由本實驗結果顯示所得到的 5D6 單株抗體，辦認日本腦炎病毒 套膜蛋白具有專一性。應用在診斷試劑開發方面，可確保偵測的準確 性，可確定偵測出日本腦炎病毒；也具有高度敏感性，低濃度的病毒 也可被偵測。當病毒在複製的初期，病毒劑量較低，即可被 5D6 抗 體結合，可達到事先預防的目的。本次實驗將 5D6 單株抗體結合上 奈米金粒子，結果顯示可縮短檢測時間，使得可較快檢驗出病毒。
本次研究所純化出的 5D6 單株抗體濃度約為 178μg/ml，濃度不 高，主要原因為抗體來源為收取細胞上清液，細胞上清液包含了細胞 激素、細胞蛋白及培養基廢物等等，而抗體的濃度在上清液中相對較 低。如提高濃度可將抗體打回實驗小鼠腹水以提高濃度：或是將單株 抗體使用表現載體表現，以利研究進行。
第二節 建議
本論文目的在診斷試劑研發，而本論文結果獲得具有專一性及中 和能力的 5D6 單株抗體，也提出奈米金在日本腦炎病毒診斷應用，
有助於新型臨床診斷開發。可應用在縮短病毒檢測的時程，有利於及 早檢測出病毒。也提高了檢測的敏感性，使病毒更容易被檢測出。
參考文獻
1. 1. Detels R, Cross JH, Huang WC, Lien JC, Chen S., Japanese encephali-tis virus in Northern Taiwan, 1969-1973. 1976.
2. Solomon T, Winter PM., Neurovirulence and host factors in flavivirus en-cephalitis--evidence from clinical epidemiology. Arch Virol Suppl, 2004(18):
p. 161-70.
3. Lin CW, Wu SC., A functional epitope determinant on domain III of the Japanese encephalitis virus envelope protein interacted with neutraliz-ing-antibody combining sites. J Virol, 2003. 77: p. 2600-6.
4. Rauscher S, Flamm C, Mandl CW, Heinz FX, Stadler PF., Secondary structure of the 3'-noncoding region of flavivirus genomes: comparative analysis of base pairing probabilities. RNA, 1997. 3(7): p. 779-91.
5. Rauscher S, Flamm C, Mandl CW, Heinz FX, Stadler PF., The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. Nature, 1995. 375(6529): p. 291-8.
6. Wu Y, Zhang F, Ma W, Song J, Huang Q, Zhang H., A plasmid encoding Japanese encephalitis virus PrM and E proteins elicits protective immunity in suckling mice. Microbiol Immunol., 2004. 8: p. 585-90.
7. Butrapet S, Kimura-Kuroda J, Zhou DS, Yasui K., Neutralizing mechan-ism of a monoclonal antibody against Japanese encephalitis virus glyco-protein E. Am J Trop Med Hyg, 1998. 58(4): p. 389-98.
8. Fan WF, Mason PW., Membrane association and secretion of the Japanese encephalitis virus NS1 protein from cells expressing NS1 cDNA. Virology 1990. 177: p. 470-476.
9. Jan LR, Yang CS, Trent DW, Falgout B, Lai CJ., Processing of Japanese encephalitis virus non-structural proteins: NS2B-NS3 complex and hetero-logous proteases. J Gen Virol, 1995. 76: p. 573-580.
10. Wengler G, Wengler G., The NS 3 nonstructural protein of flaviviruses contains an RNA triphosphatase activity. Virology, 1993: p. 265-73.
11. Koch JO, Bartenschlager R., Modulation of Hepatitis C virus NS5A hyperphosphorylation by nonstructural proteins NS3 , NS4A, and NS4B. , J Virol 1999 73: p. 7138-46
12. Kim YG, Yoo JS, Kim JH, Kim CM, Oh JW., Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA poly-merase. BMC Mol Biol., 2007 11: p. 59.
13. Mandl CW, Kroschewski H, Allison SL, Kofler R, Holzmann H, Meixner T, Heinz FX., Adaptation of tick-borne encephalitis virus to BHK-21 cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo. J Virol., 2001 75: p. 5627-37.
14. Germain N, Mérienne K, Zinn-Justin S, Boulain JC, Ducancel F, Ménez A., Molecular and structural basis of the specificity of a neutralizing ace-tylcholine receptor-mimicking antibody, using combined mutational and molecular modeling analyses. J Biol Chem, 2000 275: p. 21578-86.
15. McMinn PC., The molecular basis of virulence of the encephalitogenic fla-viviruses. J Gen Virol., 1997 78: p. 2711-22.
16. Mason PW, Dalrymple JM, Gentry MK, McCown JM, Hoke CH, Burke DS, Fournier MJ, Mason TL., Molecular characterization of a neutralizing domain of the Japanese encephalitis virus structural glycoprotein. J Gen Virol., 1989. 70: p. 2037-49.
17. Jones T, A chimeric live attenuated vaccine against Japanese encephalitis.
Expert Rev Vaccines, 2004. 3: p. 243-8.
18. Monath TP., Japanese encephalitis vaccines: current vaccines and future prospects. Curr Top Microbiol Immunol, 2002. 267: p. 105-38.
19. Takegami T, Japanese encephalitis. Uirusu, 2003. 53: p. 25-30.
20. Köhler G, Milstein C., Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 1975. 256: p. 495-7.
21. Kilpatrick KE, Cutler T, Whitehorn E, Drape RJ, Macklin MD, Withers-poon SM, Singer S, Hutchins JT., Gene gun delivered DNA-based immuni-zations mediate rapid production of murine monoclonal antibodies to the Flt-3 receptor. Hybridoma, 1998. 17: p. 569-76.
22. Bonatti S, Di Leonardo A, Mariani L, Randazzo R, Sciandrello G., Scian-drello G., Selection in HAT medium is not a reliable method for the study of reversion from 6-thioguanine resistance to sensitivity. Mutat Res., 1982. 104:
p. 377-81.
23. Kilpatrick KE, Kerner S, Dixon EP, Hutchins JT, Parham JH, Condreay JP, Pahel G., In vivo expression of a GST-fusion protein mediates the rapid generation of affinity matured monoclonal antibodies using DNA-based immunizations. Hybrid Hybridomics, 2002. 21: p. 237.
24. Price PJ., Hybridoma technology. Ric Clin Lab, 1984. 14: p. 277-86.
25. de StGroth SF, Scheidegger D., Production of monoclonal antibodies:
strategy and tactics. J Immunol Methods, 1980. 35: p. 1-21.
26. Kelly-Quintos C, Cavacini LA, Posner MR, Goldmann D, Pier GB., Cha-racterization of the opsonic and protective activity against Staphylococcus
aureus of fully human monoclonal antibodies specific for the bacterial sur-face polysaccharide poly-N-acetylglucosamine. Infect Immun, 2006. 74: p.
2742-50.
27. Doi M, Yokoyama A, Kondo K, Ohnishi H, Ishikawa N, Hattori N, Kohno N., Anti-tumor effect of the anti-KL-6/MUC1 monoclonal antibody through exposure of surface molecules by MUC1 capping. Cancer Sci, 2006. 97: p.
420-9.
28. Daniel MC, Astruc D., Gold nanoparticles: assembly, supramolecular che-mistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology. Chem Rev, 2004. 104: p. 293-346.
29. Arshady R., Preparation of polymer nano- and microspheres by vinyl poly-merization techniques. J Microencapsul., 1988. 5: p. 101-14.
30. Yasuda H., Biocompatibility of nanofilm-encapsulated silicone and sili-cone-hydrogel contact lenses. Macromol Biosci, 2006. 6: p. 121-38.
31. Traykova T, Aparicio C, Ginebra MP, Planell JA., Bioceramics as nano-materials. Nanomed, 2006. 1: p. 91-106.
32. Prato M, Kostarelos K, Bianco A., Functionalized carbon nanotubes in drug design and discovery. Acc Chem Res., 2008. 41: p. 60-8.
33. Wen C, Jin ZH, Liu XX, Li X, Guan JQ, Sun DY, Lin YR, Tang SY, Zhou G, Lin JD., Studies on nano-diamond prepared by explosive detonation by Raman and infrared spectroscopy. Guang Pu Xue Yu Guang Pu Fen Xi., 2005. 25: p. 681-4.
34. Xia XR, Monteiro-Riviere NA, Riviere JE., Trace analysis of fullerenes in biological samples by simplified liquid-liquid extraction and high-performance liquid chromatography. J Chromatogr A, 2006. 1129: p.
216-22.
35. Balzarini J., Inhibition of HIV entry by carbohydrate-binding proteins. An-tiviral Res, 2006. 71: p. 237-47.
36. Jiang HQ, Chen YF., Medical application of nano-materials. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi., 2002. 16: p. 435-7.
37. Gromowski GD, Barrett AD., Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain III (ED3) of dengue 2 virus. Virology., 2007. 366:
p. 349-60.
38. Rajamanonmani R, Nkenfou C, Clancy P, Yau YH, Shochat SG, Sukupol-vi-Petty S, Schul W, Diamond MS, Vasudevan SG, Lescar J., On a mouse monoclonal antibody that neutralizes all four dengue virus serotypes. J Gen Virol. 2009 2009. 90: p. 799-809.
39. Liu J, Liu B, Cao Z, Inoue S, Morita K, Tian K, Zhu Q, Gao GF., Charac-terization and application of monoclonal antibodies specific to West Nile vi-rus envelope protein. J Virol Methods, 2008. 154: p. 20-6.
40. Xiulan S, Xiaolian Z, Jian T, Zhou J, Chu FS., Preparation of gold-labeled antibody probe and its use in immunochromatography assay for detection of aflatoxin B1. Int J Food Microbiol, 2005. 15;99(2): p. 185-94.
41. Muscariello L, Rosso F, Marino G, Barbarisi M, Cafiero G, Barbarisi A., Cell surface protein detection with immunogold labelling in ESEM: optimi-sation of the method and semi-quantitative analysis. J Cell Physiol, 2008.
214(3): p. 769-76.
Fig.1
圖一 病毒重組蛋白純化
將大量表現的 ED3 重組蛋白使用 FPLC 純化，使用 400μM Imidazole 緩衝液析下後，分別進行 A.SDS-PAGE 使用 Coomassie blue 染劑染色 B. Western Blotting，第一抗體體為 Mouse Anti His-tag，第二抗體體 為 Goat Anti-Mouse IgG AP Conjugate (H + L)。
B A
kDa
10
Fig.2
Serum ELISA titer
0 0.05 0.1 0.15 0.2 0.25 0.3
50x 100x 200x
Serum Dilution
O.D(405 nm)
blank mice2
mice3 mice4
mice5 mice6
mice7
圖二 實驗小鼠抗體效價誘發測定
使用 ELISA 測定對抗原的效價，共有六隻實驗小鼠，犧牲前一週採 取的血清，分別稀釋 50 倍、100 倍、200 倍後為第一抗體，第二抗體 為 Horseradish Peroxidase (HRP) anti-mouse IgG
Fig.3
圖三 融合瘤細胞抗體效價測定
ELISA 測定抗體對抗原(JEV)的效價，第一抗體為融合瘤細胞上清 液，第二抗體為 Horseradish Peroxidase (HRP) anti-mouse IgG。共有 37 個生長良好的融合瘤細胞株進行分析。
ELISA titer
0 0.5 1 1.5 2 2.5 3 3.5 4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 C
sample number
OD(405nm)
Fig.4
圖四 極限稀釋後細胞抗體效價測定
取圖三的 35 號融合瘤細胞，極限稀釋後，將 25 個融合瘤單株細胞進 行 ELISA 測定抗體對抗原(JEV)的效價。第一抗體為融合瘤細胞上清 液，第二抗體為 Horseradish Peroxidase (HRP) anti-mouse IgG。圖中圓 圈箭號即為本次研究的 5D6 單株抗體。
Fig.5
圖五 5D6 對原態病毒及重組蛋白辦認能力測試
收集 5D6 的細胞上清液為第一抗體，第二抗體為 Horseradish Perox-idase (HRP) anti-mouse IgG，進行西方墨點法。第一抗體作用 2 小時 後，加入第二抗體後在作用 2 小時後，使用 Chemiluminescent HRP Substrate (MILLIPORE) 呈色
Lane 1 日本腦炎病毒 Lane 2 ED3 重組蛋白
kDa
1
70
10
2
Fig.6
圖六 5D6 不同稀釋倍數與原態病毒及重組蛋白辦認能力測試
96 孔盤分別固定 JEV(A).ED3(B)為抗原，5D6 細胞上清液，分別稀釋 為原倍、10 倍、100 倍、1000 倍、10000 倍為第一抗體，第二抗體為 Horseradish Peroxidase (HRP) anti-mouse IgG。
A.5D6 對病毒原態套膜蛋白的效價
Fig.7
圖七 5D6 辦認不同濃度的原態病毒及重組蛋白
收集 5D6 的細胞上清液為第一抗體，第二抗體為 Horseradish Perox-idase (HRP) anti-mouse IgG，進行西方墨點法。
A. Lane 1,2,3,4 分別注入 2x105,2x104,2x103,2x102 pfu/ml 的日本腦 炎病毒。
B. Lane 1,2,3,4 分別注入 20,10,5,1 ug ED3 重組蛋白
A 1 2 3 4 B 1 2 3 4
Fig.8
圖八 5D6 中和病毒複製能力測試
BHK-21 細胞培養在六孔盤中，待細胞長至八分滿後，將原倍 5D6 抗 體以 10 倍，100 倍稀釋後，加到六孔盤中，再感染日本腦炎病毒 2x105 pfu/ml，每日觀察細胞被感染的情形，當細胞病變程度達到 80%後，
加入染色液後計算病毒斑點數目。
Lane A 加入原倍 5D6 抗體 Lane B 加入 10 倍 5D6 抗體 Lane C 加入 100 倍 5D6 抗體
Cultured medium 1/10 cultured medium 1/100 cultured me-dium
A B C
Fig.9
圖九 純化 5D6 抗體
收集 5D6 細胞上清液，使用 HiTrap rProtein A FF(GE Healthcare)管柱 純化抗體。將純化後的 5D6 抗體，進行 A.SDS-PAGE 及 B.西方墨點 法分析，將純化的 5D6 抗體注入膠中，第一抗體直接以 Horseradish Peroxidase (HRP) anti-mouse IgG 作用一小時後觀察。
A B
KDa KDa
97
66 36
21
97
66 Heavy chain
Light chain
Fig.10
圖十 純化後抗體稀釋倍數與原態病毒及重組蛋白辦認能力測試 96 孔盤中，分別固定日本腦炎病毒(2x105pfu/ml)及 EDIII 重組蛋白 (10μg)進行 ELISA 分析。依照 178、17.8 跟 1.78μg/ml 濃度的抗體及 對照組(培養基)，為第一抗體，第二抗體為 Horseradish Peroxidase (HRP) anti-mouse IgG 後，使用 ABTS® 呈色試劑組(KPL)呈色，以 ELISA reader 測其 OD(405nm)之數值
A.抗原為日本腦炎原態病毒套膜蛋白
Fig.11
圖十一 純化後 5D6 中和病毒複製能力測試
BHK-21 細胞培養在六孔盤中，待細胞長至八分滿後，將 5D6 抗體濃 度 178、17.8μg/ml，加到六孔盤中，對照組為只加病毒無抗體。再感 染日本腦炎病毒 2x105 pfu/ml，每日觀察細胞被感染的情形，當細胞 病變程度達到 80%後，加入染色液後計算病毒斑點。
Lane A 為只加病毒液，病毒斑點數為 160(±11)個 Lane B 為 178μg/ml 抗體，病毒斑點數為 41(±3)個 Lane C 為 17.8μg/ml 抗體，病毒斑點數為 98(±7)個
A B C
Virus only Virus
178ug/ml
Virus 17.8ug/ml Plaque number 160±11 41 ±3 98±7
Fig.12
圖十二 免疫螢光分析抗體結合受病毒感染的細胞
計算病毒量使 MOI 分別為 10、1、0.1、0.001 後，感染 BHK-21。分 別在 0 小時、72 小時拍照觀察細胞病毒病變情形。加入 anti-mouse FITC 螢光拍照。
c MOI=10 MOI=1 MOI=0.1 MOI=0.01
0 hr
72 hr
IF
Fig.13
400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700
OD
400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700
OD
Fig.14
圖十四 5D6 接合奈米金測試對病毒的結合能力
96 孔盤固定純化的 5D6，第一抗體使用不同病毒濃度的日本腦炎病 毒，分別為 105、104、103、102、101、0.1、0.01pfu/ml 作用，兩抗為 5D6 接合奈米金的抗體。
nanogold-5D6
0 0.5 1 1.5 2
100000 10000 1000 100 10 1 0.1 0.01 0.001 0 PFU/ml
abs
Fig.15
圖十五 5D6 接合奈米金以免疫螢光分析抗體結合受病毒感染的細胞 計算病毒量以 M.O.I 為 10、1、0.1、0.01 感染 BHK-21 72 小時後，加 入 5D6 接合奈米金的抗體。直接使用螢光顯微鏡觀察。
c MOI=10 MOI=1 MOI=0.1 MOI=0.01
72 hr
IF
附表
DNA electrophoresis
DNA 5x 追蹤染劑 0.25%(w/v) bromophenol blue,0.25%(w/v) xylene cyaol FF,30%(v/v) glycerol in ddH2O
TAE buffer 40 mM Tris.Acetate, 2 mM EDTA, pH=8.5 EtBr 0.5 /ml ethidium bromide
Western Blotting 1.5M Tris-base
(pH8.8) 45.4g tris-base add ddH2O 250ml pH=8.8 0.5M Tris-HCl
(pH6.8) 7.88gtris-HCl,dd ddH2O 100ml,pH=6.8
10% SDS 10g SDS sodium dodecyl sulfate ,add ddH2O to 100ml APS 10% ammonium persulfate 1g/10ml ddH2O
Stacking gel(4%)
ddH2O 1.35ml,30%Acy/Bis 0.27ml,0.5M Tris-HCl (pH6.8)0.55ml,APS10%22.5μl,10%SDS22.5μl,TEMED
1X :25mM tris-base,250mM glycine,0.1%SDS Commassie 8.4,add methanol 300ml,10% SDS 5.6ml
10x TBS 200 mM Tris-HCl, pH 7.5, 5 M NaCl 1x TBST 1 X TBS cotaining 0.1% Tween-20 5% skim milk 2.5g skim milk/50ml TBST
Protein concentration detection
Protein Standard bovine serum albumin（1 g/ul）
Protein assay dye Bio-RAD Protein assay dye reagent concentrat, 450 ml
coating buffer 0.1 M sodium carbonate NaHCO3 pH8.8
LB Per liter：10 g Bacto tryptone,5 g Yeast extract 10 g NaCl
Top agarose
Per 100 ml ,1 g Bacto tryptone,0.5 g Yeast ex-tract,0.5 g NaCl. Add 100 ml deionized water Au-toclave 20 min
PEG/NaCl 20% (W/V)polyethylene glycol-8000,2.5M NaCl . FPLC
8x Phosphate buffer
1.42 g Na2HPO4 . 2H2O (177.99 g/mol), 1.11 g NaH2PO4 . H2O (137.99 g/mol) and 23.38 g NaCl (58.44 g/mol), add distilled water to 90 ml and dis-solve completely.Adjust pH, to 7.4. Add distilled wa-ter to 100 ml and filwa-ter through a 0.45 µm filwa-ter. This gives a final concentration of 160 mM phosphate and 4 M NaCl.
2 M Imidazole 13.62 g imidazole (68.08 g/mol) add distilled water to 90 ml and dissolve completely. Adjust pH to 7.4
us-ing conc. HCl. Add distilled water to 100 ml and filter through a 0.45 µm filter.
Cleaning buffer 20mM sodium phosphate,0.5M NaCl,0.05M ED-TA,pH7.4
Incubation buffer 1M NaOH 0.1 M NiSO4
2.63g NiSO4.6H2O (262.86/mol)add dd H2Oto 100ml filter through a 0.45 µm filter.
Denaturing buffer 10mM imidazol ,8M urea, 1 mM ß-mercaptoethanol Refolding buffer 10mM imidazol , 1mM ß-mercaptoethanol
Imidazole
附錄
Immunization Schedule:
7 BALB/cByJNarl mices . Antigen is ED3 fusion protein
Week Manipulation Adjuvant
1 Serum collection
2 Boost #1 TiterMax®
3 Serum collection
4 Boost #2 Incomplete Freund’s
adjuvant
5 Serum collection
6
Prefusion boost
8
Incomplete Freund’s adjuvant
Harvest Boost #3
none
9