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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2020, Vol. 40 Issue (4): 42-48    DOI: 10.13523/j.cb.1910008
技术与方法     
构建噬菌体展示抗体库过程中电穿孔法的条件优化
杨丽1,石晓宇2,李文蕾2,李剑2,*(),徐寒梅1,*()
1 中国药科大学 南京 211100
2 天士力生物医药股份有限公司 上海 201203
Optimization of Electroporation Conditions in Construction of Phage Display Antibody Library
YANG Li1,SHI Xiao-yu2,LI Wen-lei2,LI Jian2,*(),XU Han-mei1,*()
1 China Pharmaceutical University, Nanjing 211100, China
2 Tasly Biopharmaceuticals Co., Ltd., Shanghai 201203, China
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摘要：

目的：确定稳定且高效的电转化实验方案以达到构建高库容噬菌体展示抗体库的目的。方法：探索电压、脉冲时间、噬菌粒DNA质量与浓度、TG1大肠杆菌的生长周期、重悬洗涤缓冲液及培养基优化等方面对TG1大肠杆菌电转化效率的影响。结果：当电转杯电极间距为2mm时,设定电转仪参数为3kV、25μF、5ms、200Ω;外源DNA经纯化后加入感受态菌液中使终浓度为1ng/μl;培养基中加入20mmol/L的MgCl2,并将TG1的生长阶段调控在OD600=0.8,用无菌超纯水重悬及洗涤细胞,将感受态细胞浓度调整为4×1010个/ml。在上述条件下,电转化效率可达到4.9×109CFU/μg DNA。结论：通过多种条件优化,提高了电转化效率,为构建高库容噬菌体展示抗体库建立了基础。
关键词： 电穿孔法;  电转化效率;  TG1大肠杆菌;  噬菌体展示抗体库    
Abstract:

Objective: To determine a stable and efficient electroporation protocol to construct a high-capacity phage display antibody library. Methods: Explored the effects of voltage, pulse time, quality and concentration of phagemid DNA, growth phase of TG1 E.coli, buffer for resuspension and washing and medium optimization on the electrotransformation efficiency of TG1 E.coli. Results: Using electroporation cuvettes with electrode spacing of 2 mm, the parameters of the electrorotator were set to 3kV, 25μF, 5ms, and 200 Ω. The exogenous DNA was purified and added to the competent bacterial suspension to make the final concentration of DNA 1ng/μl. 20mmol/L MgCl2 was added to the medium, and the growth phase of TG1 was adjusted to OD600=0.8. The cells were resuspended and washed with sterile ultrapure water, and the concentration of competent cells was adjusted to 4×1010 cells/ml. Under such conditions, the electrotransformation efficiency can reach 4.9×109 CFU/μg DNA. Conclusion: Electrotransformation efficiency has been improved through multiple condition optimization, which established the basis for constructing a high-capacity phage display antibody library.
Key words: Electroporation    Electrotransformation efficiency    E.coli strain TG1    Phage display antibody library
收稿日期: 2019-10-10 出版日期: 2020-05-18
ZTFLH:  Q936  
通讯作者: 李剑,徐寒梅     E-mail: lijian16@tasly.com;1037714870@qq.com
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杨丽
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引用本文:

杨丽,石晓宇,李文蕾,李剑,徐寒梅. 构建噬菌体展示抗体库过程中电穿孔法的条件优化[J]. 中国生物工程杂志, 2020, 40(4): 42-48.

YANG Li,SHI Xiao-yu,LI Wen-lei,LI Jian,XU Han-mei. Optimization of Electroporation Conditions in Construction of Phage Display Antibody Library. China Biotechnology, 2020, 40(4): 42-48.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.1910008    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I4/42

图1  TSL与pCANTAB 5E酶切产物电泳结果 M:DL5000 Marker;1:TSL酶切产物;2:pCANTAB 5E酶切产物
图2  电压和脉冲时间对电转效率的影响
DNA 纯化
方式		 DNA 的量
(ng)		 复苏菌液体积
(ml)		 菌落数
(×104 CFU, x?±s)
未纯化 400 4.5 38.67±6.43
纯化 400 4.5 490.33±134.97
表1  DNA纯化对菌落数的影响
图3  DNA纯化对电转效率的影响
DNA的浓度
(ng/μl)		 DNA的量
(ng)		 复苏菌液
体积(ml)		 菌落数
(×105 CFU,x?±s)
1 400 4.55 183.67±14.22
5 2 000 4.75 415.67±29.02
10 4 000 4.75 442.33±131.08
表2  DNA浓度对菌落数的影响
图4  DNA浓度对电转效率的影响
图5  TG1生长阶段对电转效率的影响
感受态细胞
制备条件		 DNA的量
(ng)		 复苏菌液体积
(ml)		 菌落数
(×105 CFU, x?±s)
OD=0.5,甘油/甘露醇 100 1 36.00±7.21
OD=0.5,超纯水 100 1 291.33±34.43
OD=0.8,超纯水 100 1 368.00±63.93
表3  重悬洗涤缓冲液对菌落数的影响
图6  重悬洗涤缓冲液对电转效率的影响
培养基成分 DNA的量
(ng)		 复苏菌液体积
(ml)		 菌落数
(×105 CFU, x?±s)
无MgCl2 100 1 240.00±60.70
含20 mmol/L MgCl2 100 1 489.67±133.72
表4  培养基中添加MgCl2对菌落数的影响
图7  培养基中添加MgCl2对电转效率的影响
[1] Barderas R, Benito-Peña E . The 2018 Nobel Prize in Chemistry: phage display of peptides and antibodies. Analytical and Bioanalytical Chemistry, 2019,411(12).
[2] Frenzel A, Schirrmann T, Hust M . Phage display-derived human antibodies in clinical development and therapy. Mabs, 2016,8(7):1177-1194.
doi: 10.1080/19420862.2016.1212149 pmid: 27416017
[3] Yang H Y, Kang K J, Chung J E , et al. Construction of a large synthetic human scFv library with six diversified CDRs and high functional diversity. Molecules & Cells, 2009,27(2):225-235.
doi: 10.1007/s10059-009-0028-9 pmid: 19277506
[4] Griffiths A D, Williams S C, Hartley O , et al. Isolation of high affinity human antibodies directly from large synthetic repertoires. The EMBO Journal, 1994,13(14):3245-3260.
pmid: 8045255
[5] 沈倍奋, 陈志南, 刘民培 . 重组抗体. 北京: 科学出版社, 2005:58.
Shen B F, Chen Z N, Liu M P . Recombinant antibodies. Beijing: Science Press, 2005:58.
[6] Experton J, Wilson A G, Martin C R . Low-voltage flow-through electroporation membrane and method. Methods in Molecular Biology, 2020,2050:43-55.
doi: 10.1007/978-1-4939-9740-4_5 pmid: 31468478
[7] 蒋华波, 夏梓元, 张琼阁 , 等. 大肠埃希菌TG1电穿孔法转化条件优化研究. 生物技术进展, 2017,7(1):72-76.
doi: 10.1016/s0958-1669(96)80098-x pmid: 8791316
Jiang H B, Xia Z Y, Zhang Q G , et al. Optimization of electroporation conditions for Escherichia coli strain TG1. Current Biotechnology, 2017,7(1):72-76.
doi: 10.1016/s0958-1669(96)80098-x pmid: 8791316
[8] 王薇玮, 孙大庆, 裴世春 . 高效电转化技术在噬菌体抗体库构建中的应用. 农产品加工(创新版), 2009,4:43-46.
Wang W W, Sun D Q, Pei S C . Application of highly effective electricity transformation technology in bacteriophage immune body storehouse construction. Innovational Edition of Farm Products Processing, 2009,4:43-46.
[9] 戴建新, 周凤鹃, 孙树汉 . 电穿孔法转化大肠杆菌的影响因素. 第二军医大学学报, 1999,20(5):340-340.
Dai J X, Zhou F J, Sun S H . Effect of electroporation on transformation of Escherichia coli. Academic Journal of Second Military Medical University, 1999,20(5):340-340.
[10] André F, Mir L M . DNA electrotransfer: its principles and an updated review of it therapeutic applications. Gene Therapy, 2004,11:S33-S42.
doi: 10.1038/sj.gt.3302367 pmid: 15454955
[11] Kimoto H, Taketo A . Initial stage of DNA-electrotransfer into E. coli cells. Journal of Biochemistry, 1997,122(1):237-242.
doi: 10.1093/oxfordjournals.jbchem.a021734 pmid: 9276694
[12] Lee M J, Cho S S, Jang H S , et al. Optimal salt concentration of vehicle for plasmid DNA enhances gene transfer mediated by electroporation. Experimental and Molecular Medicine, 2002,34(4):265-272.
doi: 10.1038/emm.2002.37 pmid: 12515391
[13] Schlaak C, Hoffmann P, May K , et al. Desalting minimal amounts of DNA for electroporation in E.coli: a comparison of different physical methods. Biotechnology Letters, 2005,27(14):1003-1005.
doi: 10.1007/s10529-005-7867-z
[14] 萨姆布鲁克 J, 格林 M R . 分子克隆实验指南.第四版. 北京: 科学出版社, 2017:141.
Sambrook J, Green M R . Guide to molecular cloning. Fourth edition. Beijing: Science Press, 2017:141.
[15] 荀安营, 李娜, 邹兵 , 等. 双歧杆菌感受态细胞的制备和电转化条件的研究. 中国微生态学杂志, 2013,25(10):1128-1130.
Gou A Y, Li N, Zhou B , et al. Preparation of electrotransformation competent cells of Bifidobacterium and condition of electrotransformation. Chinese Journal of Microecology, 2013,25(10):1128-1130.
[16] 李南, 凌世淦, 吴梧桐 . 电穿孔法转化TG1大肠杆菌的条件优化. 药物生物技术, 2001,8(3):143-146.
Li N, Lin S G, Wu W T . Optimization of electroporation protocol for Escherichia coli strain TG1. Pharmaceutical Biotechnology, 2001,8(3):143-146.
[17] Nováková J, Izsáková A, Grivalská T , et al. Improved method for high-efficiency electrotransformation of Escherichia coli with the large BAC plasmids. Folia Microbiologica, 2014,59(1):53-61.
doi: 10.1007/s12223-013-0267-1
[18] Wang H, Griffiths M W . Mg 2+-free buffer elevates transformation efficiency of Vibrio parahaemolyticus by electroporation . Letters in Applied Microbiology, 2009,48(3):349-354.
doi: 10.1111/j.1472-765X.2008.02531.x pmid: 19207855
[19] Shi X, Karkut T, Alting-Mees M , et al. Enhancing Escherichia coli electrotransformation competency by invoking physiological adaptations to stress and modifying membrane integrity. Analytical Biochemistry, 2003,320(1):152-155.
doi: 10.1016/s0003-2697(03)00352-x pmid: 12895481
[20] Islam M M, Odahara M, Yoshizumi T , et al. Cell-penetrating peptide-mediated transformation of large plasmid DNA into Escherichia coli. ACS Synthetic Biology, 2019,8(5):1215-1218.
doi: 10.1021/acssynbio.9b00055 pmid: 31008591
[21] 张建琼, 张雪萍, 谢维 , 等. 高效电转化率条件的探索. 南京师大学报(自然科学版), 2000,23(2):72-75.
Zhang J Q, Zhang X H, Xie W , et al. Survey the highest of transformations-efficiency of the competent cell. Journal of Nanjing Normal University(Natural Science), 2000,23(2):72-75.
[22] 路艳艳, 皇甫永穆 . 电穿孔法对分枝杆菌进行高效基因转化. 北京医科大学学报, 2000,32(2):178-181.
Lu Y Y, Huangpu Y M . High efficiency electro-genetic transformation of Mycobacteria. Journal of Beijing Medical University, 2000,32(2):178-181.
[23] 刘麒, 王阔鹏, 于凌娇 , 等. 电转化条件对TG1大肠杆菌转化效率的影响. 吉林农业科技学院学报, 2018,27(3):7-9.
Liu Q, Wang K P, Yu L J , et al. Impact of electroporation on transformation efficiency of Escherichia (E.) coli TG1. Journal of Jilin Agricultural Science and Technology University, 2018,27(3):7-9.
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