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

作　　者：姜楠[1,2] 王琴[1] 李涛[1] 侯晓军[1] 肖乐[1] 房华丽[1] 罗森[1] 王慧[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071 [2]贵阳医学院,贵阳550001

出　　处：《生物技术通讯》2012年第5期640-643,共4页Letters in Biotechnology

基　　金：国家自然科学基金(30901278;81072677);国家重大传染病防治专项(2008ZX10004-015);北京市自然科学基金(7122134)

摘　　要：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.

关 键 词：肠出血性大肠杆菌O157∶H7 RED重组系统 espA基因 espB基因 espD基因 

分 类 号：Q78[生物学—分子生物学]