104 18, 1 1.1, (500±20) g, 1, ; BALB/c 1.2 Varian Cary 50 ; Bio-Rad model 550, BioRad, BioRad IgG(Goat-IgG) Sigma ; CNBr Sepharose 4B Amersham-Pharmac

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1 2011 1, 18(1): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J 许娜娜 1,2, 钱冬 2 1, 赵海泉 1., ; 2., 摘要 : (Lateolabrax japonicus) (Ig), -IgG(Goat-IgG), A Goat-IgG Ig, ELISA SDS-PAGE Western-blotting, Ig 30%~50%, 45%, ELISA /mg, ELISA 2.2 ; A, 0.8~1.6 ml, 76.2%, ELISA /mg, 2.57%; Goat-IgG Sephrose 4B, 1.4~2.8 ml, 72.4%, ELISA /mg, 2.63% 3 79 kd 29 kd, ; Ig AA5 Western-blotting Ig, A Goat-IgG Ig, A,, Goat-IgG, [, 2011, 18(1) ] : ; ; ; 中图分类号 : S942 文献标识码 : A 文章编号 : (2011) (Lateolabrax japonicus), (Perciformes) (Percoidei) (Percichthyidae),,, [1] (lymphocystis) [2] [3 4] [5], 6%~10% [6],,, [7 8],,,,, (Ig) 收稿日期 : ; 修订日期 : 基金项目 : (2007C10037). 作者简介 : (1986 ),,,. xunana77@yahoo.com.cn 通讯作者 :,. qdomonas@163.com;. swjs12@ahau.edu.cn

2 104 18, 1 1.1, (500±20) g, 1, ; BALB/c 1.2 Varian Cary 50 ; Bio-Rad model 550, BioRad, BioRad IgG(Goat-IgG) Sigma ; CNBr Sepharose 4B Amersham-Pharmacia ; HiTrap Protein A HP GE ; (OPD) ; IgG-HRP ; ; Tris-HCl PBS [9] Goat-IgG Goat-IgG ph 7.4 PBS,, 200 μg/ ; 3 Goat-IgG,, 3, 300 μg/ 450 μg/ 650 μg/, 3 m 3 m 3 m, (20±5) 21 d,, r/min 10 min, ; 10, 30 ml; 5, 13 ml Ig Goat-IgG 30%, 4 1 h, r/min 10 min, 30% 1, 500 μl 0.02 mol/l ph7.4 PBS ; 35%, 4 1 h,, 35% 1, 500 μl 0.02 mol/l ph7.4 PBS ;, 30% 35% 40% 45% 50% 55%, ELISA, PBS 4, (mg/ml) 1.45 OD OD Ig A Goat-IgG 1.0 ml, HiTrap Protein A HP 4 3~4 h; 0.02 mol/l ph7.4 PBS 1.0~1.5 ml/min OD 280 <0.01; 0.1 mol/l ph ~1.5 ml/min, 200 μl/,, ELISA ;, 0.02 mol/l ph7.4 PBS 4, 花鲈 Ig 的亲和层析提取 CNBr Sepharose 4B, : 0.12:1 (W/V), Sepharose 4B-Goat IgG, Amersham-Pharmacia Goat-IgG 1.0 ml, 4, 0.02 mol/l ph7.4 PBS 1.0~1.5 ml/min OD 280 < 0.01; 0.1 mol/l ph2.7 Gly-HCl, 200 μl/,, ELISA ;, 0.02 mol/l ph7.4 PBS 4, SDS-PAGE 分析 Laemmli [10] 0.75 mm SDS-PAGE, 4%, 12%, 10~20 μl, 5 min, 80 V, 150 V, 2 h, 1.4 Ig Ig,

3 1 : 105, 30 μg/ BALB/c ; 2,, 50 μg/ 100 μg/ 200 μg/ 2 Ig, sp2/0 AA5( ) 1.5 (ELISA) Goat-IgG ELISA [11], 1) Goat-IgG 5 mg/l 4, Tween %(V/V)pH7.4 PBS(PBST) 3~4 ; 2) 300 µl 0.8%~ 1% 37 3 h, PBST ; 3) Ig 100 µl, 37 1 h, ; 4) Ig ( ), 37 1 h, ; 5) 100 µl IgG-HRP(1: ), 37 1 h, ; 6) (OPD) 10 l5 min, 2 mol/l, OD 490, OD P/N (western-blot analysis) Ig SDS-PAGE, 12 V 4, 10% 37 1 h, 1:1 000 Ig 1:1 000 AA5, 37 1 h, Tween %(V/V) ph7.5 TBS(TBST) IgG-AP, 37 1 h, NBT/BCIP, PBS [12] Ig, 30% 35% 40% 45% 50% 55%, ELISA,, 30%~50%, 45%, ELISA /mg, ELISA 2.2 ; 35%, 45% ; 45%, ( 1) SDS-PAGE,, ( 1),, 2.2 A Ig Goat-IgG HiTrap Protein A HP,,, 200 μl/ Goat-IgG ( ELISA ), 2,,,, 4 8, 0.8~1.6 ml, mg, 76.2% Goat-IgG ELISA, ELISA /mg mg, 55 mg/ml, A 2.57% 2.3 Goat-IgG Ig Goat-IgG Goat-IgG Sephrose 4B, Tab. 1 item /ml total protein volume /(mg ml 1 ) protein concentration ELISA /(mg 1 pro) ELISA titer 表 1 花鲈血清 Ig 硫酸铵分级沉淀及抗体效价 Lateolabrax labrax Ig eluted with ammonium sulfate fractionation and its antibody titer /% (NH 4 ) 2 SO 4 saturation 30% 35% 40% 45% 50% 55% serum

$ammonium sulfate fractionation 1 6: (NH 4 ) 2 SO 4$ 30%, respectively. 2.

4, 3 ) Goat-IgG ( 4, 4 ), 79 kd 29 kd 2, (H ) (L

2 Protein concentration and antibody titer of

affinity column Gly-HCl, 200 μl/, Goat-IgG ( ELISA

4% Goat-IgG ELISA, ELISA 1.39 10 5 /mg 36 1.

63% 4 Ig SDS-PAGE 1: 45% SAS ; 2: ; 3: A ; 4:

4 Goat-IgG Ig Fig. 3 Protein concentration and antibody titer of Lateolabrax labrax Ig eluted from goat IgG-immobilized agarose gel 1 Ig SDS-PAGE % 2.50% 3.45% 4.40% 5.35% 6.30%. Fig. 1 SDS-PAGE of Lateolabrax labrax Ig eluted with ammonium sulfate fractionation 1 6: (NH 4 ) 2 SO 4 saturation is 1.55%, 2.50%, 3.45%, 4.40%, 5.35%, 6.30%, respectively. 2.4 Ig SDS-PAGE SDS-PAGE A Goat-IgG 45% (SAS), 4 A ( 4, 3 ) Goat-IgG ( 4, 4 ), 79 kd 29 kd 2, (H ) (L ), 2, IgM ; 45% ( 4, 1 ) 2 A Ig Fig. 2 Protein concentration and antibody titer of Lateolabrax labrax Ig eluted with protein A affinity column Gly-HCl, 200 μl/, Goat-IgG ( ELISA ), 3 ; 7 14, 1.4~2.8 ml, mg, 72.4% Goat-IgG ELISA, ELISA /mg mg, 55 mg/ml, Goat-IgG 2.63% 4 Ig SDS-PAGE 1: 45% SAS ; 2: ; 3: A ; 4: Goat-IgG; ; 5: Goat-IgG. M marker. Fig. 4 SDS-PAGE of Lateolabrax labrax serum and separated Ig 1: 45% SAS; 2: serum; 3: HiTrap Protein A HP; 4: goat-igg immobilized agarose; 5: goat IgG. M: marker.

$Goat-IgG +AA5. M. marker Fig. 5 Western-blotting of purified Lateolabrax labrax serum Ig 1. HiTrap Protein A fraction+pab; 2. goat-igg-gel fraction+ pab; 3. HiTrap Protein A fraction+aa5; 4.$ goat-igggel+aa5. M. marker 3 A Goat-IgG Sepharose 4B Ig, Ig, A Goat-IgG 1 A, 1 ml 0.8~1.2 ml ; ELISA 1.

goat-igggel+aa5. M. marker 3 A Goat-IgG Sepharose 4B Ig, Ig, A Goat-IgG 1 A, 1 ml 0.8~1.2 ml ; ELISA 1.

5 1 : 107 ( 4, 2 ),,,,, 2.5 Ig Western-blotting Ig 2 Ig ( 5) Ig Ig H L, AA5 Ig H ; A Goat-IgG 79 kd, H Western-blotting SDS-PAGE, A Goat-IgG 5 Ig Western-blotting 1. A + ; 2. Goat-IgG + ; 3. A +AA5; 4. Goat-IgG +AA5. M. marker Fig. 5 Western-blotting of purified Lateolabrax labrax serum Ig 1. HiTrap Protein A fraction+pab; 2. goat-igg-gel fraction+ pab; 3. HiTrap Protein A fraction+aa5; 4. goat-igggel+aa5. M. marker 3 A Goat-IgG Sepharose 4B Ig, Ig, A Goat-IgG 1 A, 1 ml 0.8~1.2 ml ; ELISA /mg, 15 Goat-IgG 1, A, A ;, SDS-PAGE, Goat-IgG Goat-IgG, Ig,, A Goat-IgG,, A A (Staphylococal Protein A, SPA),, 12~15 kd, Fc, A IgG, [13] A (A ) [14] A, A (Sebastes schlegeli Higendorf) (Pseudosciaena crocea) (Lutjanus argentimaculatus) (Epinephelus awoara) (Lates calcarifer) (Paralichthys olivace ) [15 18] IgM, A, (Trematomus newnesi) [19] IgG(Goat-IgG), Goat-IgG Sepharose 4B,, (Micropterus salmoides) (Sparus pagrus pagrus) IgM [20], A Goat-IgG Sepharose 4B

6 (Ig), Ig, A,, A 4 3~4 h Ig,,, IgM, 4 Ig, Ig 2 (H ) 2 (L ), H 60~81 kd, L 20~30 kd [16 29] IgM, (Sinaperca chuatsi) IgM H L 72 kd 29 kd [23] ; (Dicentrarchus labrax L.) IgM H L 78 kd 27.5 kd [19] ; (Lates calcarifer) IgM H L 81.8 kd 75.7 kd [16] Ig, Ig SDS-PAGE, Ig H L 79 kd 29 kd [28] Sephadex G-200 IgM, Ig H L 79 kd 29 kd, Western blotting, 79 kd 29 kd H L A Goat-IgG Sepharose 4B Ig, Ig Goat-IgG Ig,, Ig, 参考文献 : [1],. [J]., 2000, 2(7): [2],,,. [J]., 1998(3): [3],,,. [J]., 2000, 24(4): [4],,,. ( ) [J]., 2002, 9(1): [5] Chen S C, Lee J L, Lai C C, et al. Nocardiosis in sea bass, Lateolabrax japonicus, in Taiwan[J]. J Fish Dis, 2001, 23(5): [6],,, ( )[J]., 2007(8) [7],,,. [J]., 2003, 33(2): [8],,,. [J]., 2001(2): [9] F, R, R E,. [M]. :, 1999: [10] Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J]. Nature, 1970, 227: [11],. [M]. :, 2000: [12] E, D. [M]. :, 2002: [13] Hjelm H, Hjelm K, Sjöquist J. Protein A from Staphylococcus aureus:its isolation by affinity chromato- gramphy and its use as an immunosorbent for isolation of immunoglobulins[j]. FEBS Lett, 1972, 28(1): [14] Grodzki A C, Berenstein E. Antibody purification: affinity chromatography-protein A and protein G sepharose[j]. Methods Mol Biol, 2010, 588: [15] Shin G, Lee H, Palaksha K J, et a1. Production of monoclonal antibodies against seurm immunoglobulins of black rockfish(sebastes schlegeli Higendorf)[J]. J Vet Sci, 2006, 7(3): [16],,,. IgM [J]., 2006, 13(3): [17],. [J]., 2002, 21(4): [18] Crosbie P B, Nowak B F. Production of polyclonal antisera against barramuodi(lates calcarifer Bloch) serum immunoglobulin derived from affinity columns containing mannan-binding protein or staphylococcal protein A [J]. Aquaculture, 2002, 211: [19] Scapigliati G, Chausson F, Cooper E L, et al. Qualitative and quantitative analysis of serum immunoglobulins of four Antarctic fish species[j]. Polar Biol, 1997,18: [20] Oswaldo P, Ariadna S B, Pilar A P. Isolation and partial characterization of serum immunoglobulins from sea bass (Dicentrarchus labrax L.) and gilthead sea bream(sparus aurata L.) [J]. Fish Shellfish Immunol, 1996, 6:

7 1 : 109 [21],,,. Ig ( Ⅱ)[M]. :, 1995: [22],,,. [J]., 1998, 19 (6): [23],,,. [J]., 1998, 22 (3): [24],. [J]., 1998, 22 (2): [25],,,. [J]., 2001, 25(1): [26] 垚,,,. IgM [J]., 2003, 24(2): [27],,,. [J]., 2003, 27(6): [28],,,. [J]., 2004, 39(5): [29],,,. M [J]., 2004, 28(4): Purification and partial characterization of serum immunoglobulin in Lateolabrax japonicus XU Nana 1, 2, QIAN dong 2, ZHAO Haiquan 1 1. Faculty of Life Science, Anhui Agriculture University, Hefei , China; 2. Laboratory of Fish Disease, Zhejiang Institute of Freshwater Fisheries, Huzhou , China Abstract: The present study aims to establish antibody detection methods for further vaccine research and application. Healthy sea perches (Lateolabrax japonicus) were injected with commercial goat IgG emulsified with Freund s adjuvant four times in two months to develop good immune response. The sera of immunized sea perches were collected for immunoglobulin (Ig) isolation with saturated ammonium sulfate (SAS) precipitation, protein A-sepharose affinity chromatography and goat IgG-immobilized sepharose 4B affinity chromatography. It was shown that Ig mainly distributed in 30% 50% SAS precipitation, with the maximum ELISA titer per mg protein being 45% SAS, 2.2 times higher than that of original serum. Protein A column produced single elution peak with the elution volume of ml, accounting for 76.2% of total collected protein, with the ELISA titer being per mg protein, 15 times higher than that of original serum. Goat IgG-immobilized sepharose 4B affinity chromatography produced single elution peak with the elution volume of ml, accounting for 72.4% of total collected protein, with the ELISA titer being per mg protein. SDS-PAGE showed that Ig eluted by both protein A column and goat IgG-immobilized sepharose 4B affinity chromatography yielded two protein bands of 79 kd and 29 kd, which could be considered as H chain and L chain of sea perch Ig, while Ig precipitated with 45% SAS yielded more bands besides H and L chains. Protein bands of 79 kd and 29 kd were confirmed to be H and L chains by western-blotting with the antiserum and monoclonal antibody AA5. The Ig yield coefficients of protein A and goat-igg methods was calculated as 2.57% and 2.63%, respectively. These results indicated that both protein A column and IgG-immobilized sepharose 4B affinity chromatography could successfully isolate Ig from sea perch serum with one step of absorption and elution. Protein A column method could be considered as a more effective and less time consuming method for sea perch Ig isolation, yielding narrower elution volume and higher antibody titer, without long period of vaccination with goat IgG to fish. The current research provide a useful technical tool for further studies on humoral immune response for vaccine development and vaccine effects assessments for sea perch, which would benefit the prevention and control of sea perch diseases.[journal of Fishery Sciences of China, 2011,18(1): ] Key words: Lateolabrax japonicus; immunoglobulin; purification; affinity chromatography Corresponding author: QIAN Dong. qdomonas@163.com; ZHAO Haiquan. swjs12@ahau.edu.cn

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<453A5CCAB3C6B7BFC6D1A73131C4EA3134C6DA5C3134C6DA5C3032B7D6CEF6> 227 ELISA 刘沙洲 1,2, 桑小雪 1, 欧阳华学 3, 雷绍荣 3, 白林含 1, * (1. 四川大学生命科学学院, 四川成都 610065;2. 成都市食品药品检测中心, 四川成都 610045; 3. 四川省农业科学院分析测试中心, 四川成都 610066) 目的 : 比较