Expression, Purification Strategy and Detection Method Establishment of Anti-Fenitrothion Nanobody
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...
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| Format: | Article |
| Language: | zho |
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The editorial department of Science and Technology of Food Industry
2023-12-01
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| Series: | Shipin gongye ke-ji |
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| Online Access: | http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2023030328 |
| _version_ | 1827631077213077504 |
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| author | Pengyan GUO Zhenlin XU Zijian CHEN Xing SHEN |
| author_facet | Pengyan GUO Zhenlin XU Zijian CHEN Xing SHEN |
| author_sort | Pengyan GUO |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-09T14:15:59Z |
| format | Article |
| id | doaj.art-ad515f626eee4bc78f8967b8435e208c |
| institution | Directory Open Access Journal |
| issn | 1002-0306 |
| language | zho |
| last_indexed | 2024-03-09T14:15:59Z |
| publishDate | 2023-12-01 |
| publisher | The editorial department of Science and Technology of Food Industry |
| record_format | Article |
| series | Shipin gongye ke-ji |
| spelling | doaj.art-ad515f626eee4bc78f8967b8435e208c2023-11-29T01:58:15ZzhoThe editorial department of Science and Technology of Food IndustryShipin gongye ke-ji1002-03062023-12-01442329830510.13386/j.issn1002-0306.20230303282023030328-23Expression, Purification Strategy and Detection Method Establishment of Anti-Fenitrothion NanobodyPengyan GUO0Zhenlin XU1Zijian CHEN2Xing SHEN3College of Food Science, South China Agricultural University, Guangzhou 510642, ChinaCollege of Food Science, South China Agricultural University, Guangzhou 510642, ChinaSchool of Food Pharmaceutical Engineering, Zhaoqing University, Zhaoqing 526061, ChinaCollege of Food Science, South China Agricultural University, Guangzhou 510642, ChinaFor 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.http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2023030328fenitrothionnanobodyinduced expressionpurification strategyactivity identification |
| spellingShingle | Pengyan GUO Zhenlin XU Zijian CHEN Xing SHEN Expression, Purification Strategy and Detection Method Establishment of Anti-Fenitrothion Nanobody Shipin gongye ke-ji fenitrothion nanobody induced expression purification strategy activity identification |
| title | Expression, Purification Strategy and Detection Method Establishment of Anti-Fenitrothion Nanobody |
| title_full | Expression, Purification Strategy and Detection Method Establishment of Anti-Fenitrothion Nanobody |
| title_fullStr | Expression, Purification Strategy and Detection Method Establishment of Anti-Fenitrothion Nanobody |
| title_full_unstemmed | Expression, Purification Strategy and Detection Method Establishment of Anti-Fenitrothion Nanobody |
| title_short | Expression, Purification Strategy and Detection Method Establishment of Anti-Fenitrothion Nanobody |
| title_sort | expression purification strategy and detection method establishment of anti fenitrothion nanobody |
| topic | fenitrothion nanobody induced expression purification strategy activity identification |
| url | http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2023030328 |
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