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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  2016, Vol. 36 Issue (5): 40-45    DOI: 10.13523/j.cb.20160506
研究报告     
抗黄曲霉毒素B1单链抗体在Sf9昆虫细胞中的表达与性质分析
刘爱平1,2, 李诚1, 刘书亮1, 王小红2, 陈福生2
1. 四川农业大学食品学院 雅安 625014;
2. 华中农业大学食品学院 武汉 430070
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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摘要：

目的:在原核表达抗黄曲霉毒素B1(aflatoxin B1,AFB1)单链抗体(single chain Fv fragment,scFv)研究的基础上,为进一步了解和提高抗AFB1 scFv的活性,利用Sf9昆虫细胞表达抗AFB1 scFv,并对其活性进行探索研究。方法:构建pFastBac 1-scFv2E6VHVL重组质粒,将重组质粒转化Escherichia coli (E. coli) DH10Bac细胞,进行蓝白斑筛选,挑取阳性克隆。提取相应的重组杆状病毒穿梭载体Bacmid侵染Sf9昆虫细胞,表达scFv,利用镍亲和层析法纯化scFv,并以ELISA检测scFv活性。结果:蓝白斑筛选后,经菌落PCR和测序验证挑取的白斑阳性单克隆含有正确的单链抗体基因。提取相应的重组杆状病毒穿梭载体Bacmid侵染Sf9昆虫细胞,通过Western blot检测得知抗AFB1 scFv在Sf9昆虫细胞中成功表达。AFB1对scFv的抑制中浓度(IC50)为30μg/ml。结论:与E. coli BL21(DE3)表达系统相比,scFv灵敏度转好,但仍有较大提升空间。
关键词： AFB1;  ELISA;  scFv;  Sf9细胞    
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.
Key words: Sf9 cell    scFv    AFB1    ELISA
收稿日期: 2015-11-19 出版日期: 2015-12-14
ZTFLH:  Q511  
基金资助:

国家自然科学基金(31271876),中央高校基本科研业务费专项资金(52209-814012)资助项目
通讯作者: 王小红, 陈福生     E-mail: wxh@hzau.edu.cn;chenfs@hzau.edu.cn
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引用本文:

刘爱平, 李诚, 刘书亮, 王小红, 陈福生. 抗黄曲霉毒素B1单链抗体在Sf9昆虫细胞中的表达与性质分析[J]. 中国生物工程杂志, 2016, 36(5): 40-45.

LIU Ai-ping, LI Cheng, LIU Shu-liang, WANG Xiao-hong, CHEN Fu-sheng. Expression and Characterization of Anti-AFB1 scFv Expressed in Sf9 Cell. China Biotechnology, 2016, 36(5): 40-45.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160506    或    https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I5/40

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