中文摘要：

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。

英文摘要：

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.