Expression, Purification Strategy and Detection Method Establishment of Anti-Fenitrothion Nanobody

GUO Pengyan; XU Zhenlin; CHEN Zijian; SHEN Xing

doi:10.13386/j.issn1002-0306.2023030328

Volume 44 Issue 23

Nov. 2023

Turn off MathJax

Article Contents

Abstract

References

Science and Technology of Food Industry > 2023 > 44(23): 298-305. > DOI: 10.13386/j.issn1002-0306.2023030328

GUO Pengyan, XU Zhenlin, CHEN Zijian, et al. Expression, Purification Strategy and Detection Method Establishment of Anti-Fenitrothion Nanobody[J]. Science and Technology of Food Industry, 2023, 44(23): 298−305. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023030328.

Citation:

PDF (2046 KB)

Expression, Purification Strategy and Detection Method Establishment of Anti-Fenitrothion Nanobody

1.
College of Food Science, South China Agricultural University, Guangzhou 510642, China
2.
School of Food Pharmaceutical Engineering, Zhaoqing University, Zhaoqing 526061, China

More Information

Received Date: March 28, 2023
Available Online: October 08, 2023

Graphical Abstract

Abstract

Abstract

For the purpose of preparing anti-fenitrothion nanobodies with high-purity efficiently, the expression conditions of a recombinant anti-fenitrothion nanobody with low expression level in E. coli was optimized. The IPTG concentration and inducing temperature were selected as the independent variables and the nanobody expression level was used as the dependent variable for single-factor experiments, and the purification strategy was also investigated. The results showed that the highest expression level of 6 mg/L anti-fenitrothion nanobody was achieved at 37 ℃ without IPTG. Moreover, an interactive effect of IPTG dosage and inducing temperature was found. After a two-step purification of Ni affinity chromatography and gel filtration chromatography, the anti-fenitrothion nanobodies was finally yielded with more than 98% purity, indicating that the expression and purification strategies in this study can obtain anti-fenitrothion nanobodies efficiently. An ic-ELISA assay was then established based on the obtained nanobody, with an IC₅₀ of 5.81 ng/mL and an LOD of 0.25 ng/mL, and a detection range of 0.78~43.07 ng/mL. The assay was applied to determine fenitrothion in Chinese cabbage and lettuce samples. The recovery rate of addition was between 93.3%~111.7%, and the coefficient of variation (CV) was between 2.3%~18.2%. The proposed assay based on anti-fenitrothion nanobody has high sensitivity, which can meet the requirements for monitoring of fenitrothion under the national standard, thus can be used for rapid screening of fenitrothion. This study laid a foundation for the development of efficient and rapid immunoassays for fenitrothion.
- fenitrothion,
- nanobody,
- induced expression,
- purification strategy,
- activity identification

FullText(HTML)

References (41)

References

[1]	HU H Y, YANG L Q. Development of enzymatic electrochemical biosensors for organophosphorus pesticide detection[J]. Journal of Environmental Science and Health, Part B,2021,56(2):168−180. doi: 10.1080/03601234.2020.1853460
[2]	叶茂, 沈晓玲, 陈青舟, 等. 胶体金免疫层析法同时检测果蔬中四种农药残留[J]. 食品工业科技,2023,44(6):300−308. [YE Mao, SHEN Xiaoling, CHEN Qingzhou, et al. Simultaneous determination of four pesticide residues in fruits and vegetables by colloidal gold immunochromatography[J]. Science and Technology of Food Industry,2023,44(6):300−308. YE Mao, SHEN Xiaoling, CHEN Qingzhou, et al. Simultaneous determination of four pesticide residues in fruits and vegetables by colloidal gold immunochromatography[J]. Science and Technology of Food Industry, 2023, 44（6）: 300−308.
[3]	CANL A G, SÜRÜCÜ B, ULUSOY H İ, et al. Analytical methodology for trace determination of propoxur and fenitrothion pesticide residues by decanoic acid modified magnetic nanoparticles[J]. Molecules,2019,24(24):4621. doi: 10.3390/molecules24244621
[4]	王艳, 赵宁, 李虹佳, 等. 电沉积法构建AChE/COD@AuNPs共固定化酶生物传感器差分脉冲伏安法快速检测有机磷农药[J]. 中国食品学报,2023,23(1):356−363. [WANG Yan, ZHAO Ning, LI Hongjia, et al. Rapid detection of organophosphorus pesticides by differential pulse voltammetry with AChE/COD@AuNPs co-immobilized enzyme biosensor constructed by electrodeposition[J]. Journal of Chinese Institute of Food Science and Technology,2023,23(1):356−363. WANG Yan, ZHAO Ning, LI Hongjia, et al. Rapid detection of organophosphorus pesticides by differential pulse voltammetry with AChE/COD@AuNPs co-immobilized enzyme biosensor constructed by electrodeposition[J]. Journal of Chinese Institute of Food Science and Technology, 2023, 23（1）: 356−363.
[5]	TRINH K H, KADAM U S, SONG J N, et al. Novel DNA aptameric sensors to detect the toxic insecticide fenitrothion[J]. International Journal of Molecular Sciences,2021,22(19):10846. doi: 10.3390/ijms221910846
[6]	林珍珠. 泉州地区蔬菜有机磷农药残留检测分析[J]. 福建农业科技,2018(5):46−48. [LIN Zhenzhu. Detection on organophosphorus pesticide residue in vegetables from Quanzhou area[J]. Fujian Agricultural Science and Technology,2018(5):46−48. LIN Zhenzhu. Detection on organophosphorus pesticide residue in vegetables from Quanzhou area[J]. Fujian Agricultural Science and Technology, 2018（5）: 46−48.
[7]	杨眉, 宋明雄, 业润泽. Spe-gcms/ms法测定粮食中的10种有机磷[J]. 粮食科技与经济,2020,45(5):84−89. [YANG Mei, SONG Mingxiong, YE Runze. Determination of 10 kinds of organic phosphorus in grain by SPE-GCMS/MS[J]. Grain Science and Technology and Economy,2020,45(5):84−89. YANG Mei, SONG Mingxiong, YE Runze. Determination of 10 kinds of organic phosphorus in grain by SPE-GCMS/MS[J]. Grain Science and Technology and Economy, 2020, 45（5）: 84−89.
[8]	魏茂琼, 兰珊珊, 王丽, 等. 环境中毒死蜱的酶联免疫分析方法研究[J]. 广东农业科学,2022,49(12):82−89. [WEI Maoqiong, LAN Shanshan, WANG Li, et al. Study on enzyme-linked immunosorbent assay for chlorpyrifos in environment[J]. Guangdong Agricultural Sciences,2022,49(12):82−89. WEI Maoqiong, LAN Shanshan, WANG Li, et al. Study on enzyme-linked immunosorbent assay for chlorpyrifos in environment[J]. Guangdong Agricultural Sciences, 2022, 49（12）: 82−89.
[9]	赵志高, 骆骄阳, 付延伟, 等. 免疫分析技术在农药残留分析中的研究进展及在中药中的应用展望[J]. 分析测试学报,2021,40(1):149−158. [ZHAO Zhigao, LUO Jiaoyang, FU Yanwei, et al. Research progress on immunoassay in pesticide residue analysis and its application prospect in Chinese herbal medicine[J]. Journal of Instrumental Analysis,2021,40(1):149−158. ZHAO Zhigao, LUO Jiaoyang, FU Yanwei, et al. Research progress on immunoassay in pesticide residue analysis and its application prospect in Chinese herbal medicine[J]. Journal of Instrumental Analysis, 2021, 40（1）: 149−158.
[10]	QIE Zhiwei, HUANG Ziwei, GAO Zichen, et al. Pretreatment-integration for milk protein removal and device-facilitated immunochromatographic assay for 17 items[J]. Scientific Reports,2019,9(1):11630. doi: 10.1038/s41598-019-47692-6
[11]	JIAO Shasha, LIU Pengyan, LIU Ying, et al. Binding properties of broad-specific monoclonal antibodies against three organophosphorus pesticides by a direct surface plasmon resonance immunosensor[J]. Analytical and Bioanalytical Chemistry,2018,410(28):7263−7273. doi: 10.1007/s00216-018-1337-7
[12]	YU Jicheng, GUO Tingting, ZHANG Wei, et al. Simultaneous detection of 2, 4-dichlorophenoxyacetic acid and fenitrothion through lanthanide doped β-NaYF₄ upconversion nanoparticles with different emitting light colors[J]. Materials Research Bulletin,2019,111:133−139. doi: 10.1016/j.materresbull.2018.11.016
[13]	CHO Y A, SEOK J A, LEE H S, et al. Synthesis of haptens of organophosphorus pesticides and development of immunoassays for fenitrothion[J]. Analytica Chimica Acta,2004,522(2):215−222. doi: 10.1016/j.aca.2004.05.083
[14]	ZOU Rubing, CHANG Yunyun, ZHANG Tianyi, et al. Up-converting nanoparticle-based immunochromatographic strip for multi-residue detection of three organophosphorus pesticides in food[J]. Frontiers in Chemistry,2019,7:18. doi: 10.3389/fchem.2019.00018
[15]	CHEN Z J, ZHANG Y F, CHEN J L, et al. Production and characterization of biotinylated anti-fenitrothion nanobodies and development of sensitive fluoroimmunoassay[J]. Journal of Agricultural and Food Chemistry,2022,70(13):4102−4111. doi: 10.1021/acs.jafc.2c00826
[16]	RODRÍGUEZ-CARMONA E, CANO-GARRIDO O, DRAGOSITS M, et al. Recombinant fab expression and secretion in Escherichia coli continuous culture at medium cell densities:Influence of temperature[J]. Process Biochemistry,2012,47(3):446−452. doi: 10.1016/j.procbio.2011.11.024
[17]	OZAKI C Y, SILVEIRA C R, ANDRADE F B, et al. Single chain variable fragments produced in Escherichia coli against heat-labile and heat-stable toxins from enterotoxigenic E. coli[J]. PLoS One,2015,10(7):e131484.
[18]	何晓婷, 董洁娴, 沈兴, 等. 纳米抗体的稳定性及其结构基础研究进展[J]. 生物化学与生物物理进展,2022,49(6):1004−1017. [HE Xiaoting, DONG Jiexian, SHEN Xing, et al. Advances on the relationship between stability and structure of nanobody:A review[J]. Progress in Biochemistry and Biophysics,2022,49(6):1004−1017. HE Xiaoting, DONG Jiexian, SHEN Xing, et al. Advances on the relationship between stability and structure of nanobody: A review[J]. Progress in Biochemistry and Biophysics, 2022, 49（6）: 1004−1017.
[19]	VALDÉS-TRESANCO M S, MOLINA-ZAPATA A, POSE A G, et al. Structural insights into the design of synthetic nanobody libraries[J]. Molecules,2022,27(7):2198. doi: 10.3390/molecules27072198
[20]	SIMÕES B, GUEDENS W J, KEENE C, et al. Direct immobilization of engineered nanobodies on gold sensors[J]. ACS Applied Materials & Interfaces,2021,13(15):17353−17360.
[21]	REZAEI L, SHOJAOSADATI S A, FARAHMAND L, et al. Enhancement of extracellular bispecific anti-MUC1 nanobody expression in E. coli BL21 (DE3) by optimization of temperature and carbon sources through an autoinduction condition[J]. Engineering in Life Sciences,2020,20(8):338−349. doi: 10.1002/elsc.201900158
[22]	ADAMS A M, KAPLAN N A, WEI Z Y, et al. In vivo production of psilocybin in E. coli[J]. Metabolic Engineering,2019,56:111−119. doi: 10.1016/j.ymben.2019.09.009
[23]	TAKAYAMA Y, AKUTSU H. Expression in periplasmic space of Shewanella oneidensis[J]. Protein Expression and Purification,2007,56(1):80−84. doi: 10.1016/j.pep.2007.06.005
[24]	GOPHNA U, IDESES D, ROSEN R, et al. OmpA of a septicemic Escherichia coli O78--secretion and convergent evolution[J]. International Journal of Medical Microbiology, 2004, 294(6):373−381.
[25]	ZHOU Hui, HE Cong, LI Zhenfeng, et al. Development of a rapid gold nanoparticle immunochromatographic strip based on the nanobody for detecting 2,4-dichlorophenoxyacetic acid[J]. Biosensors,2022,12(2):84. doi: 10.3390/bios12020084
[26]	LUO Lin, LIN Shiqi, WU Zhuoyu, et al. Nanobody-based fluorescent immunoassay using carbon dots anchored cobalt oxyhydroxide composite for the sensitive detection of fenitrothion[J]. Journal of Hazardous Materials,2022,439:129701. doi: 10.1016/j.jhazmat.2022.129701
[27]	王宇, 张译丰, 沈玉栋, 等. 抗杀螟硫磷生物素化纳米抗体的制备及其在免疫检测方法中的应用[J]. 现代食品科技,2021,37(8):286−294. [WANG Yu, ZHANG Yifeng, SHEN Yudong, et al. Production of anti-fenitrothion biotinylated nanobody and the application in the development of immunoassay[J]. Modern Food Science and Technology,2021,37(8):286−294. WANG Yu, ZHANG Yifeng, SHEN Yudong, et al. Production of anti-fenitrothion biotinylated nanobody and the application in the development of immunoassay[J]. Modern Food Science and Technology, 2021, 37（8）: 286−294.
[28]	刘晓丽, 闫干干, 闫浩浩, 等. 登革病毒ns2b-ns3蛋白酶在大肠埃希菌中表达条件的优化及酶活性测定[J]. 中国生物制品学杂志, 2023:1−8. [LIU Xiaoli, YAN Gangan, YAN Haohao, et al. Optimization of expression conditions of dengue virus NS2B-NS3 protease in E.coli and determination of enzyme activity[J]. Chinese Journal of Biologicals, 2023:1−8. LIU Xiaoli, YAN Gangan, YAN Haohao, et al. Optimization of expression conditions of dengue virus NS2B-NS3 protease in E.coli and determination of enzyme activity[J]. Chinese Journal of Biologicals, 2023: 1−8.
[29]	赵烨清, 石莉, 欧阳臻, 等. 化学-渗透压法温和破碎处理下大肠杆菌细胞胞内蛋白质的释放率[J]. 江苏农业科学,2017,45(19):146−149. [ZHAO Yeqing, SHI Li, OU Yangzhen, et al. Intracellular protein release rate of Escherichia coli cells treated by chemical-osmotic pressure and mild crushing[J]. Jiangsu Agricultural Sciences,2017,45(19):146−149. ZHAO Yeqing, SHI Li, OU Yangzhen, et al. Intracellular protein release rate of Escherichia coli cells treated by chemical-osmotic pressure and mild crushing[J]. Jiangsu Agricultural Sciences, 2017, 45（19）: 146−149.
[30]	GROTE A, HILLER K, SCHEER M, et al. JCat:A novel tool to adapt codon usage of a target gene to its potential expression host[J]. Nucleic Acids Research, 2005, 33:W526-W531.
[31]	WEBSTER G R, TEH A Y, MA J K. Synthetic gene design-the rationale for codon optimization and implications for molecular pharming in plants[J]. Biotechnol Bioeng,2017,114(3):492−502. doi: 10.1002/bit.26183
[32]	SADR V, SAFFAR B, EMAMZADEH R. Functional expression and purification of recombinant hepcidin25 production in Escherichia coli using SUMO fusion technology[J]. Gene,2017,610:112−117. doi: 10.1016/j.gene.2017.02.010
[33]	HABIB I, SMOLAREK D, HATTAB C, et al. VHH (nanobody) directed against human glycophorin A:A tool for autologous red cell agglutination assays[J]. Analytical Biochemistry,2013,438(1):82−89. doi: 10.1016/j.ab.2013.03.020
[34]	DVORAK P, CHRAST L, NIKEL P I, et al. Exacerbation of substrate toxicity by IPTG in Escherichia coli BL21 (DE3) carrying a synthetic metabolic pathway[J]. Microbial Cell Factories,2015,14(1):201. doi: 10.1186/s12934-015-0393-3
[35]	MALAKAR P, VENKATESH K V. Effect of substrate and IPTG concentrations on the burden to growth of Escherichia coli on glycerol due to the expression of Lac proteins[J]. Applied Microbiology and Biotechnology,2012,93(6):2543−2549. doi: 10.1007/s00253-011-3642-3
[36]	ZHANG Z, KUIPERS G, NIEMIEC Ł, et al. High-level production of membrane proteins in E. coli BL21(DE3) by omitting the inducer IPTG[J]. Microbial Cell Factories,2015,14(1):142. doi: 10.1186/s12934-015-0328-z
[37]	WANDREY G, BIER C, BINDER D, et al. Light-induced gene expression with photocaged IPTG for induction profiling in a high-throughput screening system[J]. Microbial Cell Factories,2016,15(1):63. doi: 10.1186/s12934-016-0461-3
[38]	JIN Weixin, XING Zizhuo, SONG Yuanli, et al. Protein aggregation and mitigation strategy in low pH viral inactivation for monoclonal antibody purification[J]. MAbs,2019,11(8):1479−1491. doi: 10.1080/19420862.2019.1658493
[39]	史伟, 禹婷. 蛋白质的层析分离[J]. 内蒙古农业科技,2011(1):110−112. [SHI Wei, YU Ting. Protein chromatography separation[J]. Inner Mongolia Agricultural Science and Technology,2011(1):110−112. SHI Wei, YU Ting. Protein chromatography separation[J]. Inner Mongolia Agricultural Science and Technology, 2011（1）: 110−112.
[40]	LIU Yihua, GUO Yirong, ZHU Guonian, et al. Enzyme-linked immunosorbent assay for the determination of five organophosphorus pesticides in camellia oil[J]. Journal of Food Protection,2014,77(7):1178−1183. doi: 10.4315/0362-028X.JFP-13-465
[41]	LUO Yihui, XIA Yuxian. Selection of single-chain variable fragment antibodies against fenitrothion by ribosome display[J]. Analytical Biochemistry,2012,421(1):130−137. doi: 10.1016/j.ab.2011.10.044

Supplements (0)

Cited By

Cited by

Periodical cited type(4)

1.	黄潇漪，贾利蓉，孙玉鼎，曹月刚，冉旭. 天然香辛料对烘炒花生仁货架期品质的影响. 食品工业科技. 2024(12): 285-293 . 本站查看
2.	魏甜甜，魏勃，王承，李凯，谢彩锋，杭方学. 黄冰糖低温浸渍茉莉花制备风味糖浆工艺优化. 食品工业科技. 2022(12): 181-187 . 本站查看
3.	邹林武，姜福全，戚智胜. 白冰糖提取玫瑰花风味的工艺研究. 现代食品. 2022(15): 94-96+117 .
4.	宣晓婷，陈思媛，乐耀元，尚海涛，曾昊溟，凌建刚，张文媛. 高水分南美白对虾虾干货架期预测模型的构建. 农产品加工. 2022(19): 78-82+90 .