A novel suicide shuttle plasmid for Streptococcus suis serotype 2 and Streptococcus equi ssp. zooepidemicus gene mutation.
A novel suicide shuttle plasmid for Streptococcus suis serotype 2 and Streptococcus equi ssp. zooepidemicus gene mutation.
复制标题
用于猪链球菌血清型 2 和马链球菌亚种的新型自杀穿梭质粒。
DOI：
10.1038/srep27133
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发表时间：
2016-06-03
影响因子：
4.6
通讯作者：
Fan H
中科院分区：
文献类型：
--
作者：
Liu R;Zhang P;Su Y;Lin H;Zhang H;Yu L;Ma Z;Fan H
基于mariner的Himar 1系统已被用于创建许多革兰氏阳性细菌的突变体文库。猪链球菌血清型2（SS 2）和马链球菌亚种（SS 2）。猪细小病毒（Zoopellicus，SEZ）是威胁我国养猪业的主要病原体。为了提供一种寻找这两种病原体中毒力表型相关基因的正向遗传学技术，我们构建了一种新型的温度敏感的自杀穿梭质粒pMar 4s，该质粒含有来自pMarA的Himar 1系统转座子TnYLB-1和Himar 1 C9转座酶以及来自pSET 4s的repTA温度敏感片段。卡那霉素抗性基因位于TnYLB-1转座子中。温度敏感性和Kan抗性允许选择突变株和突变库的构建。利用pMar 4s质粒成功构建了SS 2和SEZ突变体文库。反向聚合酶链反应（Inverse-PCR）结果显示转座子插入位点的变异性很大，该文库可用于表型改变筛选。筛选了硫胺素生物合成基因apbE对SS 2抗吞噬作用的影响;同样，sagF基因被鉴定为SEZ中的溶血活性相关基因。pMar 4s适合于构建突变体库，可提供更多关于SS 2和SEZ毒力因子的信息，并阐明猪链球菌病的致病机理。
The mariner-based Himar1 system has been utilized for creating mutant libraries of many Gram-positive bacteria. Streptococcus suis serotype 2 (SS2) and Streptococcus equi ssp. zooepidemicus (SEZ) are primary pathogens of swine that threaten the swine industry in China. To provide a forward-genetics technology for finding virulent phenotype-related genes in these two pathogens, we constructed a novel temperature-sensitive suicide shuttle plasmid, pMar4s, which contains the Himar1 system transposon, TnYLB-1, and the Himar1 C9 transposase from pMarA and the repTAs temperature-sensitive fragment from pSET4s. The kanamycin (Kan) resistance gene was in the TnYLB-1 transposon. Temperature sensitivity and Kan resistance allowed the selection of mutant strains and construction of the mutant library. The SS2 and SEZ mutant libraries were successfully constructed using the pMar4s plasmid. Inverse-Polymerase Chain Reaction (Inverse-PCR) results revealed large variability in transposon insertion sites and that the library could be used for phenotype alteration screening. The thiamine biosynthesis gene apbE was screened for its influence on SS2 anti-phagocytosis; likewise, the sagF gene was identified to be a hemolytic activity-related gene in SEZ. pMar4s was suitable for mutant library construction, providing more information regarding SS2 and SEZ virulence factors and illustrating the pathogenesis of swine streptococcosis.