Activation of a refolded, berberine-specific, single-chain Fv fragment by addition of free berberine
Activation of a refolded, berberine-specific, single-chain Fv fragment by addition of free berberine
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DOI：
10.1007/s10529-006-9033-7
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发表时间：
2006-07-01
影响因子：
2.7
通讯作者：
Tanaka, Hiroyuki
中科院分区：
文献类型：
--
作者：
Kim, Jun-Sik;Masaki, Tadashi;Tanaka, Hiroyuki
在大肠杆菌中产生了针对小檗碱的特异性单链可变区片段（scFv）。从产生单克隆抗体的杂交瘤1D 5 -3B-7中克隆抗小檗碱scFv基因。使用组装PCR将重链（V-H）和轻链（V-L）基因的可变区与柔性接头连接。将V-H-linker-V-L基因克隆到pET 28 a（+）中，在E. coli BL21（DE3）。采用直接酶联免疫吸附试验（ELISA）和竞争酶联免疫吸附试验（Competitive enzyme-linked immunosorbent assay，ELISA）检测了分步透析法和人工分子伴侣法复性后scFv的活性。直接ELISA结果表明，抗小檗碱单链抗体对小檗碱具有特异性结合活性。然而，在竞争性ELISA中，活性增加取决于小檗碱的浓度。
A single-chain variable fragment (scFv) specific for berberine was produced in Escherichia coli. The anti-berberine scFv gene was cloned from hybridoma 1D5-3B-7 producing the monoclonal antibody. The variable regions of the heavy (V-H) and light chain (V-L) genes were connected with a flexible linker using an assembly PCR. The V-H-linker-V-L gene was inserted into a plasmid, pET28a (+), then overexpressed in E. coli BL21 (DE3). The active of the scFv by refolding based on stepwise dialysis methods and an artificial chaperone was determined by direct and competitive enzyme-linked immunosorbent assay (ELISA). The results of direct ELISA showed that the anti-berberine scFv retained specific binding activity to berberine. In competitive ELISA, however, activity was increased depending on the concentration of berberine.