Efficient silkworm expression of single-chain variable fragment antibody against ginsenoside Re using Bombyx mori nucleopolyhedrovirus bacmid DNA system and its application in enzyme-linked immunosorbent assay for quality control of total ginsenosides
Efficient silkworm expression of single-chain variable fragment antibody against ginsenoside Re using Bombyx mori nucleopolyhedrovirus bacmid DNA system and its application in enzyme-linked immunosorbent assay for quality control of total ginsenosides
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DOI：
10.1093/jb/mvq072
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发表时间：
2010-09-01
影响因子：
2.7
通讯作者：
Morimoto, Satoshi
中科院分区：
文献类型：
--
作者：
Sakamoto, Seiichi;Pongkitwitoon, Benyakan;Morimoto, Satoshi
利用家蚕核型多角体病毒（BmNPV）杆状病毒DNA系统，在家蚕幼虫中成功表达了抗G-Re单链抗体（G-Re）。构建了表达抗G-Re单链抗体（GRe-scFv）的杆状病毒供体载体，该载体含有蜜蜂蜂毒肽信号肽序列，以促进重组GRe-scFv分泌到家蚕幼虫血淋巴中。功能性重组GRe-scFv通过阳离子交换层析，然后通过固定化金属离子亲和层析纯化。纯化的GRe-scFv的产量为6.5mg/13只蚕幼虫，相当于650 mg/l血淋巴，表现出比在大肠杆菌中表达的产量（1.7mg/l培养基）高得多的产量。结果表明，GRe-scFv保留了亲本抗G-Re单克隆抗体（MAb-4G 10）的特性，为建立人参总皂苷的间接竞争酶联免疫吸附测定（icELISA）方法提供了可能。该方法的检测范围为0.05 ~ 10 μ g/ml。这些结果清楚地表明，蚕表达系统对于表达功能性单链抗体非常有用，而功能性单链抗体在大肠杆菌中表达时经常需要耗时且成本高昂的重新折叠。杆菌
A single-chain variable fragment (scFv) antibody against ginsenoside Re (G-Re) have been successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. The baculovirus donor vector for expression of scFv against G-Re (GRe-scFv) was constructed to contain honeybee melittin signal sequence to accelerate secretion of the recombinant GRe-scFv into the haemolymph of silkworm larvae. Functional recombinant GRe-scFv was purified by cation exchange chromatography followed by immobilized metal ion affinity chromatography. The yield of purified GRe-scFv was 6.5 mg per 13 silkworm larvae, which is equivalent to 650 mg/l of the haemolymph, exhibiting extremely higher yield than that expressed in Escherichia coli (1.7 mg/l of culture medium). It was revealed from characterization that GRe-scFv retained similar characteristic of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10), making it possible to develop indirect competitive enzyme-linked immunosorbent assay (icELISA) for quality control of total ginsenosides in various ginsengs. The detectable range for calibration of G-Re by developed icELISA shows 0.05-10 mu g/ml. These results clearly suggested that the silkworm expression system is quite useful for the expression of functional scFv that frequently required time- and cost-consuming re-folding when it expressed in E. coli.