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当前位置: 首页 > 文章 > 抗孔雀石绿单链抗体—碱性磷酸酶融合表达和活性鉴定 食品科学 2016,37 (17) 121-126
Position: Home > Articles > Fusion Expression and Characterization of Single Chain Antibody against Malachite Green-Alkaline Phosphatase FOOD SCIENCE 2016,37 (17) 121-126

抗孔雀石绿单链抗体—碱性磷酸酶融合表达和活性鉴定

作  者：
伍伟健;董洁娴;饶美芳;詹屋强;王弘;孙远明
单  位：
广东省食品质量安全重点实验室华南农业大学食品学院
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
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(Gly_4Ser)_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sc Fv-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IC_(50)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。
译  名：
Fusion Expression and Characterization of Single Chain Antibody against Malachite Green-Alkaline Phosphatase
作  者：
WU Weijian;DONG Jiexian;RAO Meifang;ZHAN Wuqiang;WANG Hong;SUN Yuanming;College of Food Science, South China Agricultural University, Guangdong Provincial Key Laboratory of Food Quality and Safety;
关键词：
malachite green;;single chain antibody;;alkaline phosphatase;;fusion expression;;direct competitive enzyme-linked immunosorbent assay
摘  要：
The variable light and heavy chain genes from anti-malachite green(MG) monoclonal cells were cloned and linked with peptide(Gly_4Ser)_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(IC_(50) = 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.

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