生物工程学报 Chinese Journal of Biotechnology DOI: 10.13345/j.cjb.170372 彭静等 / 利用抗原结合多肽嫁接抗体技术制备抗 hcg 单域抗体 Apr. 25, 2018, 34(4): 569 577 2018 Chin J Biotech, All rights reserved 医学与免疫生物技术 569 hcg 彭静, 王琼, 程小玲, 刘梦雯, 王美, 辛化伟 430065,,,. hcg., 2018, 34(4): 569 577. Peng J, Wang Q, Cheng XL, et al. Preparation of anti-hcg single domain antibody by antibody grafting technique using an antigen-binding peptide. Chin J Biotech, 2018, 34(4): 569 577. 摘要 : 本研究旨在人绒毛膜促性腺激素 (hcg) 的结合多肽的基础上应用嫁接抗体技术制备抗 hcg 单域抗体, 简化单域抗体制备过程, 提高多肽生化稳定性 利用单域抗体通用骨架 (cabbcii10), 以 hcg 结合多肽取代互补决定区 CDR1 或 CDR3, 合成 cabbcii10 嫁接抗体全基因序列并与 sfgfp 基因序列融合后, 插入到带有 His 标签的原核表达载体 pet30a(+) 中, 成功构建了 pet30a-(his6)-cabbcii10-cdr1/hcgbp1-sfgfp 与 pet30a-(his6)- cabbcii10-cdr3/hcgbp3-sfgfp 融合蛋白表达质粒 将重组质粒转化大肠杆菌 BL21 (DE3), 用 IPTG 诱导表达融合蛋白, 得到高表达量的可溶性融合蛋白 利用 Ni-NTA 亲和柱纯化得到纯蛋白, 应用 SDS-PAGE 鉴定纯化的蛋白为正确表达的目标蛋白 通过抗原抗体结合实验, 发现 hcg 结合多肽嫁接到单域抗体通用骨架的互补决定区 CDR1 或 CDR3 后都有抗原结合活性, 具有相似的抗体滴度, 且嫁接到 CDR3 后的抗原结合活性比 CDR1 要高 (2 3 倍 ) 嫁接抗体基本保留了所用单域抗体框架较为稳定的生化特性, 具有一定的热稳定性和较好的碱耐受性, 同时, 所接入的 hcg 结合片段对 hcg 具有较特异的结合活性, 为进一步优化抗原结合多肽嫁接抗体技术制备抗 hcg 单域抗体提供了可靠的实验基础 : hcg, 单域抗体, 嫁接抗体,CDR1,CDR3, 融合蛋白, 表达纯化 Received: September 21, 2017; Accepted: December 27, 2017 Supported by: National Natural Science Foundation of China (No. 31371385). Corresponding authors: Huawei Xin. Tel: +86-27-68893368; E-mail: xinhuawei@wust.edu.cn (No. 31371385) 2018-01-10 http://kns.cnki.net/kcms/detail/11.1998.q.20180109.0849.002.html
570 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech Preparation of anti-hcg single domain antibody by antibody grafting technique using an antigen-binding peptide Jing Peng, Qiong Wang, Xiaoling Cheng, Mengwen Liu, Mei Wang, and Huawei Xin College of Life Science and Health, Wuhan University of Science and Technology, Wuhan 430065, Hubei, China Abstract: We used the antibody grafting technology to prepare anti-hcg single-domain antibodies on the basis of antigen-binding peptide to simplify the single-domain antibody preparation process and improving the biochemical stability of peptide. By using a universal single-domain antibody backbone (cabbcii10), CDR1 or CDR3 was replaced by the hcg-binding peptide, and the grafted antibody gene sequences were synthesized and cloned into the prokaryotic expression vector pet30a(+) in fusion with a C-terminal sfgfp gene, i.e. pet30a-(his6)-cabbcii10-cdr1/hcgbp1-sfgfp and pet30a-(his6)-cabbcii10- CDR3/hCGBP3-sfGFP. The recombinant plasmids were transformed into E. coli BL21(DE3), and the fusion proteins were induced by IPTG. Highly soluble recombinant fusion proteins were obtained and purified by Ni-NTA affinity column. SDS-PAGE confirmed the purified protein as the target protein. The antigen-antibody binding assay showed that both the CDR1 and CDR3 grafted antibodies have hcg-binding activities. While the titers of the two grafted antibodies were similar, the binding affinity of CDR3 grafted antibody was higher than that of CDR1 grafted protein (about 2 3 times). The grafted antibodies retained the relatively high biochemical stability of the single-domain antibody backbone and were relatively thermostable and alkaline tolerant. The obtained antibodies also had a relatively high antigen-binding specificity to hcg. This study provided a reliable experimental basis for further optimization of anti-hcg single domain antibody by antibody grafting technology using antigen-binding peptide. Keywords: hcg, single domain antibody, grafted antibody, CDR1, CDR3, fusion protein, expression and purification [1-3] [4-5] [6-7] hcg [8-9] hcg [10] hcg [11-12] hcg CDR1 CDR3 1 材料与方法 1.1 材料 pet30a(+) pet30a(+)-sfgfp DP-GFP ( GFP ) LHB ( )+hcg-α 293T BL21 (DE3) Taq TaKaRa PCR Clean up Axygen Ni-NTA ( ) IPTG R-250 AMRESCO T4 DNA
彭静等 / 利用抗原结合多肽嫁接抗体技术制备抗 hcg 单域抗体 571 marker Thermo Scientific DNA marker DNA BCA JEG-3 BIOFIL SpectraMax i3x Molecular Devices 1.2 质粒的构建 anti-hcg-α [13] BamHⅠ Hind Ⅲ pet30a(+) BamHⅠ Hind Ⅲ T4 DNA anti-hcg-α pet30a(+) DNA 22 2 h BL21(DE3) BamHⅠ Hind Ⅲ DNA cabbcii10-cdr1/hcgbp1 cabbcii10-cdr3/hcgbp3 [14-15] BamH Ⅰ SalⅠ pet30a-sfgfp BamHⅠ SalⅠ T4 DNA cabbcii10-cdr1/hcgbp1 cabbcii10-cdr3/ hcgbp3 pet30a-sfgfp 22 2 h BL21(DE3) BamHⅠ SalⅠ BamHⅠ Hind Ⅲ DNA 1.3 抗体融合蛋白的诱导表达 DNA 37 OD 600 0.6 0.8 IPTG 0.25 mmol/l 3 10 000 r/min 2 min SDS-PAGE 9 Buffer H (20 mmol/l Tris-HCl ph 8.0 1 mol/l NaCl 10% 10 mmol/l β- 30 mmol/l ) 30 min 10 000 r/min 20 min 12% SDS-PAGE 1.4 抗体融合蛋白的纯化与鉴定 (16 120 r/min) Ni-NTA 12% SDS-PAGE 1.5 抗体融合蛋白浓度的测定 1.2 ml (30 mg BSA) 25 mg/ml 20 μl 25 mg/ml 980 μl 0.5 mg/ml PBS 12 50 BCA A 1 BCA B (50 1) BCA 0 1 2 4 8 12 16 20 μl 96 PBS 20 μl 96 PBS 20 μl 200 μl BCA 37 30 min 1.6 抗体融合蛋白的抗原结合活性测定 PBS 5% PBS anti-hcg-α 1 μg/ml 0.1 ml 4 PBS 3 3 min ( ) JEG-3 0.1 ml 37 1 h 010-64807509 cjb@im.ac.cn
572 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech -sfgfp cabbcii10-cdr1/hcgbp1-sfgfp cabbcii10-cdr3/hcgbp3-sfgfp 0.1 ml 37 0.5 1 h 3 GFP 0.2 ml PBS 488 nm 525 nm 1.7 cabbcii10-cdr1/hcgbp1-sfgfp cabbcii10-cdr3/hcgbp3-sfgfp DP-GFP ( GFP ) GFP 4 16 37 42 65 80 30 min 100 μl - (Britton-Robinson) ph 4.0 5.0 6.0 7.0 8.0 9.0 10.0 4 30 min 100 μl 1.8 抗体融合蛋白的滴度测定 JEG-3 0.1 ml 37 1 h cabbcii10-cdr1/ hcgbp1-sfgfp cabbcii10-cdr3/hcgbp3- sfgfp 0.1mL 37 0.5 1 h 3 GFP 1.9 抗体融合蛋白的抗原结合特异性测定 JEG-3 LHB+hCG-α 293T 0.1 ml 37 1 h -sfgfp GFP 2 结果与分析 2.1 原核表达载体的构建 pet30a-anti-hcg- 5.3 kb 400 bp pet30 cabbcii10-hcgbp1/3-sfgfp BamHⅠ SalⅠ 6.1 kb 400 bp pet30a-cabbcii10- hcgbp1/3-sfgfp BamHⅠ Hind Ⅲ 5.3 kb 1.2 kb ( 1) DNA 3 2.2 融合蛋白的诱导表达分析 pet30a-anti-hcg-α pet30a-cabbcii10- CDR1/hCGBP1-sfGFP pet30a-cabbcii10-cdr3/ hcgbp3-cabbcii10-sfgfp BL21(DE3) IPTG 图 1 重组质粒 pet30a-anti-hcg-α 和 pet30acabbcii10-hcgbp1/3-sfgfp 的构建 Fig. 1 Construction of pet30a-anti-hcg-α and pet30acabbcii10-hcgbp1/3-sfgfp recombinant plasmids. M1: 1 kb DNA marker; M2: 100 bp DNA marker; 1: pet30a-anti-hcg-α digested with BamHⅠand Hind Ⅲ; 2,3: pet30a-cabbcii10-hcgbp1/3-sfgfp digested with BamHⅠ and SalⅠ; 4,5: pet30a-cabbcii10-hcgbp1/3- sfgfp digested with BamHⅠ and Hind Ⅲ.
彭静等 / 利用抗原结合多肽嫁接抗体技术制备抗 hcg 单域抗体 573 SDS-PAGE 2 IPTG 3 2.3 融合蛋白的纯化与鉴定 pet30a-anti-hcg-α pet30a-cabbcii10-cdr1/ hcgbp1-sfgfp pet30a-cabbcii10-cdr3/hcgbp3- cabbcii10-sfgfp Ni-NTA 3 3 anti-hcg-α 25 kda 22 kda cabbcii10-hcgbp1/3-sfgfp 54 kda 55 kda 图 2 anti-hcg- 和 cabbcii10-hcgbp1/3-sfgfp 融合蛋白的表达 Fig. 2 Expression of anti-hcg- and cabbcii10- hcgbp1/3-sfgfp fusion proteins. (A) Expression of anti-hcg- usion proteins. M: protein marker; 1: before induction; 2: after induction; 3: supernatant; 4: precipitate. (B) Expression of cabbcii10-hcgbp1-sfgfp fusion proteins. M: protein marker; 1: before induction; 2: after induction; 3: supernatant; 4: precipitate. Expression of cabbcii10-hcgbp3-sfgfp fusion proteins. 5: before induction; 6: after induction; 7: supernatant; 8: precipitate. 图 3 anti-hcg- 和 cabbcii10-hcgbp1/3-sfgfp 融合蛋白的纯化 Fig. 3 Purification of anti-hcg-a and cabbcii10- hcgbp1/3-sfgfp fusion proteins. (A) Purification of anti-hcg-a fusion proteins. M: protein marker, 1 6: eluents of Ni-NTA affinity chromatography resin; 7 8: flow-through of Ni-NTA affinity chromatography resin; 9: total protein. M: protein marker. (B) Purification of cabbcii10-hcgbp1-sfgfp fusion proteins. M: protein marker; 1: total protein; 2: flow-through of Ni-NTA affinity chromatography resin; 3 9: eluents of Ni-NTA affinity chromatography resin. (C) Purification of cabbcii10- hcgbp1/3-sfgfp fusion proteins. 1: total protein; 2,3: flow-through of Ni-NTA affinity chromatography resin; 4 9: eluent of Ni-NTA affinity chromatography resin. 010-64807509 cjb@im.ac.cn
574 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech 2.4 融合蛋白的浓度测定及抗体融合蛋白的抗原结合活性分析 BSA pet30a-antihcg-α pet30a-cabbcii10-cdr1/hcgbp1-sfgfp pet30a-cabbcii10-cdr3/hcgbp3-sfgfp 0.65 2.43 3.60 mg/ml anti-hcg-a cabbcii10-cdr1/hcgbp1-sfgfp cabbcii10- CDR3/hCGBP3-sfGFP hcg 4 hcg CDR3 (hcgbp3) CDR1 (hcgbp1) 2 3 LH) 7 hcg LH LH cabbcii10- CDR3/hCGBP3-sfGFP hcg 2.5 抗体融合蛋白的稳定性 5A cabbcii10-hcgbp1/3-sfgfp 42 65 5B ph cabbcii10-hcgbp1/3-sfgfp ph<7 ph ph 4 ph 8 10 (10% 20%) 2.6 抗体融合蛋白的滴度 cabbcii10-cdr1/hcgbp1-sfgfp cabbcii10-cdr3/hcgbp3-sfgfp hcg 6 cabbcii10-hcgbp1/3-sfgfp 1 15 2.7 抗体融合蛋白的抗原结合特异性 JEG-3 LHB+hCG-α 293T cabbcii10-cdr1/ hcgbp1-sfgfp cabbcii10-cdr3/hcgbp3-sfgfp (hcg 图 4 融合蛋白 cabbcii10-hcgbp1/3-sfgfp 结合 hcg 的活性测定 Fig. 4 Determination of hcg-binding affinity of cabbcii10-hcgbp1/3-sfgfp fusion proteins. (A) HCG-binding curve against Ag relative concentration. (B) HCG-binding curve against log2 of Ag relative concentration.
彭静等 / 利用抗原结合多肽嫁接抗体技术制备抗 hcg 单域抗体 575 图 6 抗体融合蛋白的滴度测定 Fig. 6 Determination of the titers of cabbcii10- hcgbp1/3-sfgfp fusion proteins. 图 5 抗体融合蛋白的稳定性测定 Fig. 5 Determination of the stability of cabbcii10- hcgbp1/3-sfgfp fusion proteins. (A) Relative protein stability at different temperatures. (B) Relative protein stability at different ph. 3 讨论 CDR [16-17] 图 7 抗体融合蛋白的抗原结合特异性测定 Fig. 7 Determination of antigen-binding specificity of cabbcii10-hcgbp1/3-sfgfp fusion proteins. Binding affinities of cabbcii10-hcgbp1/3-sfgfp fusion proteins to hcg and LH were compared. CDR cabbcii10 [15] hcg CDR1 CDR3 CDR1 CDR3 CDR3 010-64807509 cjb@im.ac.cn
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