生物学杂志

生物学杂志

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抗黄曲霉毒素B1单链抗体在大肠杆菌和毕赤酵母中的表达和活性研究

  

  1. 1. 黄淮学院 生物与食品工程学院, 驻马店 463000; 2. 华中农业大学 食品科技学院, 武汉 430070
  • 出版日期:2018-02-18 发布日期:2018-02-18
  • 通讯作者: 王小红，教授，博士生导师，主要从事食品微生物、食品生物技术与安全研究，E-mail: wxh@mail.hzau.edu.cn
  • 作者简介:胡莉，硕士，主要从事食品生物技术与安全研究，E-mail:1361211497@qq.com
  • 基金资助:
    国家重点研究计划课题（编号2017YFC1600105）；国家自然科学基金(31772054；31271876)

Comparison of expression and activity of anti-AFB1 scFv in Escherichia Coli and Pichia Pastoris

  1. 1. College of Biological and Food Engineering， Huanghuai University， Zhumadian 463000；2. College of Food Science and Technology， Huazhong Agricultural University， Wuhan 430070， China
  • Online:2018-02-18 Published:2018-02-18

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摘要： 为了提高黄曲霉毒素 B1(aflatoxin B1, AFB1)单链抗体(single chain Fv fragment, scFv)的表达量和生物活性，将从杂交瘤细胞株2C10中反转录后经基因拼接技术获得的scFv片段基因分别构建到E.coli表达载体pET-30a中和酵母表达载体pPICZαA中进行比较。重组质粒pET-30a-pelB-scFv构建好后转化到BL21(DE3)中进行IPTG诱导蛋白表达，重组质粒pPICZαA-scFv构建完成经线性化后，经电转化法转化至毕赤酵母GS115中，甲醇诱导蛋白表达。表达蛋白经镍离子亲和层析法进行纯化，后检测其生物学活性。结果显示抗AFB1 scFv获得了高效表达，在大肠表达系统和酵母表达系统中表达量分别是34 mg/L 和132 mg/L，目的蛋白大小为29 ku左右。纯化后的蛋白经SDS-PAGE，Western blot和ELISA对其进行性质分析，得知抗 AFB1 scFv蛋白具有很好的生物学活性，且灵敏度分别为40 μg/mL 和35 μg/mL。酵母表达系统与E.coli BL21(DE3) 表达系统相比，虽然scFv灵敏度转好，但仍有较大提升空间。

关键词: AFB1, 单链抗体, 大肠杆菌, 毕赤酵母, ELISA

Abstract: In order to improve the expression yield and biological activity of aflatoxin B1 (AFB1) single chain Fv fragment (scFv), scFv genes from hybridoma cell lines 2C10 by reverse transcription method and gene splicing technology were constructed into the expression vectors pET-30a and pPICZαA respectively to compare the results. Then the constructed expression vector pET-30a-pelB-scFv was transformed into BL21(DE3) to express protein induced by IPTG. The constructed expression vector pPICZαA-scFv was linearised and transformed into Pichia pastoris by electroporation, and the transformants were induced by methanol，and the anti-AFB1 scFv was expressed. The expression proteins were purified and determined by SDS-PAGE, western blot and ELISA.The results of SDS-PAGE showed that anti-AFB1 scFvs were highly level expressed successfully, with the expression yield of about 34 mg/L in E.coli expression system and 132 mg/L in Pichia Pastoris expression system, and the goal protein is about 29 kua. The results of western blot and ELISA demonstrated that the recombinant scFv could combine with AFB1 specifically. and the sensitivity of anti-AFB1 scFv was 40 μg/mL and 35 μg/mL respectwely. Compared with anti-AFB1 scFv expressed in E.coli BL21(DE3)， the sensitivity of scFv expressed in Pichia pastoris was a little better， but there was still some room for improvement．

Key words: AFB1, single chain Fv fragment, Escherichia coli, Pichia Pastoris, ELISA