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1 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 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): 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 ,,,. hcg., 2018, 34(4): 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): 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 ). Corresponding authors: Huawei Xin. Tel: ; xinhuawei@wust.edu.cn (No )

2 570 ISSN CN /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 , 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

3 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 IPTG 0.25 mmol/l r/min 2 min SDS-PAGE 9 Buffer H (20 mmol/l Tris-HCl ph mol/l NaCl 10% 10 mmol/l β- 30 mmol/l ) 30 min r/min 20 min 12% SDS-PAGE 1.4 抗体融合蛋白的纯化与鉴定 ( 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 BCA A 1 BCA B (50 1) BCA μl 96 PBS 20 μl 96 PBS 20 μl 200 μl BCA 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 ml 37 1 h 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.

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.

4 572 ISSN CN /Q Chin J Biotech -sfgfp cabbcii10-cdr1/hcgbp1-sfgfp cabbcii10-cdr3/hcgbp3-sfgfp 0.1 ml 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 min 100 μl - (Britton-Robinson) ph min 100 μl 1.8 抗体融合蛋白的滴度测定 JEG ml 37 1 h cabbcii10-cdr1/ hcgbp1-sfgfp cabbcii10-cdr3/hcgbp3- sfgfp 0.1mL 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 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.

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. Expression of cabbcii10-hcgbp3-sfgfp fusion proteins.

3 Purification of anti-hcg-a and cabbcii10- hcgbp1/3-sfgfp fusion proteins. (A) Purification of anti-hcg-a fusion proteins.

5 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 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 cjb@im.ac.cn

6 574 ISSN CN /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 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 B 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 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.

7 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 CDR cjb@im.ac.cn

8 576 ISSN CN /Q Chin J Biotech CDR1 CDR3 CDR3 [11] Inoue CDR3 [18] CDR3 Hattori CDR3 CDR1 [16] CDR [19] cabbcii10 CDR1 GGS CDR1 CDR3 CDR2 CDR2 [16] CDR1 CDR3 CDR1 CDR3 ScFV CDR CDR ( ) [20-21] hcg hcg LH hcg REFERENCES [1] Muyldermans S. Single domain camel antibodies: current status. J Biotechnol, 2001, 74(4): [2] Cui HQ, Wang QM. Progress in single-domain antibody derived from heavy chain antibody. Chin J Biotech, 2005, 24(3): (in Chinese).,.., 2005, 24(3): [3] Liu S, Li J, Liang XG, et al. Research advance in single-domain antibody. Qianren Biol, 2015, 2(3): [4] Tanaka T, Rabbitts TH. Intracellular antibody capture (IAC) methods for single domain antibodies. Methods Mol Biol, 2012, 911: [5] Broekgaarden M, van Vught R, Oliveira S, et al. Site-specific conjugation of single domain antibodies to liposomes enhances photosensitizer uptake and photodynamic therapy efficacy. Nanoscale, 2016, 8(12): [6] Nelson AL, Dhimolea E, Reichert JM, et al. Development trends for human monoclonal antibody therapeutics. Nat Rev Drug Dis, 2010, 9(10): [7] Chi XY, Yu CM, Chen W. Single B cell monoclonal antibody technologies and applications. Chin J Biotech, 2012, 28(6): (in Chinese).,,. B., 2012, 28(6): [8] Chan JK, Tian CQ, Teoh D, et al. Survival after

9 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 577 recurrence in early-stage high-risk epithelial ovarian cancer: a gynecologic oncology group study. Gynecol Oncol, 2010, 116(3): [9] Kugasia IR, Alkayem M, Patel JB. A rare case of beta-hcg production by a solitary fibrous tumor of the pleura. Am J Case Rep, 2014, 15: [10] Nand KN, Gupta JC, Panda AK, et al. Development of a recombinant hcg-specific single chain immunotoxin cytotoxic to hcg expressing cancer cells. Protein Expr Purif, 2015, 106: [11] Yan JR, Li GH, Hu YH, et al. Construction of a synthetic phage-displayed nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications. J Trans Med, 2014, 12(1): 343. [12] Yan JR, Wang PY, Zhu M, et al. Characterization and applications of nanobodies against human procalcitonin selected from a novel naïve nanobody phage display library. J Nanobiotechnol, 2015, 13(1): 33. [13] Bond CJ, Marsters JC, Sidhu SS. Contributions of CDR3 to VHH domain stability and the design of monobody scaffolds for naive antibody libraries. J Mol Biol, 2003, 332(3): [14] Ding XK, Yang KL. Antibody-free detection of human chorionic gonadotropin by use of liquid crystals. Anal Chem, 2013, 85(22): [15] Saerens D, Pellis M, Loris R, et al. Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies. J Mol Biol, 2005, 352(3): [16] Hattori T, Umetsu M, Nakanishi T, et al. High affinity anti-inorganic material antibody generation by integrating graft and evolution technologies: potential of antibodies as biointerface molecules. J Biol Chem, 2010, 285(10): [17] Li HN, Yan JR, Ou WJ, et al. Construction of a biotinylated cameloid-like antibody for lable-free detection of apolipoprotein B-100. Biosens Bioelectron, 2015, 64: [18] Inoue H, Suganami A, Ishida I, et al. Affinity maturation of a CDR3-grafted VHH using in silico analysis and surface plasmon resonance. J Biochem, 2013, 154(4): [19] Haidar JN, Yuan QA, Zeng L, et al. A universal combinatorial design of antibody framework to graft distinct CDR sequences: a bioinformatics approach. Proteins, 2012, 80(3): [20] Govaert J, Pellis M, Deschacht N, et al. Dual beneficial effect of interloop disulfide bond for single domain antibody fragments. J Biol Chem, 2012, 287(3): [21] Zabetakis D, Anderson GP, Bayya N, et al. Contributions of the complementarity determining regions to the thermal stability of a single-domain antibody. PLoS ONE, 2013, 8(10): e ( 本文责编郝丽芳 ) cjb@im.ac.cn

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr PCR 龙平, 崔占虎, 李虔全, 徐建平, 张春红, 周立社 * *, 李旻辉 ( 包头医学院, 内蒙古包头 014060) DNA 30 28 DNA trnl-trnf PCR PCR 136 bp 323 bp 10% PCR PCR Astragalus membranaceus Fisch. Bge. var. mongho- licus Bge. Hsiao A. membranaceus