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標題: Xanthomonas albilineans 細菌素基因選殖及 Xanthomonas campestris pv. glycines 細菌素基因表現之探討
Cloning the bacteriocin genes of Xanthomonas albilineans and characterization of bacteriocin from Xanthomonas campestris pv. glycines
作者: Luo, You-Wei
關鍵字: glycinecin A;細菌素;bacteriocin;phage tail-like
出版社: 分子生物學研究所
細菌素為細菌所分泌可以抑制親緣關係接近菌種的蛋白質,Xanthomonas campestris pv. glycines YR32 (XcgYR32) 與 Xanthomonas albilineans (Xa) 為過去經由實驗室篩選會抑制其他 Xanthomonas 菌種的菌株。其中 X. albilineans 所分泌的細菌素可以利用 polyethylene glycol 6000 沉澱及使用分子篩管柱進行純化,之後經由實驗確認 Xa 細菌素為 phage tail-like bacteriocin。本研究嘗試使用另一種純化步驟,改用蔗糖梯度離心進行純化並以二維電泳分析 Xa 細菌素的蛋白組成。二維電泳可分離出 9 個蛋白點,經 trypsin 切割後以質譜儀 (LC/MS/MS) 進行分析。這些蛋白的胺基酸序列經比對後,最接近 Pseudomonas syringae pv. syringae B728a 中的 prophage 蛋白。Xa 細菌素的基因組成本研究亦藉由構築 Xa 部分基因庫來進行分析。從構築的基因庫中所篩選出的 5.8-kb 及 4.6-kb DNA 片段總共有 14 個 ORFs 可以被辨識出來。這些 ORFs 也與存在於 Pseudomonas syringae pv. syringae B728a 中的 Mu phage 基因序列具有高度的相同性。
本研究的另一個部份是將 XcgYR32 的細菌素基因 glyA 及 glyB 利用不同的 pET 載體並於不同的 E. coli 菌株中進行表現。在 BL21(DE3) 中以低濃度的 IPTG 及低溫度的誘導條件,可以表現出少量具有抑菌活性的可溶性 GlyAB-His。之後使用 Ni2+-NTA 及 Hiprep 16/60 Sephacryl S-300 分子篩管柱純化 GlyAB-His 蛋白。每 100 ml 菌液可純化的 GlyAB-His 蛋白約 50 μg,比活性為 212 x 100 AU/ml。GlyAB-His 可耐受 70℃ 1 小時活性不下降,且可耐受 trypsin、chymotrypsin 的剪切。為了提升 Glycinecin A 的耐受度,構築了 chimeric Glycinecin A N. 5~7 (chimeric A5~A7)。結果顯示 chimeric A5、chimeric A6 表現量極大,但無法偵測到殺菌活性且都為不可溶蛋白,而 chimeric A7 的構築則為可溶性且具有殺菌活性。

Bacteriocin is known to be produced by bacteria and has bactericidal activity against the closely related species. Two Xanthomonas species, Xanthomonas albilineans (Xa) and Xanthomonas campestris pv. glycines YR32 (XcgYR32), have been identified in this laboratory and have antimicrobial activity toward several tested Xanthomonas strains. Moreover, bacteriocin produced by Xa had been purified from cultural broth by polyethylene glycol 6000 precipitation and gel filtration chromatography and characterized as a phage tail-like bacteriocin. An alternative purification procedure using sucrose gradient centrifugation was applied in this study and the components of the purified protein complex were then analyzed by two-dimensional gel electrophoresis. A total of nine protein spots were isolated, digested by trypsin and analyzed by LC/MS/MS. Results of the BLAST analysis revealed that most of these proteins are closely related to prophage proteins of Pseudomonas syringae pv. syringae B728a. The genes corresponding to the Xa bacteriocin were identified from the constructed Xa partial genomic library. DNA sequence analyses revealed that a total of 14 ORFs could be identified in the cloned 5.8-kb and 4.6-kb DNA fragments from the constructed libraries. These ORFs also show high sequence similarity to the Mu phage of Pseudomonas syringae pv. syringae B728a.
In the second part of this study, the bacteriocin genes, glyA and glyB, of XcgYR32 were subcloned into various pET vectors and expressed in several E. coli strains. A very low amount of soluble GlyAB-His with bactericidal activity could be obtained from BL21(DE3) at a lower IPTG concentration and induction temperature. The GlyAB-His protein was purified through Ni2+-NTA column and Hiprep 16/60 Sephacryl S-300 column chromatography. A total of 50 μg purified GlyAB-His could be obtained from 100 ml of culture and with a specific activity of 212 x 100 AU/ml. The purified GlyAB-His was stable at temperature up to 70℃ for one hour and resistant to trypsin and chymotrypsin digestion. To promote the stability of GlyA and GlyB, chimeric proteins GlyA5 to A7 were constructed. Results showed that a very high level expression of chimeric A5 and A6 proteins could be achieved, however, most of the expressed proteins were insoluble and the chimeric protein expressed from A7 construct was found to be soluble and have bactericidal activity.
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