張照夫2006-07-262018-07-092006-07-262018-07-092003http://ntur.lib.ntu.edu.tw//handle/246246/28718豬霍亂沙門氏菌(Salmonella choleraesuis) 是兼性細胞內寄生性病原，能引起豬 隻敗血症及腸炎，死亡率甚高，為養豬業重要之傳染病，經濟損失嚴重。本菌偶 亦引發人類之敗血症、腸炎及骨髓炎，為人畜共通傳染病原，威脅公共衛生安全。 過去對細菌毒力因子之分子生物學研究，都以試管內(in vitro) 毒力基因表現為 研究方法。試管內表現之毒力因子與病原菌在動物宿主活體內(in vitro) 表現的 基因產物，未必相同。因此以試管內基因表現技術研究細菌之致病機制，仍有許 多盲點無法克服。本計畫擬以標籤式突變法分析S. choleraesuis 在豬活體內表現 之毒力因子，分三年進行。第一年為突變庫製備。以墨點雜合法篩檢282 個被 Bio-dot 濾過裝置固定在Hybond N+尼龍膜之質體。為檢查是否為交叉雜合產生 的雜合訊號，以94 個質體之標籤作成的探針雜合282 質體，將有交叉雜合的質 體刪除。其餘的質體以相同的方式持續進行雜合，作為探針質體之數目則遞減 為48、24 和8。結果得到82 個pUT mini-Tn5Km2 質體含有獨特標籤序列。一 組48 個含有強烈雜合訊號標籤之質體被重複應用構築成20 個S. choleraesuis 突 變庫。Salmonella choleraesuis is a facultative intracellular pathogen that causes a septicemic disease of pigs. S. choleraesuis is host-adapted for swine. S. choleraesuis occasionally also causes human infection, so it is a zoonotic pathogen. Although S. choleraesuis causes an economic loss in swine industry, the information about the virulence genes of this organism is very limited. The hypothesis is that there are many proteins which are only present when the bacteria are growing in the animal host (in vivo) and not when the pathogen is grown under laboratory conditions (in vitro), and that these proteins play a critical role in the disease process. The goal is to identify genes encoding potential virulence factors that are expressed only in vivo in the swine host. These proteins may prove to be important in the pathogenesis of S. choleraesuis infection in pigs. Generation of the S. choleraesuis mutant bank was the purpose of the first year project. A series of identical membranes for dot blot hybridizations was prepared by transferring of 282 plasmids onto Hybond N+ membranes using the Bio-dot Microfiltration Apparatus. To test whether the hybridization signals resulted from cross-hybridization between tags, amplified tags from 94 plasmids were used to probe a dot blot of 282 plasmids, which included the 94 used to generate the probe. There were cross-hybridization to the probe. These plasmids were deleted. The same method was repeatedly for the other plasmid. Cross-hybridization was continued but the amount of plasmid was used to probe were 48, 24 and 8. Eighty-two pUT mini-Tn5Km2 plasmids containing unique tag were found. A set of 48 plasmids containing strongly-hybridizing tags was repeatedly used to construct 20 libraries of S. choleraesuis.application/pdf843574 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活體內毒力因子Salmonella choleraesuissignature-tagged mutagenesisin vivo virulence factor[SDGs]SDG3reporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/28718/1/912313B002386.pdf