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Kyushu University Institutional Repository

抗ジンセノシドRe小型化抗体を用いたイムノアッセイの開発

ベンヤカン, ポンキウィトゥーン

https://doi.org/10.15017/1441172

出版情報：Kyushu University, 2013, 博士（薬学）, 課程博士バージョン：

権利関係：Fulltext available.

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（様式

5)

氏名：ベンヤカンポンキウィトゥーン論文題名

区分

: Development of immunoassays using single chain variable fragment antibodies against ginsenoside Re

（抗ジンセノシド

Re

小型化抗体を用いたイムノアッセイの開発）

：甲

論文内容の要旨

Ginsenosides are well known as major bioactive compounds mainly produced in Panax ginseng. So far, mo^陀 than30 ginsenosides have been identified

企

omvarious ginsengs. Due to various activities reported, a number of commercial ginseng preparations have been available all over the world. Since the variety of concentrations of ginsenosides in the market products has been critical issue from the point of view of quality control, a sensitive, speedy, and simple method for standardizing gins如 gsamples is required.

Gi田町個!de R1 R2 Ri F剛叩楓出国市1

Gin揖E儲IdeRe 日 Rha1‑'Glc..(). Glc・ GI附明白ldeRg, H Glc‑0‑ Ole・ Pm top出血xadiol

Ginsen由lcleRb, Glc1‑2Glc‑ H Gluc1‑'Glc‑ Gins en田！deRe Glc1‑2Glc・日・Arneη1・6Glc‑ Glnsen田IdeRd Glc1‑2Glc・ H Glc・

Figure 1 Structures of dammarane

^肘

neginsenofil̲des

Previously, the monoclonal antibody against ginsenoside Re (MAb‑4GlO) has been reported to be potentially used in immunoassay to determine ginsenosides contents. In the present study, single chain variable fragment (scFv) against ginsenoside Re (GRe‑scFv) was constructed. The variable heavy chain

O

沼）

and light chain (VL) genes were cloned directly

合

omthe cDNA of the 4Gl0 hybridoma cell line and assembled by splicing by overlapping extension PCR (SOEPCR) using specific primers designed to have flexible peptide (Gly4Ser)3 between VH and VL domains. The constructed scFv gene was ligated in

^加

the pET28a vector and transformed

旭

toE.coli BL21 (DE3

） . 百i

eexpressed GRe‑scFv containing the His6‑tag at its N‑termini was purified by immobilized metal ion affinity chromatography (IMAC), and refolded by stepwise dialysis.The yield of GRe‑scFv after purification was 1. 7 mg per liter of culture medium. The purified GRe‑scFv had retained the characteristics of the parental monoclonal antibody which had wide cross‑reactivities with ginsenosides.τ'he use of GRe‑scFv expressed in E. coli in ELISA was validated to be a precise, accurate, and sensitive method for determination of ginsenosides

拍

variousginsengs [1].

Besides bacterial expression system, BmNPV bacmid DNA system w^回 usedfor expression of

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GRe‑scFv in the haemolymph of silkworm larvae. The baculovirus donor vector for expression of GRe

・

scFvwas constructed to contain honeybee melittin signal sequence to accelerate secretion of the recombinant GRe‑scFv. Functional recombinant GRe‑scFv was purified by cation exchange chromatography followed by IMAC. The yield of purified GRe‑scFv was 0.5 mg per one silkworm. GRe‑scFv expressed in silkworm also retained similar characteristic of the parental MAb

・

4G10and GRe‑scFv expressed in E. coli, making it possible to develop indirect competitive ELISA for quality control of total ginsenosides in various ginsengs. The results suggested that different hosts for expression did not affect the characteristics of GRe‑scFv. Silkworm expression system could be alternatively used to prepare probe for immunoassay for determination of ginsenosides content [2].

For an alternative way of determination of ginsenosides, green fluorescent protein (GFP) was fused with GRe‑scFv to produce a fluorescent single domain antibody (fluobody) against ginsenoside Re. GRe‑scFv was

白

sedat

血

eC‑tenninus of GFP extracted

企

omAequorea coerulescens (AcGFP

），担

the case of C‑fluobody, and at the N‑tenninus of AcGFP, in the case ofN‑fluobody, with a flexible peptide linker (Gly4Ser)2. Both C‑fluobody and N

・

fluobodywere successfully expressed in E. coli and purified using IMAC. After stepwise dialysis, reactivity and cross‑reactivies of both fluobodies were analysed. Interestingly, both fluobodies have shown more specificity to ginsenoside Re and ginsenoside Rgl, which are protopanaxadiol type. Subsequently, fluorescence intensity was measured from both fluodies. Fluorescence intensity of C

・

fluobodyhas shown to be 600

・

foldhigher than也atofN‑fluobody. Therefore, C‑fluobody was used to develop a fluorescence‑linked immunosorbent assay (FLISA) to determine

仰

senosidescontent. Since it required shorter time to perform an FLISA than an ELISA, a simple, sensitive, and speedy immunoassay to determine ginsenosides content could be developed using C‑fluobody as a probe [3].

Table 1 Crossreactivities of MAb‑4G10. GR，かscFvexnressed in

E .

coli. GRe‑scFv exnressed in silkworm. C‑fluobodv. and N‑fluobodv a^笠ainst!!insenosides

Cro自由−r自activities(%)

Compounds MAb・4G10 GRe・scFv GRe・scFv C・fluobody N・fluobody

(E.coli) (silkworm) Prot

ロ

panaxa:

凶

.ol

Ginsenoside Re 100 100 100 100 100 Ginsenoside Rgl 70.9 67.2 73.5 72.3 78.6 Pro旬pan田adiol

Ginsenoside Rd 76.2 73.5 71.8 15.3 12.9 Ginsenoside Rbl 0.04 <0.001 <0.001 0.4 0.4 Ginsenoside Re 0.05 <0.001 <0.001 0.4 0.4

[1] p阻止itwitoonB. et al., J. Nat. Med., 65 (1) 25

・

30,2011.

[2] Sakamoto S. Pon!?:kitwitoon B. et al., J. J3iochem., 148 (3) 335

・

340,2010.

[3] Sakamoto S. Tanizaki Y. Pon2kitwitoon B. et al., Protein Expr. Purif., 77 (1) 124‑130, 2011.

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