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研究生:黃仲德
研究生(外文):Chung-Ter Huang
論文名稱:胃幽門螺旋桿菌之核糖核酸酶P基因的蛋白質表現,純化與功能鑑定
論文名稱(外文):EXPRESSION, PURIFICATION AND CHARACTERIZATION OF RNASE P PROTEIN COMPONENT FROM HELICOBACTER PYLORI
指導教授:黃海美
指導教授(外文):Haimei Huang
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:51
中文關鍵詞:胃幽門螺旋桿菌酸誘導核糖核酸酶
外文關鍵詞:H.pyloriacid-inducedRNase P
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首先利用資料庫進行基因序列的比對與搜尋,鎖定的範圍為相似度(homology)小於30%的基因; 另外,我又從Ang et al所著的文獻中得知,胃幽門螺旋桿菌在酸性環境下會誘導或抑制某些基因的mRNA的表現量,所以從中挑選另外6個基因來進行研究,因此HP0270, HP0555, HP0746, HP1493, HP1074. HP1209 與 HP1448為研究的目標基因。首先將這7個基因分別建構入在5’含有6個Histidine的pQE30載體中。接下來將建構好的載體轉形(transformation)至SG13009菌株中,並使用相對應的引子做PCR分析與使用IPTG去誘導細菌在37℃,3個小時環境下表現蛋白質。之後發現所選擇的7個基因中,帶有HP1448基因的質體,編號為10號的菌株,能成功表現出可溶性的蛋白質,所以接下來便以HP1448基因為我研究的目標基因。
轉型成功後的菌株,使用(a).0.5 mM IPTG誘導細菌在25℃下9個小時,或在(b).20℃下20小時的環境下所表現的蛋白質,比在37℃下3小時較多。接下來使用(b)的方式去誘導1升的細菌做蛋白質表現。使用Ni-NTA親和力管柱純化細菌內所表現的重組蛋白質HP1448,最後約可得到1.5 mg的蛋白質。然而,發現純化後的HP1448蛋白質,在pH等於8時,會發生蛋白質沉澱的現象,因此改變儲存HP1448蛋白質緩衝液的pH值,將pH值降到6.5,即可避免蛋白質的沉澱,並可保存一個月。所以,此重組蛋白質HP1448可以穩定儲存在此緩衝液中,並可進一步對蛋白質結構與蛋白質功能作深入研究與探討。
重組蛋白質HP1448可以當做抗原(antigen)並選用兔子來進行免疫實驗以製備多株抗體.從第5次注射後所得到的兔子血清經過處理後,得到的抗體(anti-HP1448)經過校價的測試,發現稀釋比例1:4000可以偵測到16 ng的重組蛋白質HP1448。因此,便可利用所製作的抗體與西方點墨法去分析胃幽門螺旋桿菌在不同環境下,HP1448蛋白質的表現量。
胃幽門螺旋桿菌在pH 5.5與pH 7.2的Brucella agar plates培養48小時後,HP1448蛋白質在pH5.5的表現量比在pH7.2的環境下略少,但在含有尿素且pH 5.5的環境下,HP1448蛋白質表現量比在pH 7.2高出1.7倍。將胃幽門螺旋桿菌培養在pH 5.5且含有尿素的Brucella broth medium下發現,HP1448蛋白質的表現量比在pH7.2的環境下高出5倍。但是在沒有加入尿素在Brucella broth medium,在pH 5.5所表現的HP1448蛋白質為在pH7.2時的一半。
HP1448蛋白質在資料庫中被預測為RNase P的protein component,此蛋白質所扮演的角色,可以藉由RNase P的酵素實驗來證實。這實驗中的受質與RNase P 的RNA component可以利用 in-vitro的轉錄實驗得到,所以一開始要先將受質與RNA component的基因分別建構入pGEM的載體中。而RNase P 的RNA component在高離子強度的緩衝液下,具有修飾受質的功能。而蛋白質HP1448是否能增加RNA component在低離子強度緩衝液下的活性,還需要進一步的證實。
HP1448蛋白質的製備流程在這研究中已經建立,且在4℃蛋白質能穩定儲存在pH 6.5的緩衝液中,並應用去製作多株抗體,便能去觀察當幽門桿菌在不同環境下,HP1448蛋白質的表現量。在未來也可應用在RNase P酵素活性的測試與結構上的分析。
HP0270, HP0555, HP0746, HP1493, HP1074. HP1209 or HP1448 gene of H. pylori 26695 was cloned into the 5’ position of histidine-tag site in pQE30 vector, respectively. After transforming individual recombinant vector into E. coli SG13009, antibiotic resistant clones were screened with corresponding primers of various targeted genes by PCR and 1 mM IPTG-induction in individual culture at 37℃ for 3 hours. Since clone #10 contains the right sequence of the HP1448 gene in vector and can produce more soluble protein than the others, it was used in the rest of this study.
More recombinant (rec)- HP1448 protein was induced from bacteria culture after 0.5 mM IPTG addition (a) at 25℃ for 9 h, or (b) at 20℃ for 20 h , in comparison with those at 37℃ for 3 h. Approximately 1.5 mg HP1448 protein could be purified through Ni-NTA affinity column from 1 liter of bacteria culture containing IPTG. Newly prepared protein aggregated easily in Tris imidazole NaCl elution buffer at pH 8.0. If this elution buffer was replaced to pH of 6.5 for protein storage at 4℃, HP1448 protein would not aggregate for at least 1 month. Therefore, the HP1448 protein was stored in this buffer for further functional and structural studies in this research.
The purified HP1448 protein was used as an antigen to produce antibodies in rabbits. Anti-HP1448 antibody from rabbit sera were prepared and tittered. About 16 ng of HP1448 protein could be detected in 1:4000 of anti-sera from rabbit after the 5th boosted injection. HP1448 protein expression was recognized in the H. pylori culture at different conditions by using antibodies and western analysis,.
In comparison with those at pH 7.2, HP1448 protein expression, was found in the H. pylori, was slightly down-regulated after growth on Brucella agar plates at pH 5.5 for 48 hours. In contrast, more than 1.7-fold HP1448 protein appeared from samples cultured on plates containing 5mM urea at pH 5.5 than those at pH 7.2. Without urea in the medium at pH 5.5, the HP1448 protein was expressed only one half of those from control in the H. pylori. However, cultured in a medium containing urea for 24 h, bacteria at pH 5.5 showed 4-fold HP1448 expression to those samples at pH of 7.2.
Since the HP1448 gene was annotated as a protein component of RNase P, its role was determined by means of in-vitro RNase P activity measurement. In this study, the substrate pre-tRNAPhe and the RNA component for RNase P assay were prepared by means of in-vitro transcription from the constructed pGEM vector inserted with a gene for pre-tRNA and a gene for RNA component of RNase P, respectively. This study indicated that the RNase P RNA component cleaved pre-tRNAPhe in the high salt buffer for RNase P assay. More studies should be conducted to determine the role of the recombinant HP1448 protein (as a cofactor for the assay of RNase P activity) by its ability to induce cleavage activity on pre-tRNAPhe in low salt condition.
謝誌
English abstract
Chinese abstract
Introduction 1
Materials and methods 7
Results 20
Discussion 26
Reference 30
Tables 32
Figures 36
Appendix 48
Ang, S., C.Z. Lee, K. Peck, M. Sindici, U. Matrubutham, M.A. Gleeson, and J.T. Wang. 2001. Acid-induced gene expression in Helicobacter pylori: study in genomic scale by microarray. Infect Immun. 69:1679-86.
Beebe, J.A., and C.A. Fierke. 1994. A kinetic mechanism for cleavage of precursor tRNA(Asp) catalyzed by the RNA component of Bacillus subtilis ribonuclease P. Biochemistry. 33:10294-304.
Blaser, M.J., G.I. Perez-Perez, H. Kleanthous, T.L. Cover, R.M. Peek, P.H. Chyou, G.N. Stemmermann, and A. Nomura. 1995. Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach. Cancer Res. 55:2111-5.
Dunn, B.E., H. Cohen, and M.J. Blaser. 1997. Helicobacter pylori. Clin Microbiol Rev. 10:720-41.
Guerrier-Takada, C., K. Gardiner, T. Marsh, N. Pace, and S. Altman. 1983. The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell. 35:849-57.
Harris, M.E., and E.L. Christian. 2003. Recent insights into the structure and function of the ribonucleoprotein enzyme ribonuclease P. Curr Opin Struct Biol. 13:325-33.
Kazantsev, A.V., A.A. Krivenko, D.J. Harrington, R.J. Carter, S.R. Holbrook, P.D. Adams, and N.R. Pace. 2003. High-resolution structure of RNase P protein from Thermotoga maritima. Proc Natl Acad Sci U S A. 100:7497-502.
Kraus, G., R. Geffin, G. Spruill, A.K. Young, R. Seivright, D. Cardona, J. Burzawa, and H.J. Hnatyszyn. 2002. Cross-clade inhibition of HIV-1 replication and cytopathology by using RNase P-associated external guide sequences. Proc Natl Acad Sci U S A. 99:3406-11.
Kurz, J.C., S. Niranjanakumari, and C.A. Fierke. 1998. Protein component of Bacillus subtilis RNase P specifically enhances the affinity for precursor-tRNAAsp. Biochemistry. 37:2393-400.
Lage, A.P., E. Godfroid, A. Fauconnier, A. Burette, J.P. Butzler, A. Bollen, and Y. Glupczynski. 1995. Diagnosis of Helicobacter pylori infection by PCR: comparison with other invasive techniques and detection of cagA gene in gastric biopsy specimens. J Clin Microbiol. 33:2752-6.
Lock, R.A., G.W. Coombs, T.M. McWilliams, J.W. Pearman, W.B. Grubb, G.J. Melrose, and G.M. Forbes. 2002. Proteome analysis of highly immunoreactive proteins of Helicobacter pylori. Helicobacter. 7:175-82.
Marshall, B.J., and J.R. Warren. 1984. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet. 1:1311-5.
Megraud, F. 1997. How should Helicobacter pylori infection be diagnosed? Gastroenterology. 113:S93-8.
Niranjanakumari, S., J.C. Kurz, and C.A. Fierke. 1998. Expression, purification and characterization of the recombinant ribonuclease P protein component from Bacillus subtilis. Nucleic Acids Res. 26:3090-6.
Raj, S.M., and F. Liu. 2003. Engineering of RNase P ribozyme for gene-targeting applications. Gene. 313:59-69.
Spitzfaden, C., N. Nicholson, J.J. Jones, S. Guth, R. Lehr, C.D. Prescott, L.A. Hegg, and D.S. Eggleston. 2000. The structure of ribonuclease P protein from Staphylococcus aureus reveals a unique binding site for single-stranded RNA. J Mol Biol. 295:105-15.
Stams, T., S. Niranjanakumari, C.A. Fierke, and D.W. Christianson. 1998. Ribonuclease P protein structure: evolutionary origins in the translational apparatus. Science. 280:752-5.
Tomb, J.F., O. White, A.R. Kerlavage, R.A. Clayton, G.G. Sutton, R.D. Fleischmann, K.A. Ketchum, H.P. Klenk, S. Gill, B.A. Dougherty, K. Nelson, J. Quackenbush, L. Zhou, E.F. Kirkness, S. Peterson, B. Loftus, D. Richardson, R. Dodson, H.G. Khalak, A. Glodek, K. McKenney, L.M. Fitzegerald, N. Lee, M.D. Adams, J.C. Venter, and et al. 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature. 388:539-47.
Wen, Y., E.A. Marcus, U. Matrubutham, M.A. Gleeson, D.R. Scott, and G. Sachs. 2003. Acid-adaptive genes of Helicobacter pylori. Infect Immun. 71:5921-39.
Xiao, S., F. Scott, C.A. Fierke, and D.R. Engelke. 2002. Eukaryotic ribonuclease P: a plurality of ribonucleoprotein enzymes. Annu Rev Biochem. 71:165-89.
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