食品科学

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抗孔雀石绿单链抗体-碱性磷酸酶融合表达和活性鉴定

伍伟健,董洁娴,饶美芳,詹屋强,王 弘*,孙远明   

  1. 广东省食品质量安全重点实验室,华南农业大学食品学院,广东 广州 510642
  • 出版日期:2016-09-15 发布日期:2016-09-22

Fusion Expression and Characterization of Single Chain Antibody against Malachite Green-Alkaline Phosphatase

WU Weijian, DONG Jiexian, RAO Meifang, ZHAN Wuqiang, WANG Hong*, SUN Yuanming   

  1. College of Food Science, South China Agricultural University, Guangdong Provincial Key Laboratory of Food Quality and Safety, Guangzhou 510642, China
  • Online:2016-09-15 Published:2016-09-22

摘要:

采用重叠延伸的方法成功将抗孔雀石绿(malachite green,MG)单克隆细胞的轻链VL和重链VH用连接肽(Gly4Ser)3连接,形成单链抗体(single chain variable fragment,scFv)基因,并将其酶切连接进入含有碱性磷酸酶(alkaline phosphatase,PhoA)基因的载体plip6/GN中,成功构建重组质粒plip6/GN-MG-scFv。随后重组质粒转入大肠杆菌BL21,经诱导表达后,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和Western blotting鉴定结果表明所获得的融合蛋白scFv-PhoA大小约72 kD。利用scFv与MG特异性结合的活性和PhoA催化对硝基苯磷酸二钠的显色机制,经过直接竞争酶联免疫吸附法测定融合蛋白scFv-PhoA的IC50为9.81 ng/mL。本方法操作简单、灵敏度高且检测时间短,这为进一步进行MG免疫法快速检测提供了参考。

关键词: 孔雀石绿, 单链抗体, 碱性磷酸酶, 融合表达, 直接竞争酶联免疫吸附法

Abstract:

The variable light and heavy chain genes from anti-malachite green (MG) monoclonal cells were cloned and
linked with peptide (Gly4Ser)3 to construct a single chain variable fragment (scFv) antibody gene. The gene was inserted into
the vector plip6/GN containing alkaline phosphatase (PhoA) gene to construct the recombinant plasmid plip6/GN-MG-scFv.
After the induction of E. coli BL21 containing the recombinant plasmid with isopropyl β-D-thiogalactoside (IPTG), the
protein from the periplasm was detected by SDS-PAGE and Western blotting. The results demonstrated that the fusion
protein scFv-PhoA (about 72 kD) was expressed successfully. A one-step enzyme-linked immunosorbent assay (ELISA)
was developed (IC50 = 9.81 ng/mL) by using scFv-PhoA fusion protein with disodium 4-nitrophenyl phosphate (pNPP) as
the substrate. These results demonstrate that fusion protein scFv-PhoA can be a homologous reagent which simplifies and
accelerates the detection of malachite green.

Key words: malachite green, single chain antibody, alkaline phosphatase, fusion expression, direct competitive enzyme-linked immunosorbent assay

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