| Summary: | 碩士 === 嘉南藥理科技大學 === 生物科技系暨研究所 === 97 === Vibrio vulnificus a halophilic gram-negative marine bacterium that is survied in ocean, estuarine water. V. vulnificus causes both fatal primary septicemia and wound infection in human, mortality was up to 50% in septic patients, with most of them dying within 48 h. Recently, Several putatie virulence factors of V. vulnificus are demonstrated, such as capsule, metalloprotease, phospholipase, cytolysin, iron acquisition systems and RTX toxin. To identify the genes required for killing of macrophage, we screened an insertional mutant library of V. vulnificus generated by transposon mutagenesis for reduced cytotoxicity toward macrophage. Here we reported that among 867 Tn5 mutants screened, 7 Tn5 mutants exhibited decreased cytotoxic activity toward marcrophage. Four genes inserted by transposon were cloned and sequenced. Sequence analysis revealed that the transposon was inserted into the genes : rtxA, rtxE, argD and VV3039. RtxA was composed of 5,206 amino acids with a theoretical molecular mass of ~572 kDa and is a member of RTX family. RtxE was composed of 722 amino acids with a theoretical molecular mass of ~79 kDa. The RtxE is an ABC transporter (ATP-binding cassette transporter) that belongs to a type I secretion system. ArgD was composed of 403 amino acids with a theoretical molecular mass of ~44 kDa and was involved in an urea cycle pathway and amino acid metabolism. VV3093 was composed of 636 amino acids with a theoretical molecular mass of ~23 kDa and probably was a transcription regulator. In this study, we cloned and sequenced four genes: rtxA, rtxE, argD and VV3093 and these genes probably were involved in cytotoxicity towards macrophages.
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