以冷休克蛋白作為放線菌分類上質譜分析之標的蛋白

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研究生：	賴怡卉 Yi-Hui Lai
論文名稱：	以冷休克蛋白作為放線菌分類上質譜分析之標的蛋白 A cold shock protein as the mass spectrometric biomarker for classification of actinomycetes
指導教授：	許宗雄博士 Dr. Tzong-Hsuing Hseu
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物科技研究所 Biotechnology
論文出版年：	2003
畢業學年度：	91
語文別：	英文
論文頁數：	54
中文關鍵詞：	放線菌、質譜分析、冷休克蛋白
外文關鍵詞：	actinomycetes, mass spectrometric analysis, cold shock protein
相關次數：	點閱：2 下載：0
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利用質譜分析的方法，可以找到放線菌其中兩屬，分別為Microbispora 和Dactylosporangium，所特有的標的蛋白 (biomarker)，並且能夠作為菌種分類的依據。這個蛋白質經過資料庫搜尋比對後發現可能是一種cold shock protein。為了更進一步了解這個cold shock protein，我們利用PCR把其中一個cold shock protein由另一隻放線菌株，S. ceolicolor中複製出來，並使用重組蛋白的方法製作出所需要的抗體。我們從Microbispora 和Dactylosporangium兩種菌屬中各挑選一隻菌，以及S. ceolicolor，進行低溫誘導(cold shock induction)的實驗，由蛋白質以及基因的層次來研究菌株裡cold shock protein的表現情形。結果發現這三隻菌的cold shock protein在低溫處理之下並沒有產生預期的大量表現，而是constitutive expression。另一方面，cold shock protein 的mRNA 卻會受到低溫誘導而增加表現量，然後隨著低溫處理的時間增加而慢慢減少終至消失。但是質譜分析的結果卻顯示這些菌的冷休克蛋白大小不能完全符合預期。所以如果要以冷休蛋白作為放線菌分類的依據，必須建立一個較嚴謹且標準化的放線菌培養條件，才能在質譜分析上達到較高的再現性。

A mass spectrometrically identified biomarker, possibly “cold shock protein” by database homologous searching, can be helpful to classify two genus of actinomycetes, Microbispora and Dactysporangium. To further characterize cold shock proteins, we amplified the gene of a cold shock from another genus of actinomycetes, S. coelicolor and make specific antibody against it. Cells were maintained at low temperature for 0~24 h, cold shock proteins and their mRNAs isolated from S.coelicolor, D. fusco-aurantiacum and M. amethystogenes were monitored by Western and Northern blot. The finding suggested cold shock proteins didn’t change as much as expected. Nevertheless, their mRNAs increased abruptly upon temperature downshift (cold shock after 1 h), started to decrease after 2 h and finally disappeared at 24 h. However, findings of MALDI-LTOF mass analysis of these strains were not analogous to our expected results. The m/z values of their cold shock proteins were different from that found by a previous report. Therefore, whether cold shock protein is suitable to be a biomarker in mass analysis for actinomycetes classification should be further evaluated. We should improve the mass spectrometric reproducibility by strictly establishing more standardized actinomysetes culture condition.

List of Figures---------------------------------------------------------------------- ii
一、 中文摘要-------------------------------------------------------------------- 1

二、 英文附錄

1. Abstract----------------------------------------------------------2

2. Introduction----------------------------------------------------- 3

3. Materials and Methods---------------------------------------- 7

4. Results---------------------------------------------------------- 15

5. Discussion------------------------------------------------------ 20

6. Acknowledgments--------------------------------------------- 23

7. Reference--------------------------------------------------------24

List of Figures

Fig. 1.     (A) Alignment of rScoF2 with CspA in E.coli and all cold shock proteins in S.coelicolor  (B) Cluster analysis of rScoF2 and cold shock proteins in S.coelicolor.--------------------------------------------------------------------------------------32

Fig. 2.     A 204-bp DNA fragment was amplified by S. coelicolor genomic PCR with (A) no DMSO；(B) 5％ DMSO；(C) 10％ DMSO. -----------------------------------------------33

Fig. 3.     A 204-bp DNA fragment was amplified by D. fusco-aurantiacum genomic PCR

with 10％ DMSO. ------------------------------------------------------------------------------34

Fig. 4.     A 204-bp DNA fragment was amplified by M. amethystogene genomic PCR with

10％ DMSO.-------------------------------------------------------------------------------------35

Fig. 5.     15％ SDS-PAGE analysis of GST-ScoF2 overexpression in BL21 (DE3) cells. ----- 36

Fig. 6.     12％ SDS-PAGE analysis of GST-ScoF2 purification. -----------------------------------37

Fig. 7.     12％ SDS-PAGE analysis of GST-ScoF2 purification (thrombin treated). -------------38

Fig. 8.     15％ SDS-PAGE analysis of concentrated GST-ScoF2 and rScoF2. --------------------39

Fig. 9.     Alignment of rScoF2 with CspA in E. coli and ScoF2-like proteins in

D. fusco-aurantiacum and M. amethystogenes-----------------------------------------------40

Fig. 10.    Western blot of recombinant ScoF2 recognized by (A) Anti-6x His-tag

monoclonal antibody；(B) purified Anti-ScoF2 polyclonal------------------------------41

Fig. 11.    Western blot of ScoF2 in S. coelicolor. (A)15％ SDS-PAGE analysis of

S. coelicolor total protein. (B) ScoF2 expression upon cold shock. ----------------------42

Fig. 12.    Western blot of ScoF2-like cold shock protein in D. fusco-aurantiacum.

(A) 15％ SDS-PAGE analysis of D. fusco-aurantiacum total protein.

(B) ScoF2-like protein expression upon cold shock. --------------------------------------- 43

Fig. 13.    Western blot of ScoF2-like cold shock protein in M. amethystogenes.

(A) 15％ SDS-PAGE analysis of M. amethystogenes total protein.

(B) ScoF2-like protein expression upon cold shock. --------------------------------------- 44

Fig. 14.    Northern Blot of scof2 mRNA in S.coelicolor. (A) Ribosomal RNAs.

(B) scof2 mRNA changes upon cold shock. ------------------------------------------------- 45

Fig. 15.    Northern Blot of scof2-like mRNA in D. fusco-aurantiacum. (A) Ribosomal RNAs.

(B) scof2-like mRNA changes upon cold shock. ------------------------------------------- 46

Fig. 16    MALDI-LTOF spectrum of S. coelicolr cell extract.---------------------------------------- 47

Fig. 17    MALDI-LTOF spectrum of D. fusco-aurantiacum cell extract.--------------------------- 48

Fig. 18    MALDI-LTOP spectrum of D. aurantiacum cell extract.------------------------------------49

Fig. 19    MALDI-LTOF spectrum of M. amethystogenes cell extract.-------------------------------50

Fig. 20    MALDI-LTOF spectrum of M. chromogenes cell extract.----------------------------------51

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