EXPRESSION OF HEAT - LABILE ENTEROTOXIN B SUBUNIT FROM ESCHERICHIA COLI IN TRANSGENIC WATERCRESS (NASTURTIUM OFFICINALE L.)
Nguyen Hoang Loc1, Nguyen Van Song1, Nguyen Quang Duc Tien1, Tang Thuy Minh1, Pham Thi Quynh Nga1, Truong Thi Bich Phuong1, KimTae-Geum2, Yang Moon-Sik2
1Institute of Resources, Environment and Biotechnology, Hue University, Hue, Vietnam
2Division of Biological Sciences and the Research Center of Bioactive Materials, Chonbuk National University, Jeonju, Chonbuk 561-756, Korea
ABSTRACT
The synthetic LTB (B subunit of Escherichia coli heat - labile enterotoxin) gene was fused to the endoplasmic reticulum retention signal SEKDEL to enhance its expression level and protein assembly in plants. This gene was then inserted into a plant expression vector under the control of CaMV 35S promoter and was introduced into watercress plant (Nasturtium officinale L.) by biolistic transformation method. The integration of synthetic LTB gene into genomic DNA of transgenic plants was confirmed by PCR using genomic DNA. The assembly of plant - produced LTB was detected by Western blot analysis. Enzyme - linked immunosorbent assay indicated that plant - synthesized LTB protein bound specifically to GM1 - ganglioside, which is receptor for LTB on the cell surface, suggesting that the LTB subunits formed biological active pentamers.
Keywords: Escherichia coli heat-labile enterotoxin B subunit (LTB), expression, transgenic watercress, Nasturtium officinale.
INTRODUCTION
The enterotoxigenic heat-labile enterotoxin B subunit (LTB) from Escherichia coli was found to be of enterotoxigenic E. coli (ETEC) that is a potent mucosal immunogen and immunoadjuvant for co- administered antigens. Numerous investigations shown that LTB is a promising candidate to be a vaccine antigen against LT-producing ETEC (Kang et al., 2003, Kang et al., 2004, Kim et al., 2007). It was already expressed in several bacterial and plant systems (Mason et al., 1998, Kozuka et al., 2000, Chikwamba et al., 2002, Rezaee et al., 2005, Kang et al., 2006, Rosales-Mendoza et al., 2007, Ravin et al., 2008, Lim et al., 2009, Loc et al., 2010). ETEC is the most common cause of diarrhea, especially in many developing countries and the major disease agent of ETEC is the heat-labile enterotoxin (LT).
The LT is a plasmid - encoded, high molecular weight toxin, and immunologically and physicochemically related to cholera toxin (CT) (Guidry et al., 1997, Fleckenstein et al., 2000). The LT is composed of one A subunit (LTA) (27 kDa) and five non-covalently associated B subunits (LTB) (11.6 kDa each) forming a ring - like pentamer.
Watercress plant (Nasturtium officinale,
synonym: N. microphyllum), a member of Brassicaceae family, is an important horticultural crop in many parts of the world, and one of the oldest known leaf vegetables consumed by human beings (Jin et al., 1999). Watercress is a perennial herb, hardy aquatic creeper that grows naturally in slowly flowing clean streams, preferably coming from chalky or limestone soils (Philip, Rix, 1995).
Watercress plant is very rich in vitamins and minerals, and has long been valued as a food and medicinal plant. Considered a cleansing herb, its high content of vitamin C makes it a remedy that is particularly valuable for chronic illnesses (Chevallier, 1996). The leaves are antiscorbutic, depurative, diuretic, expectorant, purgative, hypoglycaemic, odontalgic, stimulant and stomachic (Brown, 1995). The freshly pressed juice has been used internally and externally in the treatment of chest and kidney complaints, chronic irritations and inflammations of the skin (Launert, 1981). Applied externally, it has a long - standing reputation as an effective hair tonic, helping to promote the growth of thick hair. A poultice of the leaves is said to be an effective treatment for healing glandular tumours or lymphatic swellings (Phillips, Foy, 1990).
The aim of this study was to efficiently produce
recombinant LTB in watercress plant The gene encoding LTB was introduced and expressed in watercress plant by biolistic transformation, and the product was found to have a pentameric form with the ability to bind the cell receptor, monosialoganglioside GM1.
MATERIALS AND METHODS Plant materials and culture conditions
Watercress (N. officinale L.) seeds were incubated in warm water for 10 min to stimulate the ability of germination, then sterilized with 70%
EtOH for 5 min and followed by 0.1% HgCl2 for 7 min. After sterilization, the seeds were washed five times with sterile distilled water, and germinated on MS (Murashige, Skoog, 1962) medium containing 3% sucrose and 0.8% agar. Internode segments (1 cm in length) of 3-weeks old in vitro growing plants were cultured on the callus induction medium including MS salts and B5 vitamins (Gamborg et al., 1968), supplemented with 1.3 µM 2,4- dichlorophenoxyacetic acid (2,4-D) and 3.94 µM 6-
benzylaminopurine (BAP). Ten days-old calli were then subcultured on the shoot regeneration medium including MS salts and B5 vitamins, supplemented with 3.15 µM BAP every 2 weeks, and after 10 weeks of culture shoots were regenerated from callus. The in vitro shoots (2-3 cm in length) were rooted on the MS medium supplemented with 0.5 µM napthaleneacetic acid (NAA). The pH of medium was adjusted to 5.8, and then it was autoclaved at 121oC for 15 min. The cultures were incubated at 25 ± 2oC under an intensity of 2,000- 3,000 lux with a photoperiod of 10-h day light.
Construction of plant expression vector
The synthetic LTB (sLTB) gene was modified from the coding sequence of LTB gene (GenBank Locus ABO11677) by Kang et al. (2004) based on optimized codon usage of a plant using the overlap extension PCR method. The plant expression vector used in our study, pMYO51, consists of a sLTB, a signal peptide, and the ER retention signal (SEKDEL), under the control of CaMV 35S (Cauliflower Mosaic Virus 35S RNA) promoter (Fig 1).
Figure 1. Structure of pMYO51 vector used for Nasturtium officinale transformation. The synthetic LTB gene was fused with SEKDEL under the control of the CaMV 35S promoter. An NPT II (neomycin phosphotransferase II) gene expression cassette was used for kanamycin selection of transgenic plants. LB and RB are the left and right borders of T-DNA sequence. pNOS promoter and t-NOS terminator were obtained from the Agrobacterium tumefaciens nopaline synthase gene.
Plant transformation
Internode segments (1 cm in length) of in vitro watercress were cut and placed on filter paper on the top of MS agar medium before bombardment.
Internode segments were then bombarded with gold (0.6 µm) particles using a Biolistic PDS 1000/He Particle Delivery System (Bio-rad, Hercules, CA) following the manufacturer’s instructions and placed in a culture room at 25oC. Two days after
bombardment, the explants were transferred on the callus induction medium without antibiotic for 1 week, and then on the selection medium containing 25 mg/l kanamycin. The explants were subcultured on the same medium for each 2 weeks. The shoots regenerated from the selection medium after 8 weeks were isolated and cultured on the rooting medium.
The putative transgenic watercress plantlets formed roots in 1 weeks of culture.
Genomic DNA isolation and PCR analysis
Total genomic DNA was extracted from leaves of putative transgenic and wild-type watercress plant by the method of Kang and Fawley (1997). PCR analysis was accomplished by using the forward primer (5’-GGA TCC GCC ACC ATG GTG AAG GTG AAG-3’) and the reverse primer (5’-GGT ACC TCA TAG CTC ATC TTT C-3’), which were specific for sLTB gene. PCR amplification was performed as follows: 95oC/5’, 30×(95oC/1’, 55oC/1’, 72oC/1’), 72oC/5’. PCR products were separated by electrophoresis on 0.8% agarose gel and stained with ethidium bromide.
Immunoblot detection of LTB protein in transgenic plants
Leaf samples (approximately 0.5 g) from watercress plants were ground in liquid nitrogen with a mortar and pestle, and resuspended in 1 ml of extraction buffer (50 mM HEPES, pH 7.5, 10 mM potassium acetate, 5 mM magnesium acetate, 1 mM EDTA, 1 mM dithiothreitol, and 2 mM phenylmethanesulfonyl fluoride). An aliquot (20 µg) of total soluble protein, as determined by Bradford protein assay (Bio-rad), from watercress plants were separated on 12% SDS-polyacrylamide gel electrophoresis. Purified bacterial LTB was also loaded in the range of 1 µg. The separated protein bands were transferred from the gel to Hybond C membrane (Bio-rad) using a Trans-blot® SD semi-dry transfer cell (Bio-rad) at 15 V for 30 min. Nonspecific antibody reactions were blocked by incubating the membranes in 25 ml of 3% casein in TBST buffer (TBS plus 0.05% Tween-20) with gentle agitation overnight. The membrane was incubated for 2 h with gentle agitation in 10 ml of 1:2000 dilution of rabbit anti-LTB antiserum (Immunology Consultants Lab.
Inc., OR) in TBST antibody dilution buffer containing 1.5% casein and then washed three times with TBST buffer. The membrane was incubated for 2 h in 1:7000 dilution of anti-rabbit IgG conjugated with alkaline phosphatase (Promega S3731, Madison, WI) in TBST buffer and washed three times with TBST buffer, and once with TMN buffer. After washing, the color was developed with BCIP/NBT in TMN buffer.
GM1 - ganglioside binding assay
To determine the binding ability of plant- produced LTB to gangliosides, microtiter plate was coated with monosialoganglioside GM1 (Sigma) by incubating the plate with 100 µl/well GM1 (3 µg/ml) in bicarbonate buffer, pH 9.6 at 4oC overnight. As a control, plate was coated with 100 µl/well BSA (3 µg/ml). After washing three times with PBST, the plate was blocked with 1% BSA in 10 mM PBS (300 µl/well) at 37oC for 2 h. The plate was then washed three times with PBST, and was incubated with various concentrations of total soluble protein from transgenic and wild-type watercress plants in PBS (100 µl/well) for 2 h at 37oC. The primary and secondary antibody treatments were carried out as follows: the plate was incubated with a 1:2000 dilution of rabbit anti-LTB antibody (Immunology Consultants Lab. Inc., OR) (100 µl/well) in 10 mM PBS containing 0.5% BSA for 2 h at 37oC, and washed four times with PBST. The wells were incubated with 1:4000 dilution of goat anti - rabbit IgG conjugated with horseradish peroxidase (Sigma G-7641, St. Louis, MO) (100 µl/well) in 10 mM PBS containing 0.5% BSA for 2 h at 37oC, and washed four times with PBST. The plate was finally incubated with 100 µl/well TMB substrates (Pharmingen 2606KC and 2607KC, San Diego, CA) for 30 min in the dark to maximize the reaction rate.
After incubation, the reaction was measured at 405 nm in an automated ELISA system (Bio-rad).
RESULTS AND DISCUSSION Construction of expression vector
The sLTB gene was inserted into the plant expression vector pMY27 (Lee et al., 2010) at BamHI/KpnI site, yielding pMYO51 (Fig. 1). The ligation reaction mixture was used to transform E.
coli strain TOP10 (Invitrogen, Carlsbad, CA), and kanamycin-resistant colonies were isolated after overnight incubation at 37°C. The recombinant vector, pMYO51, was confirmed by PCR amplifition with specific primers of LTB gene (Fig 2).
Figure 2. PCR analysis of pMYO51 DNA. The PCR product was separated on 1% agarose gel. SM: DNA size marker (λ/EcoRI+HindIII); 1: sLTB gene.
Transformation of sLTB gene and PCR analysis Internode segments of watercress plants were cultured on the selection medium with kanamycin after bombardment. Our results showed that shoot regeneration occurred from nearly 1.33% of explants (Fig 3). Oszvald et al. (2008) obtained a transformation efficiency of 2.9% from LTB trangenic rice plants by biolistic method.
Four plants resulting from independent transformation events were selected and maintained in in vitro condition. The presence of the sLTB gene in transgenic plants was confirmed by genomic DNA PCR amplification followed by gel electrophoresis of the amplified fragments (Fig 4). The expected PCR product (414 bp) was presented (lanes 1-4) in all transformed plants. The PCR product with same size was obtained with pMYO51 vector as a positive control template (PC). The PCR product was not detected in non-transgenic plants (NC).
Figure 3. In vitro shoot regeneration from internode segments of Nasturtium officinale plant. A: non-transgenic plant, A:
sLTB transgenic plant.
Figure 4. PCR analysis of the genomic DNA of sLTB transgenic Nasturtium officinale plants. The PCR products were separated on 1% agarose gel. SM: DNA size marker (λ/HindIII); PC: pMYO51 vector harboring sLTB gene (positive control);
NC: genomic DNA of non-transgenic plant (negative control); 1-4: genomic DNAs of sLTB transgenic plants (template).
A B
Immunoblot analysis of plant - synthesized LTB protein
The total soluble proteins (TSPs) were extracted from leaves of the four individual transgenic watercress plants. Purified bacterial LTB was used as a positive control to detect antibody-specific protein in the transgenic plants. Immunoblot analysis of these plants revealed an oligomeric LTB protein with a molecular weight of approximately 45 kDa (five non-covalently associated B subunits (LTB) of 11.6 kDa forming a ring-like pentamer) in three plants (#1, 2 and 4) (Fig 5). This result is similar with the results obtained from different plant expression systems, such as that of potato (Mason et al., 1998), maize (Chikwamba et al., 2002), tobacco (Kang et al., 2003), lettuce (Kim et al., 2007), carrot (Rosales-Mendoza et al., 2007), rice (Oszvald et al., 2008) and Peperomia pellucida (Loc et al., 2010).
Non-transgenic plant did not cross-react with LTB antibody, therefore the specific signal band corresponding to LTB protein was not detected on the membrane.
Figure 5. Western blot analysis of LTB protein in transgenic Nasturtium officinale plants with protein weight marker (97- 14.4 kDa). (A) Unboiled TSPs of transgenic plants with LTB (lines 1-4); (B) Boiled TSPs of sLTB transgenic plants (lines 1-4). PC: purified bacterial LTB protein (positive control);
NC: protein extract of non-transgenic plant (negative control); P: pentamer; M: monomer.
Binding assay of LTB protein to GM1-ganglioside To study the oligomerization of LTB protein produced in transgenic plant, its binding ability to GM1-ganglioside receptor was tested using 96-well plates coated with GM1-ganglioside. GM1- ganglioside has been shown to be the receptor for biological active LTB protein in vivo, and pentameric structure is required for appreciable receptor binding. In the GM1-ELISA binding assays, LTB protein produced in transgenic plants demonstrated a strong affinity for GM1-ganglioside, but not for BSA (Fig 6). Based on the absorbance measurement to determine GM1 binding, the expression levels of LTB protein in the three transgenic plants (#1, 2 and 4) were similar. The strong significantly binding efficacy of plant- produced LTB to GM1 indicates that plant-derived LTB subunit interacts with GM1.
Figure 6. GM1-ELISA analysis of LTB pentamer formation in transgenic Nasturtium officinale leaf tissues. GM1 ganglioside or BSA was bound to the wells of a microtiter plate. Protein extracts from sLTB transgenic plants were added to the wells and then anti-LTB antiserum was added to identify ganglioside-bound LTB pentamers. Lanes 1, 2 and 4 were protein extracts of transgenic plants expressing LTB protein.
CONCLUSION
We have successfully transformed sLTB gene into watercress plant (Nasturtium officinale L.) with strong relative expression. This edible transgenic watercress is a useful system for expressing other antigen proteins for mucosal immunization by oral A
B
consumption of the raw plant material. The ability of the LTB protein produced in watercress to generate both immunogenicity and adjuvanticity will be the subject of analysis in future mucosal immunization experiments in animals.
Acknowledgements: This research was supported by a grant of Higher Education Project from Vietnam Ministry of Education and Training (2008-2011).
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BIỂU HIỆN TIỂU PHẦN B NỘI ĐỘC TỐ KHÔNG BỀN NHIỆT CỦA ESCHERICHIA COLI (LTB) TRONG CÂY CẢI XOONG (NASTURTIUM OFFICINALE L.) CHUYỂN GEN
Nguyễn Hoàng Lộc1,∗∗∗∗, Nguyễn Văn Song1, Nguyễn Quang Đức Tiến1, Tăng Thúy Minh1, Phan Thị Quỳnh Nga1, Trương Thị Bích Phượng1, Tae-Geum Kim2, Moon-Sik Yang2
1Viện Tài nguyên, Môi trường và Công nghệ sinh học, Đại học Huế
2Khoa Sinh học và Trung tâm Nghiên cứu hoạt chất sinh học, Đại học Quốc gia Chonbuk, Hàn Quốc
TÓM TẮT
Gen LTB được dung hợp với tín hiệu ghi nhớ của mạng lưới nội sinh chất (endoplasmic reticulum retention signal, SEKDEL) để tăng mức độ biểu hiện và lắp ráp protein trong cây. Sau đó, gen này được chèn vào trong vector biểu hiện thực vật dưới sự điều hòa của promoter CaMV 35S và được biến nạp vào cây cải xoong (Nasturtium officinale L.) bằng kỹ thuật vi đạn. Sự hợp nhất của gen LTB trong genome của cây cải xoong chuyển gen đã được xác định bằng khuếch đại PCR. Sự tự lắp ráp của protein LTB do cây sản xuất được phát hiện bằng kỹ thuật Western blot. Phân tích GM1 - ELISA cho thấy protein LTB đã liên kết đặc hiệu với GM1 - ganglioside, một receptor của LTB trên bề mặt tế bào, gợi ý rằng các tiểu đơn vị LTB đã tạo thành cấu trúc pentamer có hoạt tính sinh học.
Từ khóa: biểu hiện gen, cải xoong chuyển gen, tiểu phần B nội độc tố không bền nhiệt của E. coli (LTB), Nasturtium officinale
∗ Author for correspondence: E-mail: [email protected]
