一种新的编码霍乱毒素的丝状噬菌体CTAKΦ

A new type of filamentous phage CTAKΦ encodes cholera toxin

目的 探讨介导霍乱弧菌毒素基因水平转移的一种独特的遗传结构。方法 从O139群霍乱弧菌菌株FJ9712 9上清中分离出丝状噬菌体颗粒CTAKΦ ,并进行电镜观察。从丝状噬菌体颗粒CTAKΦ中纯化出DNA ,并进行链型分析。对pCTAK进行酶谱分析。用ctxAB、zot引物对pCTAK进行PCR扩增检测 ,用RS1探针对pCTAK进行Southernblot检测 ;用pCTAK转化DH5α和IEM10 1得到DX2 9和 432 9,将pCTAK克隆于pUC18载体并转入DH5α得到UD2 9,用GM1 ELISA检测DX2 9、432 9、UD2 9的CT表达。对pCTAK的ctxAB、zot基因片段进行测序 ,并用DNASTAR软件和BLAST算法 ,在国际互联网上对测序结果进行序列分析。结果 发现和分离了一种编码霍乱毒素的质粒pCTAK ,它以稳定的高拷贝数存在于 1株天然的O139霍乱弧菌FJ9712 9中 ;酶谱分析发现其明显不同于CTX元件酶谱 ,在CTX元件中相当保守的酶切位点 :BglⅡ、EcoRⅤ、PstⅠ、EcoRⅠ ,在pCTAK中没有切点。ctxAB、zot引物对pCTAK的PCR扩增检测呈阳性 ,RS1探针杂交呈阳性 ;pCTAK的ctxAB、zot基因序列与已报道的序列有很高的同源性。pCTAK能直接或经载体转化DH5α和IEM10 1并表达CT。从FJ9712 9培养上清中分离出丝状噬菌体颗粒CTAKΦ ,电镜观察并计算其直径大小为 7nm左右。其全基因组大小为 8.5kb ,为?

Objective To study and identify the organism to donate the genetic element to nontoxinogenic strains of V. cholerae and to induce the horizontal transfer of cholera toxin gene. Methods Southern blot. PCR examination. cholera toxin of pCTAK was expressed and studied in E.coli with ELISA. The ctxAB and zot gene of pCTAK was sequenced. CTAKΦ phage particles were isolated from supernatants of pCTAK strains and examined by electron microscopy. Phage DNA was isolated from CTAKΦ particles. CTAKΦ particles and DNA were analyzed with S1 nuclease. Results A plasmid (designed pCTAK) was identified harboring at high copy number in a V. cholerae O139 strain FJ97129. It encoded cholera toxin operon (ctxAB), zoluna toxin gene (zot), RS sequence. Extracellular filamentous phage particles (designed CTAKΦ) were isolated from supernatants of the strain FJ97129. Single strand DNA purified from CTAKΦ particles was about 8.5kb in size. Conclusion A new type of filamentous phage encoding cholera toxin was found and identified. [