臺灣博碩士論文加值系統

第二型豬環狀病毒 (Porcine circovirus 2，PCV2)，是一種會感染豬隻的病毒。目前與 PCV2 相關的綜合疾病症狀被稱為豬罹患離乳後多系統消耗性綜合症(postweaning multisystemic wasting syndrome，PMWS)。 Capsid蛋白質 (Cap) 為 PCV2 的衣殼蛋白。具有抗原決定位，是生產疫苗的主要標的。本研究嘗試利用大腸菌高密度發酵生產Cap蛋白。
Cap 蛋白成功地在大腸菌 Rosetta 2 (DE3) 以 SUMO-Cap 融合蛋白型式表達，而且這個融合蛋白透過陽離子交換層析進行純化。經由 SUMO protease 可將融合伴剪切，再透過管柱層析，可以純化到 Cap 蛋白。另外，本研究也嘗試高密度發酵培養，並以乳糖當作誘導劑進行 SUMO-Cap 融合蛋白誘導表達。結果顯示，12X ZYP 饋料培養基中含有 0.42% 的甘油及0.16% 的乳糖時，有較好的表達量，比利用異丙基-β-D-硫代半乳糖苷 (IPTG)作為誘導劑的結果更好。

Porcine circovirus 2 (PCV2) is the main viral pathogen that causes the desease called powtweaning multisystemic wasting syndrome. Capsid protein (Cap) of PCV2 containing the epitopes to elicit immune reaction, is the main target of subunit vaccine. In this study, the high cell density fermentation to produce the Cap protein of PCV2 were performed.
The Cap protein was successfully expressed in E. coli Rosetta 2 (DE3) as a SUMO-Cap fusion protein, and the fusion protein was purified to homogenity by cation exchange chromatography. Through the digestion by SUMO protease, followed by coloum chromatrogaphy, Cap protein can be purified. The high cell density fermentation, coupled with lactose induction, for the production of SUMO-Cap were performed in this study. The results indicated that 12X ZYP feeding medium, containing 0.42% glycerol and 0.16% lactose as inducer, showed the highest fusion protein production, with production level even higher than that using IPTG as inducer.

中文摘要 ...I
英文摘要 ...II
本文目錄 ...III
表目錄....VII
圖目錄...VIII
縮寫表..XII
第一章　緒論...1
1.1　豬環狀病毒...1
1.2　大腸菌表達系統...2
1.3　融合伴系統...3
1.4　高密度培養...4
1.5　研究動機...5
第二章　材料與方法 ...6
2.1　菌株 ...6
2.2　抗生素...6
2.3　儀器 ...6
2.4　方法 ...8
2.4.1　構築載體...8
2.4.2　蛋白質表達分析...8
2.4.2.1 SUMO-Cap Rosetta 蛋白質誘導...8
2.4.2.2 SUMO-Cap 蛋白萃取....9
2.4.2.3 Glycine SDS-PAGE分析.....10
2.4.3　SUMO-Cap 蛋白質純化...11
2.4.3.1 蛋白質萃取......11
2.4.3.2 SP sepharose管柱純化...11
2.4.4 SUMO protease 誘導表達..12
2.4.5　SUMO protease 純化..12
2.4.5.1 蛋白質萃取...12
2.4.5.2 Ni sepharose管柱純化...12
2.4.6　SUMO protease 剪切測試....13
2.4.7剪切後之 Cap 蛋白質純化....14
2.4.8 SUMO-Cap蛋白質與Cap蛋白質結合力測試...14
2.4.9高密度培養與誘導...15
第三章　結果....17
3.1　搖瓶測試....17
3.1.1 SUMO-Cap Rosetta 蛋白質誘導結果...17
3.1.2 SUMO protease 誘導結果...17
3.2　蛋白質純化....18
3.2.1 SUMO-Cap 蛋白質純化...18
3.2.2 SUMO portease 純化...18
3.3　SUMO protease 剪切SUMO-Cap 測試...18
3.4 剪切後之Cap蛋白質純化..19
3.5 SUMO-Cap蛋白質與Cap蛋白質結合力測試...19
3.6　發酵槽測試....21
3.6.1.1 IPTG 誘導..21
3.6.1.2 乳糖誘導....21
3.6.1.3 改變培養基乳糖誘導....23
3.6.1.4 指數饋料....24
3.6.1.5 改以葡萄糖饋料....25
3.6.1.6 發酵測試比較....26
第四章　討論..28
4.1　搖瓶測試..28
4.2　蛋白質純化..28
4.3　SUMO protease 剪切SUMO-Cap 測試 ..28
4.4 剪切後之 Cap 蛋白質純化..29
4.5 SUMO-Cap蛋白質與Cap蛋白質結合力測試....29
4.6　發酵槽高密度培養..30
第五章　結論..32
參考文獻....33
圖表....36
附錄.......72
附圖...74

1. Faurez F, Dory D, Grasland B, Jestin A. Replication of porcine circoviruses. Virol J. 2009;6:60.

2. Tischer I, Mields W, Wolff D, Vagt M, Griem W. Studies on epidemiology and pathogenicity of porcine circovirus. Arch Virol. 1986;91(3-4):271-276.

3. Tischer I, Bode L, Peters D, Pociuli S, Germann B. Distribution of antibodies to porcine circovirus in swine populations of different breeding farms. Arch Virol. 1995;140(4):737-743.

4. Misinzo G, Delputte PL, Meerts P, Lefebvre DJ, Nauwynck HJ. Porcine circovirus 2 uses heparan sulfate and chondroitin sulfate B glycosaminoglycans as receptors for its attachment to host cells. J Virol. 2006;80(7):3487-3494.

5. Khayat R, Brunn N, Speir JA, et al. The 2.3-angstrom structure of porcine circovirus 2. J Virol. 2011;85(15):7856-7862.

6. Baneyx F, Mujacic M. Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol. 2004;22(11):1399-1408.

7. Walsh G. Biopharmaceutical benchmarks 2006. Nat Biotechnol. 2006;24(7):769-776.

8. Fang L, Jia KZ, Tang YL, Ma DY, Yu M, Hua ZC. An improved strategy for high-level production of TEV protease in Escherichia coli and its purification and characterization. Protein Expr Purif. 2007;51(1):102-109.

9. Esposito D, Chatterjee DK. Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol. 2006;17(4):353-358.

10. Hay RT. SUMO: a history of modification. Mol Cell. 2005;18(1):1-12.


11. Malakhov MP, Mattern MR, Malakhova OA, Drinker M, Weeks SD, Butt TR. SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. J Struct Funct Genomics. 2004;5(1-2):75-86.

12. Kapust RB, Waugh DS. Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci. 1999;8(8):1668-1674.

13. Terpe K. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol. 2003;60(5):523-533.

14. Shiloach J, Fass R. Growing E. coli to high cell density--a historical perspective on method development. Biotechnol Adv. 2005;23(5):345-357.

15. Riesenberg D, Schulz V, Knorre WA, et al. High cell density cultivation of Escherichia coli at controlled specific growth rate. J Biotechnol. 1991;20(1):17-27.

16. Lee SY. High cell-density culture of Escherichia coli. Trends Biotechnol. 1996;14(3):98-105.

17. Jung G, Denefle P, Becquart J, Mayaux JF. High-cell density fermentation studies of recombinant Escherichia coli strains expressing human interleukin-1 beta. Ann Inst Pasteur Microbiol. 1988;139(1):129-146.

18. Paalme T, Tiisma K, Kahru A, Vanatalu K, Vilu R. Glucose-limited fed-batch cultivation of Escherichia coli with computer-controlled fixed growth rate. Biotechnol Bioeng. 1990;35(3):312-319.

19. Luli GW, Strohl WR. Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations. Appl Environ Microbiol. 1990;56(4):1004-1011.

20. Kleman GL, Strohl WR. Acetate metabolism by Escherichia coli in high-cell-density fermentation. Appl Environ Microbiol. 1994;60(11):3952-3958.
21. Yang YG, Tong Q, Hu TS, Qian YC, Yang SL, Gong Y. The Application of a Novel Lytic System to the Recovery of Recombinant Proteins in E.coli. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2000;32(3):211-216.

22. Yin S, Sun S, Yang S, Shang Y, Cai X, Liu X. Self-assembly of virus-like particles of porcine circovirus type 2 capsid protein expressed from Escherichia coli. Virol J. 2010;7:166.