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研究生:方榮華
研究生(外文):Fang Rong Hwa
論文名稱:洛德因乳酸桿菌抗藥質體的鑑定與選殖
論文名稱(外文):Identification and cloning of antibiotic resistant plasmids from Lactobacillus reuteri
指導教授:張登欽張登欽引用關係
指導教授(外文):Chung Tung Ching
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:1994
畢業學年度:82
語文別:中文
論文頁數:131
中文關鍵詞:洛德因乳酸桿菌
外文關鍵詞:Lactobacillus reuteri
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到目前為止,能成功的以高效率被轉形於洛德因乳酸桿菌(reuteri-pro-
ducing Lactobacillus reuteri;RPLR )之 plasmid,大部分都是源自
RPLR 本身之 Homologous plasmid(如pLUL631、pLUL612,...等)。有鑑
於 RPLR 仍未有可用之 cloning vector , 因此若要建造該菌之有效
vector,勢必得由 Homologous plasmid之各控制因子(如R-gene,promo-
ter,replication origin,....等)下手才易成功因此本實驗由本省分離
RPLR菌,並篩選具紅黴素(Erythromycin;Em)或氯黴素(chloramphenicol;
Cm)抗性菌株五株,經以 CsCl-EtBr gradient centrifugation 方法抽出
其所含質體後,採用電孔(electroporation)方法,轉殖入 RPLR 的標準株
DSM20016( 不含任何質體 ), 最後得以鑑定出四個抗紅黴素質體,分別是
pTE31(4.4 kb),pTE32(7.0 kb),pTE33(7.0 kb),pTE36(7.0 kb)和一個抗
氯黴素質體 pTC88(7.0 kb)。 為證明獲得抗藥能力之 transformant 其
抗藥質體確是由上述 Donor strain 而來, 本實驗分別進行了1. 抗生素
最小抑菌濃度(MIC)測定, 2. 限制酵素分析法 3. 南方雜合法(Southern
blotting hybridization) 而得以確認之 。本實驗更進一步訂出五個質
體的限制酵素圖譜. 經以適當限制酵素切出 R-gene 片段 , 並以 pUC19
為載體選殖表現於 E. coli TG1 及 E. coli DH5α. 試驗結果, 計得到
五個含抗藥基因的 recombinant plasmids, 其分別是 1. pUE3116:(含
em R-gene 片段 2.8 kb) , pUE3220 (含 em R-gene 片段 2.0 kb) ,
pUC3320(含 em R-gene 片段 2.0 kb) , pUE3628( 含 em R-gene 片段
2.8 kb), pUC8835(含 cm R-gene 片段 3.5 kb),這些轉殖成功的抗藥基
因片段, 將可以進一步行定序及作為將來建造 RPLR 載體時使用。
Up to now , only homologous plasmids , such as pLUL631 、
pLUL612 , ...etc.,were be able to be transformed into reuterin-
producing Lactobacillus reuteri;(RPLR) at high frequencies.Also
for the time being, no efficient cloning vector is avaiable for
RPLR.Under such a circumstance,identifying parts(i.e.,replicon
、 promoter and R-gene)directly from homologous plasmids of
RPLR is the best initiate way for constructing cloning vector
for RPLR. Five local RPLR strains,among them four were
resistant to erythromycin(EM) and one was resistant to
chloramphenicol(CM) , were selected for this studies. We
isolated plasmids from these antibiotic resistant strains by
CsCl-EthBr gradient centrifuga- tion method and transformated
them into RPLR type strain DSM20- 016, which is free from any
plasmid, by electroporation method. By analyzing antibiotic
resistant transformants acquired in this experiment. We were
able to identify four EM resistant plasmids(which were pTE31
【4.4 kb】、pTE32【7.0 kb】、pTE33【7. 0 kb】,and pTE36【15.0 kb
】) and one CM resistant plasmid(which was pTC88【7.0 kb】;
Further confirmation for these R-plasmids as originating from
donor strains were conducted by ① minimum inhibitory
concentration test,②restriction DNA length analysis, and ③
southern blotting hybridization . We also cloned and expressed
R-gene fragments from these five plasmids into E. coli TG1 and
E. coli DH5α by pUC19 cloning system; Five recombinant
plasmids including pUE3116(4.3 kb;containing a 1.6kb e■DNA
fragment),pUE3220(4.7kb;containing a 2.0 kb e■DNA fragment),
pUE3320(4.7 kb;containing a 2.0 kb e■ DNA fragment),
pUE3628(5.5 kb;containing a 2.8kb e■DNA fragment), and
pUC8835(6.2 kb; containing a 3.5 kb c■ DNA fragment )were
obtained in this experiment. This is the first success of
cloning and expressing c■ gene fragment from RPLR into E.
coli. These R-gene fragments, after further sequencing,will be
a good source of marker parts for constructing cloning vector.
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