王金和臺灣大學：獸醫學研究所邱振豪(Chenhao Chiu)Chenhao Chiu2007-11-282018-07-092007-11-282018-07-092006http://ntur.lib.ntu.edu.tw//handle/246246/59925中文摘要 鴨疫里莫氏桿菌 （Riemerella anatipestifer, RA） 引起水禽及火雞之多發性漿膜炎，造成嚴重經濟損失。RA共有21種血清型，其中台灣以第2血清型為最主要。不活化菌苗在不同血清型別間無交叉保護，亟需發展活菌疫苗預防本病發生。RA之分子生物基礎研究報告不多，毒力因子尚未完全明瞭。近年有多種細菌曾利用跳躍子 (transposon) 和穿梭載體 (shuttle vector) 進行基因突變且成功選殖具免疫力之突變株。本實驗利用基因相減雜交法分析RA菌株之基因多樣性及利用跳躍子和穿梭載體技術製備RA突變株，以供深入探討RA之致病因子及發展疫苗之用。應用基因相減雜交法區分DNA片段僅存在於台灣第二血清型RA臨床分離株而不存在於RA第二血清型疫苗弱毒株。選殖36個DNA片段，以BLAST 程式分析中，有9個選殖片段與RA質體相關，2個與Bacteroides fragilis 基因相關，1個與Yersinia基因相關，1個與Eubacterium partial 16S rRNA相似，其餘23片段沒有與任何巳知基因相似。蛋白質比對結果，有5個為未知功能的假設性蛋白質 (hypothetical protein)，9個選殖片段分別為β-lactamase 負調控蛋白 (negative regulator of β-lactamase)、複製酶(replicase)、尿紫質元脫梭酶 (uroporphyrinogen decarboxylase)、整合酶(integrase)、溶菌酶 (lysozyme)、複製性蛋白 (replication protein)、ATP酶 (ATPase)、成隱性毒素 (addiction module toxin)，16個片段則與細菌蛋白相似性低，另外6個無相似性蛋白。實驗結果顯示基因相減雜交法可確認存在於第二血清型RA野外分離株之DNA片段，進而提供RA基因差異研究的開始。在製作 RA 突變株方面，以三類跳躍子 (1) Mini-Tn5 Km2, Mini-Tn5 Cm, pLOF/Km 或 pLOF/Cm 及 (2) pEP4351, R751::Tn4351 (3) pSC189/Km 或pSC189/Cm 進行 RA 之突變，篩選出陽性轉型株以沙忍氏墨點轉漬法確定是否有轉位 (transposition) 發生。另外利用其他菌種之穿梭載體： pCP23, pCP29, pGP704Sac28, pGP704Sac38，做特定基因之置換，進而達到突變作用。在 Mini-Tn5 Km2，Mini-Tn5 Cm， pLOF/Km 或 pLOF/Cm, pSC189/Km 或 pSC189/Cm 等跳躍子皆未篩得突變株，非 RA 來源之穿梭載體亦未發現 RA 突變株。由Proteobacteria 和 CFB group 來源之跳躍子皆無法在 RA 菌體上有功用？推測原因可能為： 1. RA 無法認識跳躍子抗藥基因的起動子。 2. RA 本身之內切限制系統阻斷了外來基因進入。 3. RA 無法複製和保留整個帶有抗藥基因的移動序列。4. 跳躍子之 transposase gene 無法被 RA 的 RNA polymerase 辨認，因而無轉位的動作。Abstract Riemerella anatipestifer infection is an economic important disease in waterfowl and turkey industries. Twenty-one different serovars of R. anatipestifer have been reported and serovar 2 is the major type in Taiwan. PCR-based subtractive hybridization was employed to isolate DNA fragments in a tested R. anatipestifer strain. Serovar 2 of Taiwan local strain presented the specific fragments but was absent in reference avirulent vaccine strain. Thirty six specific DNA fragments in the local wild strain were obtained. The fragments were sequenced and their DNA sequences were analyzed by BLAST program. Among them 9 DNA fragments (genes) related to R. anatipestifer plasmids, 2 DNA fragments related to B. fragilis genes, 1 DNA fragment related to Yersinia, 1 DNA fragment related to eubacterium partial 16S rRNA gene and the other 23 DNA fragment had no DNA sequence homologs in the Genbank. Protein analysis showed that 5 clones had homology to hypothetic proteins, 9 clones were similar to negative regulator of β-lactamase, replicase, uroporphyrinogen decarboxylase, integrase, lysozyme, replication protein, ATPase, and addiction module toxin, respectively and 16 clones had a low similarity to bacterium protein. The other 6 clones encode proteins with unknown function. Subtractive hybridization technique is successfully identifying genes in field isolated R. anatipestifer that are absent in the reference avirulent vaccine strain. These results provide new insights into R. anatipestifer genetic diversity. Attempts to generate mutants by using transposons and shuttle vectors, but failed. The reasons that we are unable to generate mutants in R. anatipestifer with the broad-host-range plasmids and transposons are: (1). the plasmids and transposons were unable to transfer into the R. anatipestifer. (2). the plasmids and transposons might be degraded by R. anatipestifer restriction system. (3). the antibiotic gene from the plasmids or transposons could not express in R. anatipestifer and (4). The promoter of the transposase gene (tnp) could not be recognized by R. anatipestifer’s RNA polymerase and no transposase was expressed.目錄 目錄-------------------------------------------------------------------------------------------- Ⅰ 圖次-------------------------------------------------------------------------------------------- Ⅳ 表次-------------------------------------------------------------------------------------------- Ⅴ 中文摘要-------------------------------------------------------------------------------------- Ⅵ 英文摘要-------------------------------------------------------------------------------------- Ⅷ 第一章 前言------------------------------------------------------------------------------- 1 第二章 文獻探討------------------------------------------------------------------------- 2 第一節 Riemerella anatipestifer之病原性---------------------------- 2 2.1.1細菌分類沿革--------------------------------------------- 2 2.1.2形態學與生化特性--------------------------------------- 3 2.1.3血清學------------------------------------------------------ 4 第二節 Riemerella anatipestifer感染症------------------------------- 5 2.2.1流行病學--------------------------------------------------- 5 2.2.2臨床症狀和病理變化------------------------------------ 6 2.2.3診斷與治療------------------------------------------------ 7 2.2.4預防與控制------------------------------------------------ 7 2.2.5 RA 毒力因子研究及疫苗開發------------------------ 8 第三節 基因抑制相減雜交法------------------------------------------ 11 2.3.1引言--------------------------------------------------------- 11 2.3.2原理及應用------------------------------------------------ 12 第四節 跳躍子及穿梭載體--------------------------------------------- 17 2.4.1 跳躍子----------------------------------------------------- 17 2.4.2 穿梭載體-------------------------------------------------- 18 第三章 材料與方法---------------------------------------------------------------------- 19 第一節 細菌與載體------------------------------------------------------ 19 3.1.1 R. anatipestifer菌株-------------------------------------- 19 3.1.2選殖用E. coli --------------------------------------------- 20 3.1.3載體--------------------------------------------------------- 21 第二節 質體DNA 之抽取---------------------------------------------- 22 3.2.1小量質體DNA製備------------------------------------- 22 3.2.2小量質體DNA抽取套組 ------------------------------ 22 第三節 基因抑制相減雜交法 ----------------------------------------- 23 3.3.1 R. anatipestifer-------------------------------------------- 23 3.3.2 E.coli ------------------------------------------------------- 24 3.3.3基因體DNA製備---------------------------------------- 24 3.3.4基因抑制相減雜交試驗--------------------------------- 25 3.3.5 菌量 PCR----------------------------------------------- 28 3.3.6 DNA墨點法----------------------------------------------- 28 3.3.7沙忍氏墨點轉漬分析------------------------------------ 29 3.3.8核酸之定量------------------------------------------------ 32 3.3.9 DNA定序及分析----------------------------------------- 32 第四節 轉型作用--------------------------------------------------------- 33 3.4.1氯化鈣法--------------------------------------------------- 33 3.4.2電穿孔轉型法--------------------------------------------- 33 第五節 接合反應--------------------------------------------------------- 34 第四章 結果------------------------------------------------------------------------------- 35 第一節 R. anatipestifer菌株之鑑定----------------------------------- 35 第二節 抑制相減雜交法選殖片段的之確認------------------------ 35 第三節 選殖片段DNA序列分析-------------------------------------- 36 第四節 R. anatipestifer 突變株製備---------------------------------- 37 第五節 沙忍氏墨點轉漬分析跳躍子之插入位--------------------- 37 第五章 討論------------------------------------------------------------------------------- 55 第六章 參考文獻------------------------------------------------------------------------- 591973738 bytesapplication/pdfen-US鴨疫里莫氏桿菌基因體相減雜交跳躍子穿梭載體Riemerella anatipestiferGenome subtractive hybridizationTransposonShuttle vector[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/59925/1/ntu-95-R92629001-1.pdf