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目的霍乱弧菌(VC)O139群CTXφ和nct-CTXφ基因组共存新类型的结构研究。方法分别扩增CTXφ或nct-CTXφ基因组串联体的第一个基因组末端和第二个起始端之间的基因,扩增片段再经RsaI和BglII酶切、型特异引物PCR方法,区分rstR-ig2类型;扩增TLC和RS区之间片段并区分rstR-ig2类型;扩增RTX与CTXφ或nct-CTXφ核心区之间片段。结果O139群VC代表菌株及CTXφ和/或nct-CTXφ的rstR-ig2类型(以上标表示)如下,FJ9510株CTX ETφ、FJ98352株CTXETφ+nct-CTX calcφ、FJ99364株CTXETφ+nct-CTX newO139φ+nct-CTX calcφ、JS9803株CTXETφ+nct-CTX newO139φ、JX94484株CTXETφ+nct-CTX newETφ;不同rstR-ig2类型CTXφ和nct-CTXφ基因组zot基因3′末端46bp序列中20个位点不同,推测的15个氨基酸序列中11个位点不同;O139群VC CTXφ基因组附近序列均为TLC和RTX基因簇,而nct-CTXφ基因组附近均为未知或新类型序列。结论阐明O139群VC CTXφ和nct-CTXφ基因组共存新类型主要变异区及染色体插入位点附近结构。
Objective To study the coexistence of CTXφ and nct-CTXφ genomes of V. cholerae (VC) O139 genome. Methods The genes between the first genomic end and the second start of CTXφ or nct-CTXφ genome were amplified respectively. The amplified fragments were digested with RsaI and BglII, and the PCR was performed by specific primer PCR to distinguish rstR-ig2 Type; amplify fragments between TLC and RS regions and distinguish rstR-ig2 types; amplify fragments between RTX and CTXφ or nct-CTXφ core regions. Results The representative strain of O139 VC and the rstR-ig2 type of CTXφ and / or nct-CTXφ (indicated by the above standard) were as follows. FJ9510 strain CTX ETφ, FJ98352 strain CTXETφ + nct-CTX calcφ, and FJ99364 strain CTXETφ + nct-CTX newO139φ + nct CTXetφ + nct-CTX newO139φ and CTXetφ + nct-CTXnewETφ of strain JX94484 were different between the two strains. The 20 different sites in the 46bp sequence of the 3 ’end of the zot gene in CTXφ and nct-CTXφ genomes of rstR-ig2 were 15 11 amino acid sequences of different positions; O139 VC CTXφ genome near the sequence are TLC and RTX gene cluster, and nct-CTXφ genome near unknown or new type of sequence. Conclusion The coexistence of new types of CTXφ and nct-CTXφ genomes in the O139 group and the vicinity of the chromosome insertion site are elucidated.