fraction of the cells.IFA showed that espH was distributed on cell membrane in the position of pedestal formation, and the fusion protein of espH and EGFP on cell membrane.Infection test proved that espH gene-deleted EPEC mutant formed pedestal and caused the functional disturbance of Caco-2 cell barrier as well as the abscission of microvilli of TC-7 cells.Conclusion The espH gene-deleted EPEC mutant was successfully constructed, and espH was proved as a translocated effecter of EPEC dependent on T3SS and was distributed on cell membrane in position of pedestal formation after translocation.However, no evidence showed that espH played an important role in A / E lesion.更多还原