利用噬菌体展示技术从矮牵牛中分离和鉴定针对二氢黄酮醇4-还原酶的单链可变片段。

Use of phage display for isolation and characterization of single-chain variable fragments against dihydroflavonol 4-reductase from Petunia hybrida.

作者信息

De Jaeger G, Buys E, Eeckhout D, Bruyns A M, De Neve M, De Wilde C, Gerats T, Van Montagu M, Fischer R, Depicker A

机构信息

Departement Genetica, Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Universiteit Gent, Belgium.

出版信息

FEBS Lett. 1997 Feb 17;403(2):116-22. doi: 10.1016/s0014-5793(97)00025-2.

DOI:10.1016/s0014-5793(97)00025-2

PMID:9042949

Abstract

To isolate specific single-chain variable (scFv) fragments against dihydroflavonol 4-reductase (DFR) from Petunia hybrida the phage display technology was used. DFR was overproduced in Escherichia coli, purified and used for immunization. From DFR-immunized mice, a phage display library was made starting from spleen mRNA using an optimized set of primers for V(H) and V(L) amplification. Several rounds of panning against recombinant DFR yielded five different scFv fragments, confirmed by subsequent DNA sequencing. They all specifically bound to recombinant DFR in ELISA and DFR in flower extracts on Western blot. These results show that phage display is a promising technology in plant molecular biology to obtain specific recombinant antibodies not only for ELISA and Western blot but also for in vivo applications in the long run.

摘要

为了从矮牵牛中分离出针对二氢黄酮醇4-还原酶（DFR）的特异性单链可变（scFv）片段，采用了噬菌体展示技术。DFR在大肠杆菌中过量表达、纯化后用于免疫。从经DFR免疫的小鼠脾脏mRNA出发，使用一组优化的引物扩增V(H)和V(L)，构建了一个噬菌体展示文库。经过几轮针对重组DFR的淘选，得到了五个不同的scFv片段，后续DNA测序证实了这一点。它们在ELISA中均能特异性结合重组DFR，在蛋白质印迹法中能与花提取物中的DFR特异性结合。这些结果表明，噬菌体展示技术在植物分子生物学中是一项很有前景的技术，不仅可用于ELISA和蛋白质印迹法，从长远来看还可用于体内应用，以获得特异性重组抗体。