大肠杆菌vpr基因突变株的构建及其特性鉴定

Construction and Characterization of a vpr Isogenic Knockout E.co li Mutant

将重组自杀性质粒 p YYvpr转化到含全 vpr基因的 E.coli MC10 6 1感受态细胞中 ,利用卡那霉素抗性筛选到 6个菌落 ,经 PCR和 Southern blot进一步验证 ,得到一个可靠的 vpr同源基因突变株 ,即 MC10 6 1△ vpr。该突变株与亲本 MC10 6 1具有相似的生长特征。噬菌体裂解试验表明 ,突变株对 VT2噬菌体 C43b不敏感 ,而 MC10 6 1敏感 ,可见大量的蚀菌斑。噬菌体溶原试验表明 ,MC10 6 1和突变株 MC10 6 1△vpr对噬菌体的敏感性无差别。所有这些表明 ,vpr基因与 VT2噬菌体的裂解性有关 ,可能是编码 VT2噬菌体受体蛋白的基因。

The recombinant suicide plasmid pYYvpr was tran sformed into the wild type E.coli strain,MC1061,which possesses the full-le ngth vpr gene.The recombinant suicide plasmid could not replicate in MC1061. Six resultant colonies grown on the plates of kanamycin were obtained.Analysed by P C R and Southern blot with two different Dig-probes,vpr and Kan. resistant ge ne probes,an isogenic knockout mutant,named MC1061△vpr,was confirmed.The mutan t had similar growth characteristics to the wild type strain and supported lysog enic infection of VT2 recombinant phage,C43b,but did not support lytic infection .All results showed that the vpr gene should be related with the lytic infec tion of VT2 phage,and might code for a receptor of VT2 phage.