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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 investigate a unique genetic structure that mediates horizontal transfer of Vibrio cholera toxin genes. Methods The filamentous phage particles CTAKΦ were isolated from the supernatant of Vibrio cholerae O139 strain FJ97129 and observed under electron microscope. DNA was purified from filamentous phage particles CTAK [Phi] and subjected to a strand analysis. Enzymatic analysis of pCTAK. PCR amplification of pCTAK was carried out using ctxAB and zot primers, and Southern blot was performed on pCTAK using RS1 probe; DH5α and IEM101 were transformed into pXD9 and pGEM9 by pCTAK; pCTAK was cloned into pUC18 vector and transformed into DH5α to obtain UD2 9 The CT1 expression of DX2 9, 432 9 and UD2 9 was detected by GM1 ELISA. The ctxAB and zot gene fragments of pCTAK were sequenced and the sequencing results were sequenced on the Internet using DNASTAR software and BLAST algorithm. Results A cholera toxin-encoding plasmid, pCTAK, was found and isolated in a stable high copy number in a native strain of V. cholerae FJ97129; zymogram analysis revealed significant differences from the CTX element zymogram and in CTX Components quite conservative sites: Bgl Ⅱ, EcoRⅤ, Pst Ⅰ, EcoR Ⅰ, no point in the pCTAK. The ctxAB and zot primers were positive for PCR amplification of pCTAK and the RS1 probe was positive for hybridization. The sequences of ctxAB and zot genes of pCTAK were highly homologous with the reported sequences. pCTAK can transform DH5α and IEM101 directly or via vectors and express CT. Filamentous phage particles CTAKΦ were isolated from the culture supernatant of FJ97129. The diameter of the filamentous phage was about 7nm by electron microscopy. The whole genome size is 8.5kb, for?