научная статья по теме SINGLE-CHAIN VARIABLE FRAGMENT (SCFV) EXPRESSION IN TOBACCO PLANTS VIA AGROINOCULATION Биология

Текст научной статьи на тему «SINGLE-CHAIN VARIABLE FRAGMENT (SCFV) EXPRESSION IN TOBACCO PLANTS VIA AGROINOCULATION»

ФИЗИОЛОГИЯ РАСТЕНИЙ, 2015, том 62, № 3, с. 432-438

ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ

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SINGLE-CHAIN VARIABLE FRAGMENT (scFv) EXPRESSION IN TOBACCO

PLANTS VIA AGROINOCULATION1 © 2015 X. F. Wang*, L. Li**, T. Yang*, J. Liu***, Y. Fan****, X. Zhu*, X. Z. Wang*

* Northeast Normal University, Institute of Genetic and Cytology, Changchun, Jilin, China ** Hebei University of Technology, Tianjincity, China *** College of Life Sciences, Northwest A&F University, Yangling, China **** Department of Biology, Changchun Normal University, Changchun City, Jilin, China

Received June 6, 2014

The acidic fibroblast growth factor (aFGF) is a member of FGF family which is composed of 154 amino acids. In order to explore the functional anti-aFGF single-chain variable fragment antibody (scFv) expression in eukaryote system and to enhance the binding activity, the functional scFv specific for human aFGF was expressed in tobacco plants via TMV vector mediated by Agrobacterium. The engineered bacterial EHA105: p35S-30B-scFv was inoculated into the leaves of Nicotiana benthamiana plants by needle-less injection. Green fluorescence protein was used as a reporter gene and accumulated at the highest level on the 8th day of post-inoculation. Expression of scFv was identified by RT-PCR and Western-blot. The yield of anti-aF-GF-scFv protein was reached to 0.5% of the plant total soluble proteins and a fair binding activity with its antigen aFGF was detected.

Keywords: Nicotiana benthamiana - human acidic fibroblast growth factor (aFGF) — scFv — transient expression — TMV

DOI: 10.7868/S0015330315030197

INTRODUCTION

The aFGF is a member of FGF family which has broad biological functions that can affect the growth of nervous cells, differentiation and oncogenic transformation. The aFGF is structurally related mitogens belonging to a family of proteins with a number of biological activities [1]. Yamanaka et al. [2] proved that aFGF was overexpressed in the pancreas cancer tissue contrasted with the normal tissue. Chow et al. [3] found that fibroblast growth factor-1 appeared to play a certain role in hepatocarcinogenesis. Therefore, using antibody specific for aFGF is an efficient way to therapy the cancer. The scFv is a gene engineering antibody that is only 1/6 of the intact IgG in size which was reported frequently in recent years [4]. The small size of scFv allows increased diffusion into a solid tumor mass and more rapid clearance from the blood [5]. As scFv quickly equilibrates, it readily penetrates into solid tumors, and may show much greater target specificity for tumor localization compared with intact antibody [6]. Furthermore, scFv greatly simplifies the

1 This text was submitted by the authors in English.

Corresponding authors: Xiaojuan Zhu and Xingzhi Wang. Northeast Normal University, Institute of Genetic and Cytology, no. 5268 Renmin Street, Changchun, Jilin 130024, China; e-mails: zhuxj720@nenu.edu.cn, wangxz543@nenu.edu.cn

design of genetically modified constructs for tumor therapy.

The anti-aFGFscFv has already been expressed in Escherichia coli in our lab. However, the production often lost the biological function due to the formation of inclusion bodies. Plant bioreactor has become an ideal expression system for foreign proteins which attracts researchers in recent years [7]. The driving forces behind the rapid developing of plant bioreactors include product safety, low production cost and easy scale up. There are various approaches for the heterologous protein expression in plants: including nuclear or plastid transformed, transient or stable expression, seed [8], whole plant or plant cell suspension culture expression system [9]. Transient expression is generally achieved by using pathogen Agrobacterium tumefaciens which is the most commonly used vector for the transformation of a variety of dicots and a number of monocots. A. tumefaciens has a naturally Ti plasmid which is able to integrate its T-DNA and transform host cells. It is an efficient infection way by using the Ti plasmid to transfer the plant virus to the wound cell in the plant which avoids the direct operation of RNA virus. RNA virus particle can spread, replicate, amplify quickly as well as the foreign gene inserted into the virus genome when reaches the high level expression [10].

In this paper, we first reported the expression of anti-aFGFscFv by using the TMV virus vector (p35S-30B) mediated by A. tumefaciens. The foreign protein anti-aFGFscFv expressed in systemic leaves takes up to 0.5% of the total soluble leaf proteins, which had a fair binding activity with its antigen aFGF, demonstrating that Agroinoculation could be used as an efficient and simple way to express foreign proteins in plants.

MATERIALS AND METHODS

Plasmids and bacteria. p35S-30B and p35S-30B-GFP were provided by the Institute of Microbiology, Chinese Academy of Sciences. p35S-30B-scFv was constructed in our laboratory. EHA105 and JM109 cells were preserved in our laboratory.

Reagents. Anti his-tag monoclonal antibody was purchased from Biolab company; mouse anti-scFv polyclonal antibody was produced in our laboratory; HRP labeled goat anti mouse IgG was purchased from "Sigma".

p35S-30B-scFv construction. The scFv gene was amplified from pET-28a-scFv using a forward primer P1 (5' - CCTTAATTAAACCATGGGCAGCAGC -CAT-3') that contained 6 his-tag and a reverse primer P2 (5'- CCGCTCGAGCTAGAGCTCATCTTTT-GAGGAGACG-3') that fused with KDEL and inserted into the cloning sites of p35S-30B cDNA to form the p35S-30B-scFv which was then introduced into Agrobacterium strain EHA105.

Agroinoculation of tobacco plants. Nicotiana benthamiana plants were grown in pots at 25°C in a growth chamber under 16 h light/8 h dark cycles. Agrobacterium EHA105 harboring p35S-30B-scFv, abbreviated to Agro35S-30B-scFv, was grown overnight at 28°C in 5 mL of LB media containing kana-mycin (50 |g/mL) and rifampicin (25 |g/mL). Then 1 mL of the overnight culture was diluted with 50 mL of LB media containing the antibiotics, 10 mM MES (pH 5.6) and 20 |M acetosyringone. The culture was incubated overnight at 28 °C, with shaking at 300 g. Agrobacterium cells were collected by centrifugation at 4300 g, re-suspended in solution MMA (10 mM MgCl2, 10 mM MES (pH 5.6), 100 |M acetosyringone), adjusted to a final OD600 of 0.8 and left at room temperature for 3 h without shaking, and then pressure-infiltrated into the leaves of N. benthamiana plants using a 2 mL syringe without needles.

Evaluation of the expression system by GFP. As an

infection control A. tumefaciens EHA105 containing 35S-30B-GFP was transfected in N. benthamiana plants under the same condition. The green fluorescence protein was visualized using a 100-W, long-wave UV lamp. The scFv accumulated in plants should be similar with GFP since the two proteins were expressed in the same expression system. The scFv accumulation was estimated by observing GFP in plants.

Confirmation of scFv RNA in N. benthamiana plants by RT-PCR. The total RNA was isolated from plants infected by EHA105-p35S-30B-scFv, EHA105-p35S-30B and wild type materials separately with the RNAgents® Total RNA Isolation System ("Promega"). First-strand cDNA was synthesized using avian myeloblastosis virus (AMV) Reverse Transcriptase ("Promega"), the cDNA of scFv was then amplified by PCR. PCR products were separated on a 1% agarose gel and visualized by ethidium bromide staining.

Western-blot analysis. The p35S-30B-scFv-infect-ed plant leaves were frozen in liquid nitrogen and pulverized. The powder was stirred in extraction buffer (20 mM PBS buffer, pH 7.4, containing 0.6 M NaCl), and centrifuged at 13000 g for 20 min at 4°C. The su-pernatants were analyzed by 15% SDS-PAGE, and then transferred to nitrocellulose membranes for im-muno-blotting. The recombinant protein was visualized by chemiluminescent detection (The SuperSignal® WestPico Trial Kit, "PIERCE"). The protein extracts from p35S-30B infected plants were used as the negative control.

Determination of scFv amount in N. benthamiana plants by ELISA. The total soluble proteins were extracted by grinding the infiltrated plants (0.5 g fr wt) in liquid nitrogen. The powder was homogenized with a 1.0 mL bicarbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6), centrifuged and the supernatant concentration was determined by Bradford [11] with bovine serum albumin as a standard. The different amount protein scFv purified from Escherichia coli was added into the 96-well polystyrene plate to get the OD 570 nm protein standard curve. The amount of scFv protein from p35S-30B-scFv infected plants was assessed by ELISA according to the protein standard curve.

Activity analysis of scFv. The 96-well polystyrene plate was coated with 100 ^L of 10 ^g/mL aFGF solution in carbonate buffer (pH 9.6). After blocking with 10% (w/v) instant milk/PBS, 100 |L of series dilution of scFv expressed in N. benthamiana plants was added, followed by incubation at 37°C for 1 h. After washing with PBS containing 0.05% Tween-20, 100 |L of diluted anti-scFv sera was added, followed by incubation at 37°C for 1 h. After washing with PBS containing 0.05% Tween-20, 100 |L of HRP-conjugated sheep anti-mouse IgG antibody was added, followed by incubation at 37°C for 1 h. After washing, peroxidase reaction was carried out using o-phenylenediamine (OPD) and hydrogen peroxide as the substrate at room temperature for 20 min. The reaction was terminated by adding 50 |L of 2 M H2SO4, and the absorbance at 570 nm was measured with Microplate Reader.

LB

35SP RdRp MP scFv TMGM VCP 35SP

RB

p35S-30B::scFv

Fig. 1. The scheme of recombinant vector p35S-30B-scFv.

LB — left border; RB - right border; 35SP — CaMv35S promoter; RdRp - RNA dependent RNA polymerase; MP — movement protein; TMGMVCP - coat protein; scFv — 850 bpscFv insert. Arrow: foreign gene promoter.

Fig. 2. Construction of p35S-30B-scFv.

a — clone the fragment of scFv from pET-28a-scFv (+) vector crea

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