Chloramphenicol acetyltransferase as a selection marker for chlamydial transformation.
Chloramphenicol acetyltransferase as a selection marker for chlamydial transformation.
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DOI：
10.1186/1756-0500-6-377
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发表时间：
2013-09-23
影响因子：
1.8
通讯作者：
Fan H
中科院分区：
文献类型：
--
作者：
Xu S;Battaglia L;Bao X;Fan H
衣原体是一种常见的细菌病原体，可导致许多疾病。利用β-内酰胺酶作为选择标记转化这种重要生物的方法最近才发展起来。然而，美国国立卫生研究院关于重组DNA分子研究的指南不允许用含有β-内酰胺酶基因的载体对某些人类衣原体病原体进行转化实验。因此，迫切需要一种不同的选择标记来实现这些衣原体的转化。将携带β-内酰胺酶和氯霉素乙酰转移酶基因与绿色荧光蛋白基因融合的无质粒沙眼衣原体用pGFP:SW2转化后，通过氨苄西林或氯霉素选择获得转化子。使用不含β-内酰胺酶的pGFP:SW2衍生物获得稳定的耐氯霉素但对氨苄西林敏感的转化子。所有转化子均表达绿色荧光蛋白，糖原合成活性恢复。氯霉素耐药性可作为衣原体遗传实验的选择标记。这消除了使用β-内酰胺酶的需求，β-内酰胺酶传播到一些沙眼衣原体血清型可能危及孕妇衣原体感染的临床治疗。氯霉素乙酰转移酶也可以作为β-内酰胺酶转化的衣原体菌株遗传分析的有用的二次选择标记。
Chlamydia is a common bacterial pathogen responsible for many diseases. Methods for transforming this important organism using a β-lactamase as a selection marker have been developed very recently. However, the National Institutes of Health Guidelines for Research Involving Recombinant DNA Molecules do not permit transformation experiments with β-lactamase gene-containing vectors for certain human chlamydial pathogens. Therefore, a different selection marker is urgently needed for transformation of those chlamydiae. After transformation of plasmid-free Chlamydia trachomatis with pGFP:SW2, which carries a β-lactamase and a chloramphenicol acetyltransferase gene fused to a green fluorescence protein gene, transformants were obtained by selection with either ampicillin or chloramphenicol. Stable chloramphenicol-resistant, but ampicillin-sensitive, transformants were obtained using a pGFP:SW2 derivative without the β-lactamase. All transformants expressed green fluorescence protein and had glycogen synthesis activity restored. Chloramphenicol resistance may be used as a selection marker for genetic experiments in Chlamydia. This eliminates the requirement for the use of β-lactamase, of which dissemination to some C. trachomatis serovars may jeopardize clinical treatment of chlamydial infections in pregnant women. Chloramphenicol acetyltransferase may also serve as a useful secondary selection marker for genetic analyses in β-lactamase-transformed chlamydial strains.