肠出血性大肠杆菌毒力相关基因突变株的构建

Construction of EHEC Virulence-Associated Gene Mutant

目的:利用Red重组系统敲除肠出血性大肠杆菌O157∶H7的毒力基因espA、espB、espD,构建3株突变株。方法:以肠出血性大肠杆菌O157∶H7为模板,PCR扩增基因两翼的同源序列;将PCR产物插入pEASY-T1载体并测序,将测序正确的上、下游同源序列分步酶切,构建于pUC19-kan质粒上,经PCR获得两端同源序列中间嵌合卡那霉素抗性基因标记的线性片段,利用质粒pKD46介导的重组技术,敲除espA、espB、espD基因,之后转入pCP20质粒以去除抗性标记,最后测定突变株及野生菌株的生长曲线。结果:敲除了肠出血性大肠杆菌O157∶H7的毒力基因espA、es pB、espD,获得3株突变株,突变株与野生株的生长曲线相近。结论:为进一步研究espA、espB、espD基因在肠出血性大肠杆菌O157∶H7致病过程中的作用奠定了基础。

Objective： To knockout enterohemorrhagic E.coli O157∶H7 espA,espB,espD gene with Red recombi nant system.Methods： Two wings of target genes were cloned from EHEC O157∶H7 by PCR.Products of PCR were inserted into pEASY-T1 cloning vector.Gene cloning plasmids were sequenced,and correct both ends of the arms were cut down by endonucleases.Then they were in sequence linked with the pUC19-kan vector which was cut down by same endonucleases.Both ends of the arms and the chimeric kanamycin resistance gene marker of linear fragment were cloned by PCR.Using plasmid pKD46-mediated recombination,the genes of espA,espB,espD were knocked out.Plasmid pCP20 was introduced into the recombinant to remove the kanamycin resistant relevant DNA fragment.The mutants and wild-type strain curves were surveyed.Results： The mutants of espA,espB and es pD deletion of E.coli O157∶H7 were constructed,and the growth curves of mutants and wild-type strain were simi lar.Conclusion： The mutants will be the foundation for the further research of the function of these gene.