摘要
采用PCR法合成了stx2基因上游和下游的2个同源臂序列分别为1635,1655bp。2个同源臂序列被克隆到载体pCR2.1-TOPO上,两臂中间插入庆大霉素抗性基因Gm(855bp),构建了stx2基因敲除盒子pCR2.1-B-Gm-A。将含2个同源臂和Gm抗性基因片段的扩增子电转化到含质粒pKM208的感受态细胞EHECO157:H7中,通过λ-Red重组系统产生了stx2基因敲除突变菌株。用Verocell试验检测了突变株的产毒能力并进行了细胞黏附试验。结果表明,合成同源臂利用λ-Red重组系统可以有效的敲除EHEC毒素基因,stx2基因敲除后的突变株不产stx2毒素,对真核细胞CaCo-2和Hep-2的黏附能力明显降低。
To knockout E.coli O157：H7 stx2 gene using λ-Red recombination system,a pair of primers,which was the homologous sequences of upstream and downstream regions of stx2 gene,was amplified from wild type EHEC O157：H7 strain by PCR.The two fragments and a gentamycin （Gm） resistant gene （855 bp） were cloned into pCR2.1-TOPO constructing a stx2 gene deleted cassette pCR2.1-B-Gm-A.PCR amplicons containing two homologous sequences and Gm gene were electroporated into competent EHEC O157：H7 strain cells containing pKM208 and get the stx2 deleted mutant.The mutant was detected by Vero cell assay and cell adherence assay with CaCo-2 cells and Hep-2 cells.The result showed that the λ-Red recombination system worked efficiently in knockout the stx2 gene of EHEC.The stx2 gene deleted mutant did not produce Shiga toxin and the ability to adhere to CaCo-2 and Hep-2 cells obviously decreased compared with the wild type strain.This study provided a theoretical basis for the EHEC pathogenesis research.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2010年第7期926-930,935,共6页
Chinese Journal of Veterinary Science
基金
西北农林科技大学青年启动基金资助项目(08080230)
