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| Expression and Characterization of Anti-AFB1 scFv Expressed in Sf9 Cell |
| LIU Ai-ping1,2, LI Cheng1, LIU Shu-liang1, WANG Xiao-hong2, CHEN Fu-sheng2 |
1. College of Food Science, Sichuan Agricultural University, Yaan 625014, China;
2. College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China |
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Abstract Objective:In order to investigate and improve activity of anti-aflatoxin B1(AFB1) single chain Fv fragment (scFv), anti-AFB1 scFv was expressed in Sf9 insect cells and then further studied according to previous research on prokaryotic expression of anti-AFB1 scFv. Methods:The recombinant plasmid pFastBac 1-scFv2E6VHVL was constructed, and then transformed into competent cell of Escherichia coli DH10Bac. Blue-white selection was performed for positive clone selection. Subsequently, the corresponding Bacmid-scFv from positive clones were extracted to infect Sf9 cells, and anti-AFB1 scFv was expressed, purified and determined by ELISA. Results:After blue-white selection, the positive clones were confirmed by colony-polymerase chain reaction (PCR) and sequencing, and Bacmid-scFv from positive clones were extracted to infect Sf9 insect cells. Successful expression was confirmed by Western blot. After purification, the scFv from Sf9 insect cells was analyzed by ELISA. The results suggested that the sensitivity of scFv obtained was 30μg/ml. Conclusion:Compared with anti-AFB1 scFv expressed in E. coli BL21(DE3), the sensitivity of scFv expressed in Sf9 insect cells was little better, but there was still room for improvement.
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Received: 19 November 2015
Published: 14 December 2015
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