張照夫2006-07-262018-07-092006-07-262018-07-092004http://ntur.lib.ntu.edu.tw//handle/246246/28728以標籤式突變法(STM) 鑑定豬霍亂沙門氏菌在豬隻活體內存活有關基因。將獨 特DNA 序列標籤(tags) 築入具抗康黴素基因標誌(marker) 之自殺性質體載體- pUTminiTn5Km2, 以接合作用將此載體送入對康黴素有感受性之豬霍亂沙門氏 菌體內，構築STM 基因庫。帶有獨特DNA 序列標籤之自殺性載體藉跳躍子突 變(transposon mutation) 插入抗康黴素豬霍亂沙門氏菌體內形成突變株。960 個 突變株組合為20 群(pool), 每群含48 個突變株。每群突變株接種2 頭豬隻, 篩 檢活體內存活有關基因。經活體篩檢後, 有34 株不能由腸系膜淋巴結回收，此 34 株可被認定為毒力弱化之突變株。利用逆向PCR 增幅嵌入康黴素基因兩端區 域并定序之。定序結果顯示，此34 株突變區域各異，可歸為八類基因：(1) 細 胞封套，(2) 第三型分泌系統，(3) 毒力質體，(4) 運送，(5) Gifsy-1 前噬菌體蛋 白，(6) 調控基因，(7) 代謝，及(8) 未知功能。A signature-tagged mutagenesis (STM) system was adapted to identify the genes required for in vivo survival of Salmonella enterica serovar Choleraesuis (Sal. Choleraesuis) in pigs. An STM library was constructed in Sal Choleraesuis (kanamycin sensitive) by conjugation with a suicide plasmid vector (pUTminiTn5Km2) which contains unique DNA sequence tags and a kanamycin resistance marker. Sal Choleraesuis (kanamycin-resistant) mutants were obtained by transposon mutation, resulting in gene inactivation by insertion of the suicide vector carrying a unique tag. 960 mutants of Sal Choleraesuis were pooled together (48 mutants/pool) and screened in a pig model. Out of 960 mutants, 34 of them were not recovered from the mesenteric lymph node. These 34 mutants were considered as virulence-attenuated mutants. In order to identify the virulence genes, the regions flanking the kanamycin gene of the attenuated mutants were amplified by inverse PCR and sequenced. The sequence analysis showed that the 34 mutants had insertions in 34 different genes that could be grouped in eight classes: (i) genes encoding cell envelope; (ii) type III secretion system; (iii) gene encoded in a virulence plasmid; (iv) genes involved in transport; (v) genes coding for Gifsy-1 prophage protein; (vi) genes encoding proteins involved in regulatory; (vii) genes involved in metabolic pathways; and (viii) DNA sequences that showed similarity to hypothetical genes with unknown function.application/pdf181729 bytesapplication/pdfzh-TW國立臺灣大學獸醫學系暨研究所豬霍亂沙門氏菌標籤式突變法毒力因子活體內表現突變種Samonella Choleraesuissignature-tagged mutagenesisvirulence factorin vivo expressionmutant[SDGs]SDG3reporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/28728/1/922313B002136.pdf