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测定和分析霍乱弧菌分型噬菌体VP3基因组序列,并为ElTor型霍乱弧菌两类菌株的分型方法原理提供研究基础。鸟枪法构建VP3噬菌体全基因组随机文库;测序拼接成最小重叠群,引物步移法填补缝隙序列,拼接后获得VP3全基因组序列。PCR随机扩增噬菌体DNA片段并酶切鉴定;预测可能存在的开放读码框(ORF);对VP3和相关噬菌体的DNA聚合酶基因作进化树分析,协助判定VP3的分类;对预测的部分启动子区利用报道基因进行活性分析。VP3全基因组为环状双链DNA,长度39504bp;酶切鉴定结果与序列一致。确定了49个ORF,注释了27个ORF的编码产物,其中有20个基因产物与T7样噬菌体同源,包括RNA聚合酶(RNAP)、参与DNA复制的蛋白、衣壳蛋白、尾管及尾丝蛋白、DNA包装蛋白等。DNA聚合酶(DNAP)进化树分析表明VP3与T7样噬菌体有同源性。将预测的10个启动子序列克隆到lacZ融合质粒pRS1274上,经检测均具有启动子活性。测定和分析VP3的基因组序列,基因组结构与进化树分析提示VP3属于T7噬菌体家族。
To determine and analyze the genomic sequence of VP3 phage VP3 and to provide the basis for the genotyping method of two strains of V. cholerae ElTor. The whole genome of VP3 phage was constructed by shotgun method. The minimal contigs were sequenced and the gap sequences were filled by primer walking method. The whole VP3 genome sequence was obtained after splicing. PCR was used to amplify the phage DNA fragments and identified by restriction enzyme digestion. The possible open reading frames (ORFs) were predicted. Phylogenetic tree analysis of DNA polymerase genes of VP3 and related phages was performed to help determine the classification of VP3. Subdomains utilize reporter genes for activity analysis. VP3 whole genome circular double-stranded DNA, length 39504bp; restriction enzyme digestion results consistent with the sequence. A total of 49 ORFs were identified and 27 ORFs were annotated. Among them, 20 were homologous to T7-like phages, including RNA polymerase (RNAP), proteins involved in DNA replication, capsid protein, tail tube and tail Silk protein, DNA packaging protein and so on. Phylogenetic tree analysis of DNA polymerase (DNAP) showed that VP3 has homology with T7-like phage. The predicted 10 promoter sequences were cloned into the lacZ fusion plasmid pRS1274 and all of them had promoter activity. The genomic sequence of VP3 was determined and analyzed. Genome structure and phylogenetic tree analysis suggested that VP3 belonged to the T7 phage family.