INTRODUCTION OF THE EMM6 GENE INTO AN EMM-DELETED STRAIN OF STREPTOCOCCUS-PYOGENES RESTORES ITS ABILITY TO RESIST PHAGOCYTOSIS
INTRODUCTION OF THE EMM6 GENE INTO AN EMM-DELETED STRAIN OF STREPTOCOCCUS-PYOGENES RESTORES ITS ABILITY TO RESIST PHAGOCYTOSIS
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DOI：
10.1016/0923-2508(92)90112-2
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发表时间：
1992-07-01
影响因子：
2.6
通讯作者：
SCOTT, JR
中科院分区：
文献类型：
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作者：
PEREZCASAL, J;CAPARON, MG;SCOTT, JR
为了完成正式的证明，M蛋白的功能，以保护A组链球菌从吞噬作用，并奠定基础的分子遗传解剖的这一主要毒力因子，我们已经重新引入了一个野生型等位基因的M蛋白基因（emm）到染色体的菌株，从它以前被删除。为此，我们使用同源重组与工程质粒，引入到化脓性链球菌染色体的电穿孔技术，我们开发的。质粒pJRS 120 E在大肠杆菌中构建，并含有S.化脓性链球菌JRS 4和选择性标记（ermAM）之间的染色体区域，侧翼的emm6在S。化脓将该质粒导入S.化脓性链球菌菌株JRS 75，由于卡那霉素抗性基因（aphA3）等位基因取代emm6基因，该菌株对吞噬作用没有抗性。DNA杂交分析表明，在红霉素抗性菌株JRS 115中，同源重组导致JRS 75的aphA 3基因被引入的emm6等位基因所取代。菌株JRS 115产生的M6量与亲本emm6+菌株JRS 4大致相同（通过蛋白质印迹分析测定），并且对吞噬作用具有抗性。
To complete the formal proof that the M protein functions to protect the group A streptococcus from phagocytosis and to lay the groundwork for the molecular genetic dissection of this major virulence factor, we have reintroduced a wild-type allele of the M protein gene (emm) into the chromosome of a strain from which it had previously been deleted. For this purpose, we used homologous recombination with an engineered plasmid that was introduced into the Streptococcus pyogenes chromosome by an electroporation technique that we developed. The plasmid pJRS120E was constructed in Escherichia coli and contains the structural gene for the antiphagocytic M6 protein (emm6) of S. pyogenes JRS4 and a selectable marker (ermAM) between the chromosomal regions that flank emm6 in S. pyogenes. This plasmid was introduced into S. pyogenes strain JRS75, a strain that is not resistant to phagocytosis as a result of the allelic replacement of the emm6 gene by a kanamycin resistance gene (aphA3). DNA hybridization analysis indicated that in erythromycin-resistant strain JRS115, homologous recombination resulted in the replacement of the aphA3 gene of JRS75 with the introduced emm6 allele. Strain JRS115 produces approximately the same amount of M6 as does the parental emm6+ strain JRS4, as assayed by Western blot analysis, and is resistant to phagocytosis.