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1 4 4 Vol. 4 No Journal of Food Safety and Quality Aug., 2013 B 1 冯 凡 1 1,, 许杨 2*, 王丹 1, 雷达 1, 孙澄浩 1 (1., ; 2., ) 摘要 : 目的 F7 方法 pet22b-f7, E. coli BL21(DE3), 结果 F7 AFB1, ELISA AFB1 结论 关键词 : B 1 ; ; ; ; Prokaryotic expression and renaturation of anti-idiotype nanobody against aflatoxin B 1 FENG Fan 1, XU Yang 1, 2*, WANG Dan 1, LEI Da 1, SUN Cheng-Hao 1 (1. State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang , China; 2. Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang , China) ABSTRACT: Objective To rapidly produce a large amount of anti-idiotype nanobody against aflatoxin B 1 (AFB 1 ). Methods Prokaryotic expression vector pet22b-f7 were constructed, and then transformed into E.coli BL21(DE3) for expression of anti-idiotype nanobody against aflatoxin B 1 (AFB 1 ). The conditions such as temperature, concentration of inducer, and induction time were optimized. Results The expression quantity of anti-idiotype nanobody F7 increased, with the main form of inclusion body. Anti-idiotype nanobody against AFB1 with bioactivity were obtained after dissolution and renaturation. ELISA results indicated that the renatured protein had the properties of combination with AFB 1 antibody. Conclusion It provides a reference for further study of properties and subsequent transformation. KEY WORDS: aflatoxin B 1 ; anti-idiotype antibody; nanobody; prokaryotic expression; renaturation B 1 (aflatoxin B 1, AFB 1 ), (Aspergillus flavus) (Aspergillus parasiticus) (Aspergillus nomius) [1],, [2], [3,4] [5,6] AFB 1 AFB 1,,, AFB 1 (anti-idiotype antibody, AId) V ( ) AId, * 通讯作者 :,,, [email protected] *Corresponding author: XU Yang, Professor, Doctoral Tutor, State Key Laboratory of Food Science and Technology, Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang , China. [email protected]

2 4, : B ,,, [7] 1993, Hamers [8], (heavychain antibodies, HCAbs) VHH (variable domain of heavy chain of heavy-chain antibody, VHH), [9],, (Mr) 15000, (nanobody, Nb) [10],,,, [11],,, VHH,, AFB 1 AFB 1, F7 F7 B 1,, AFB 1, F7,, ( ),,,, AFB 1 AFB 1 1 材料与方法 1.1 材料 质粒与菌种 pet22b Novagen ; E.coli DH5α E. coli BL21(DE3) ; F7 phen AFB 试剂与仪器 ( Thermo ); ( BIO-RAD ); Mulotifuge X1R ( Thermo ); SDS-PAGE ( BIO-RAD ); Taq DNA (NotⅠ NcoⅠ ) TaKaRa ; PCR ; M13 Parmacia 1.2 方法 表达载体 pet22b-f7 的构建 pet22b-f7 1 pet22b NotⅠ NcoⅠ F7 phen F7, F7 pet22b, E.coli DH5α, PCR,, pet22b-f7 E.coli BL21(DE3), PCR F7 独特型纳米抗体的诱导表达条件优化 pet22b-f7 37, 1% LB-A, 37 OD ~0.8, IPTG 0.4 mmol/l h, SDS-PAGE IPTG , 20, mmol/l IPTG , 20, IPTG 0.4 mmol/l h , IPTG 0.4 mmol/l 20 6 h, r/min 4 10 min,,, SDS-PAGE F7 独特型纳米抗体的溶解和复性 ph [12],

3 r/min 4 10 min, 50 mmol/l Tris HCl (ph 8.0), 2 mmol/l EDTA, 100 mmol/l NaCl, 0.5% TritonX-100(v/v), 2 mol/l, 6, 10 s, r/min 4 10 min, 1 50 mmol/l Tris HCl (ph 8.0), 8 mol/l, 10 mmol/l DTT, 2 mmol/l EDTA, 50 mmol/l Tris-HCl (ph 8.0), 0.5mmol/L EDTA, 50 mmol/l NaCl, 5% Glycerol (v/v), 3 mmol/l (GSH), 1 mmol/l (GSSG) 0.5 mol/l, 1 mg/ml F7 独特型纳米抗体的活性鉴定 ELISA AFB 1 (5 µl/ml) 96, 4 PBST 3, 4% 37 1 h PBST 3, (PBS ) AFB 1 ( ng/ml), 100 µl/, 37 1 h PBST 3, M13, 37 1h PBST 6, TMB, 100 µl/, 37 5 min, 50 µl/, H 2 SO 4 (2 mol/l), 450 nm OD 2 结果与分析 2.1 pet22b-f7 表达载体的构建和验证 pet22b-f7 NcoI NotI, 2 pet22b-f7( 2) 400 bp 5450bp, F7 pet22b-f7, Fig. 1 1 pet22b-f7 Scheme of construction of the prokaryotic expression vector pet22b-f7 2 Fig. 2 Identification of the recombinant plasmids by agarose gel electrophoresis M1: λ-hind III digest DNA Marker M2: DL 2000 DNA marker 1: the recombinant plasmids digested by NcoI and NotI

4 4, : B F7 独特型纳米抗体表达条件的优化 pet22b-f7 E.coli BL21(DE3), IPTG, SDS-PAGE 3, 0.4 mmol/l IPTG, ,, 20 4, 20, mmol/l IPTG, 5, 0.4 mmol/l IPTG h 1~6 h, 6 h, 6 h, 0.4 mmol/l IPTG 20 6 h, r/min 4,,, SDS-PAGE 6,, 80% pet22b-f7 N (pelb),,,,,, [13],, [14-16],, [17] :, ;, ;, 2.3 F7 独特型纳米抗体的体外复性及复性后活性鉴定 SDS-PAGE, 80% ( 6) ph 8.0,, 8 mol/l 0.5 mol/l,, 1 mg/ml 3 SDS-PAGE F7 Fig. 3 SDS-PAGE analysis of the expression of F7 at different temperature. M: protein molecular weight marker (low); 1: total cell protein (TCP) under non-induced; 2, 3, 4, 5: total cell protein at 20, 25, 30, 35 4 SDS-PAGE IPTG F7 Fig. 4 SDS-PAGE analysis of the expression of F7 at different concentrations of IPTG M: protein molecular weight marker (low); 1: TCP under non-induced condition 2, 3,4, 5, 6,7: TCP at 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mmol/l IPTG

5 F7,,, 5 SDS-PAGE F7 Fig. 5 SDS-PAGE analysis of the expression of F7 at different induced time M: protein molecular weight marker (low); 1: TCP under non-induced condition 2, 3,4, 5, 6: TCP at 1, 3, 6, 12, 24 h 7 ELISA F7 Fig. 7 Analysis of the F7 activities by indirect competition ELSA F7 from phage; F7 from pet22b-f7 3 讨论 6 SDS-PAGE F7 E.coli BL21(DE3) Fig.6 SDS-PAGE analysis of the expression of F7 in E.coli BL21(DE3) M: protein molecular weight marker (low); 1, 2: TCP under non-induced and induced condition; 3: soluble protein; 4: insoluble protein ELISA AFB 1 (5 µl/ml) 96, AFB ng/ml 7 AFB 1 F7 F7 AFB 1 AFB 1, 10 ng/ml AFB 1, AFB 1 F7 AFB 1, pet22b-f7 E.coli BL21(DE3), ( 3) IPTG ( 4) ( 5),, ELISA, AFB 1 AFB 1, AFB 1,,, [18,19] AFB 1

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