Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
The attached
copy is furnished to the author for internal non-commercial research
and education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. Authors requiring further information
regarding Elseviers archiving and manuscript policies are
encouraged to visit:
http://www.elsevier.com/copyright
a r t i c l e
i n f o
Article history:
Received 26 August 2009
and in revised form 2 October 2009
Available online 8 October 2009
Keywords:
Cholera toxin
Edible vaccine
Mucosal carrier
Rabies glycoprotein
a b s t r a c t
The pentameric B subunit of cholera toxin (CtxB) is an efcient mucosal adjuvant for vaccines. We report
the expression of a chimeric protein comprising the synthetic cholera toxin B subunit fused at its C-terminal with rabies surface glycoprotein (G protein) in tobacco plants. The 80.3 kDa fusion polypeptide
expressed at 0.4% of the total soluble protein in leaves of the selected transgenic lines. The fusion protein
formed a 403 kDa pentameric protein which was functionally active in binding to GM1 receptor. The
plant-made protein had a higher afnity for GM1 receptor than the native bacterial CtxB. The pentameric
fusion protein was recognized by the anti-cholera toxin as well as anti-rabies antibodies. Its immunoprotective ability against rabies remains to be examined.
2009 Elsevier Inc. All rights reserved.
Introduction
Transgenic plants expressing foreign proteins of industrial and
pharmaceutical value are suggested to be economically viable alternatives to fermentation-based production systems. However, there
is a need to evaluate plants for the expression of a variety of functionally active eukaryotic proteins that undergo complex folding
and post translational modications. One such group of pharmaceutically important proteins is the subunit vaccine antigens. When
expressed in edible plant parts, these have been reported to be
effective as oral and mucosal antigens [13]. The expression level
of these antigens expressed in plants, as nuclear genes is often
low, ranging from 0.001% to 0.3% of total soluble protein (TSP)1
[4]. The low level expression limits the extent of immune response
and the development of an effective plant-based oral vaccine. Strategies like co-administration with an adjuvant [5] and fusion of antigens with an effective carrier molecule, either chemically or
genetically, can increase the immunogenicity of antigens [6,7].
In several studies, cholera toxin B subunit (CtxB) has been used
as a carrier for antigens [811]. CtxB is a homopentameric
(Mr 58,000) protein comprising the polypeptides (Mr 11,600)
arranged in a ring-like conguration. The native pentameric structure of CtxB and its binding to GM1 receptors are crucial for its
function as mucosal carrier and the resultant immunological re-
sponse. Conjugation of antigens with CtxB can reduce the dose required for T-cell activation by more than 10,000-fold as compared
to the free antigen [12]. Rabies virus genome encodes ve major
proteins- nucleoprotein (N), phosphoprotein (P), matrix protein
(M), glycoprotein (G) and RNA-dependent RNA polymerase (L)
[13,14]. The surface glycoprotein (G) plays an important role in viral pathogenesis and functions as a protective antigen [15]. One of
the strategies to control rabies in wild animals is to develop oral
vaccine that can be given as bait in the wild. This requires enhancing immunogenicity and expression of the G protein at high level
in plant tissue. With an aim to enhance immune response and
achieve high level expression of the G protein, plant codon optimized synthetic ctxB and G protein (rgp) genes were fused and
transformed into tobacco plants. The plants expressed the chimeric
CtxB-G protein which was puried from leaves of the transgenic
tobacco plants and analyzed for functional integrity.
Materials and methods
Construction of ctxB and rgp fusion and cloning into plant expression
cassette
We have earlier reported designing and cloning of plant codon
optimized synthetic ctxB gene of Vibrio cholerae O139 strain 1854
[16] and rgp gene of rabies virus glycoprotein [17] and their
expression in tobacco leaves. In this study, a glycineproline hinge
was used at the point of fusion of translational frames of the CtxB
and G proteins. The signal sequence PR-S, of the pathogenesis
induced tobacco protein PR-1a was used to facilitate transport of
185
HindIII
RB
pSM31
Pnos
nptII
BglII
XbaI
pr-s
Tnos PECaMV35S
SacI
LB
Tnos
ctxB
SEKDEL
HindIII
RB
Pnos
pSA5
nptII
HindIII
Tnos PECaMV35S
Apa1
pr-s
XhoI SacI
LB
Tnos
rgp
SEKDEL
HindIII
RB
pSR1241
Pnos
nptII
Tnos PECaMV35S
XbaI
pr-s
BglII
ApaI
ctxB
Hinge of GPGP
LB
Tnos
SEKDEL
2040bp
Fig. 1. Gene constructs showing cloning of the fusion gene ctxB-rgp in pBI101. The pr-s-ctxB and rgp fragments were PCR amplied from pSM31 and pSA5, respectively.
Amplied PCR fragments were digested with respective enzymes and triple ligated in pBI101 to obtain pSR1241 with two glycine-proline repeats as hinge at the 30 end of
ctxB.
186
6 NT
187
bp
2027
1584
1375
Fig. 2. Detection of synthetic ctxB-rgp gene in T0 transgenic tobacco plants by PCR (A) and Southern hybridization analysis of the transgenic (lane 1) and non-transgenic (lane
2) plants (B). Genomic DNA was isolated from transgenic and wild-type plant leaf. PCR was performed using gene specic primers. The PCR products were separated on 1.0%
agarose gel. M, EcoR1/HindIII digested k DNA markers, PCR products with DNA template from six independent T0 transgenic (lanes 16) and non-transgenic (NT) plants. For
Southern hybridization, genomic DNA from T0 transgenic plant #1 was digested with XhoI, electrophoresed in 0.8% agarose gel and blotted onto Hybond N+ membrane. Since
the fragment between T-borders has one XhoI site, the number of fragments hybridizing with 570 bp rgp probe from 30 region of the gene represents copy number of the
insert. A single band (lane 1) indicates the presence of single copy in the genome.
GM1, the order of binding afnity was: plant-CtxB > CtxB-G > bacterial CtxB at both the temperatures. Thus, the plant-expressed
CtxB showed higher afnity towards GM1 as compared to the bacterial CtxB and CtxB-G. The CtxB-G showed higher afnity towards
GM1 as compared to bacterial CtxB but lower afnity than plantexpressed CtxB at both the temperatures.
Purication of CtxB-G protein and pentamerization
logically active against both CtxB and the rabies antibodies was
established by indirect ELISA using both the antibodies.
The transgenic plants (#1, 2 and 3) showing high expression of
the CtxB-G protein were selected for analysis on immunoblot. As
expected, a 403 kDa (66 kDa glycosylated G + 14.6 kDa glycosylated CtxB polypeptide pentamer) protein was observed in the
non-reduced and un-boiled sample when detected by both CtxB
(Fig. 5A) and G protein antibodies (Fig. 5B). No band was detected
in the non-transgenic plants. The highest expression of CtxB-G protein was noticed in plant #1 (Fig. 6A, lane1 and Fig. 6B, lane 1).This
is consistent with the results of real-time PCR and ELISA. Under the
denaturing conditions, a band of 80.6 kDa, representing the
monomeric fusion polypeptide was detected using equine anti-rabies antibodies (Fig. 5C).
Binding afnity of the tobacco-expressed proteins to GM1 receptors
Functionality of CtxB and its fusion derivatives was correlated
with the ability to bind to GM1 ganglioside.The binding afnity
of the plant derived CtxB, CtxB-G and bacterial CtxB to GM1 was
determined by GM1-ELISA carried out at 4 or 37 C (Fig. 6). Within
the linear range of binding at variable molar concentrations of
188
0.45
0.8
0.35
0.7
0.3
0.25
0.2
0.15
0.1
Plant # 1
NT plants
0.9
0.4
CtxB-G (Absorbance)
CtxB-G ( %TSP)
0.6
0.5
0.4
0.3
0.2
0.05
0.1
Ab1
Ab2
Indirect ELISA
#1 # 3
T0 plants
Ab1
Ab2
GM1-ELISA
Fig. 4. CtxB-G protein expression in leaf in T0 transgenic tobacco plants #1 and #3 by GM1-ELISA (A) and assay of immunological activity of CtxB-G protein against rabies
antibodies by indirect and GM1-ELISA (B). The expression level of CtxB-G protein is given as %TSP. In indirect ELISA and GM1-ELISA for plant # 1, the plates were developed
using peptide anti-G (Ab1) and equine anti-rabies (Ab2) antibody. NT, non-transgenic plants.
B
M
kDa
403
NT
NT
kDa
NT
205
130
95
72
55
250
80.6 kDa
36
28
Fig. 5. Western blot analysis of transgenic plants under non-denaturing conditions using anti-cholera antibody (A), anti-rabies antibody (B) and under denaturing condition
using anti-rabies antibody (C). Crude protein (30 lg) prepared from the leaves of non-transgenic (NT) and transgenic T0 plants #1, 2 and 3 was loaded along with molecular
weight markers (M).
0.5
0.45
Bacterial CtxB
0.4
A 405 nm
0.35
plant CtxB
0.3
at 370C
CtxB-G
0.25
BSA
0.2
CtxB-G
0.15
plant CtxB
0.1
bacterial CtxB
0.05
at 40C
BSA
0
0
10
after intra-muscular challenge with the rabies virus [26]. The fusion of CtxB with G protein studied here is expected to provide
CtxB as a receptor specic carrier. This may reduce the dose required for oral mucosal uptake of CtxB-G. The approach may en-
Total
protein
(lg)
CtxBG
(lg)
Recovery
(%)
Purity
(%)
45,100
60
180
56
100
31
0.4
93
The above values are based on the purication initiated with 10 g fresh leaves. Total
protein was calculated by Bradford assay, CtxB-G was determined by ELISA.
A
kDa
205
S
403
97
66
205
45
97
Fig. 7. SDSPAGE analysis of the afnity puried CtxB-G protein. Puried protein
(200 ng) was loaded for silver staining (A). The puried protein was pentamerized
in vitro and checked on silver stained gel following non-denaturing SDSPAGE (B).
M, molecular weight markers and S, puried protein sample.
189
ic fusion protein maintained immunologically active G protein domains. However, the absorbance values of CtxB-G protein in GM1ELISA were lower than the absorbance values in indirect ELISA for
the two antibodies. This is be due to higher binding of CtxB to GM1
receptors coated on the ELISA plates as compared to its binding to
the plates in indirect ELISA.
The binding assay showed that a pentameric CtxB was formed
with functionally correct folding and assembly of the chimeric
CtxB-G expressed in tobacco leaves. These results suggest that
the folding of the fusion protein was driven by CtxB though this
is the smaller (14.6 kDa) partner in the 80.6 kDa chimeric protein.
Earlier, we reported that the plant-synthesized CtxB shows a higher afnity for binding to GM1 as compared to the bacterial CtxB
[16]. The glycosylation of the plant-expressed CtxB was predicted
to facilitate functionally more favorable folding of the CtxB in tobacco cells. Here we show that the binding afnity of CtxB-G protein to GM1 was lower than that of CtxB expressed in tobacco
leaves. This may be due to the bulky nature of the G protein in
CtxB-G protein pentamer (403 kDa) which may cause steric hindrance to the binding of CtxB to GM1 receptor as compared to
the binding of CtxB pentamer (73 kDa). The G protein has three potential N-linked glycosylation sites at Asn37, Asn247 and Asn319
positions [37]. The chaperones, other folding enzymes [38,39]
and the glycosylation in plant cells may lead to an increase in afnity of the CtxB for GM1 receptors as compared to the non-glycosylated bacterial CtxB. The binding afnity of the three proteins to
GM1 was higher at 37 C than at 4 C. Maximum binding of the
cholera toxin to GM1 is reported to occur within 1 h at 370C
[40]. Though the binding assay was carried out using crude extract
of the proteins, binding curves, nevertheless, demonstrate that the
three pentemeric proteins bound GM1 with different afnities.
This study describes the expression and assembly of the CtxB-G
protein fusion in transgenic tobacco plants. The assembly of CtxBG protein monomers into biologically active pentamers in transformed tobacco leaf tissue suggests that CtxB-G protein antigen expressed in plants may show efcient internalization through the
mucosal receptors. This may enhance immunogenic ability of the
CtxB-G protein fusion against rabies following oral and mucosal
immunization. Differences in binding constants of the CtxB expressed in different cellular systems suggest the need to examine
the folding and functional behavior of proteins obtained from heterologous expression systems.
Acknowledgments
We greatly acknowledge the nancial support provided by
Council for Scientic and Industrial Research and to the Department of Science and Technology for J.C. Bose Fellowship to Rakesh
Tuli. We thank Shadma Ashraf for the construct of the rgp and S.
Mishra for the ctxB genes, reported earlier.
References
[1] C.J. Arntzen, High-tech herbal medicine: plant-based vaccines, Nat. Biotechnol.
15 (1997) 221222.
[2] X.L. Jiang, Z.M. He, Z.Q. Peng, Y. Qi, Q. Chen, S.Y. Yu, Cholera toxin B protein in
transgenic tomato fruit induces systemic immune response in mice, Trans. Res.
16 (2007) 169175.
[3] N. Tomonori, T. Hidenori, Y. Yoshikazu, Y. Lijun, M. Takehiro, M. Mio, M. Ushio,
N. Akiko, U. Akihiro, H. Takachika, M. Shigeto, T. Kunisuke, T. Fumio, K. Hiroshi,
Rice-based mucosal vaccine as a global strategy for cold chain and needle free
vaccination, Proc. Natl. Acad. Sci. USA 104 (2007) 1098610991.
[4] J. Yu, W.H. Langridge, Novel approaches to oral vaccines: delivery of antigens
by edible plants, Curr. Infect. Dis. Rep. 2 (2000) 7377.
[5] W. Tao, C. Jian-Ping, L. Hong, Z. Ke-Qian, Z. Lei, Y. Chun-Lei, T. Da-Chang, Coexpression and immunity of Legionella pneumophila mip gene and
immunoadjuvant ctxB gene, Acta Biochim. Biophys. Sin. 37 (2005) 199204.
[6] K. Jin, H. So-Chon, Y. Moon-Sik, J. Yong-Suk, Expression of synthetic
neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic
190
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23] R.F. Lathe, M.P. Kieny, D. Schmitt, P. Curtis, J.P. Lecocq, M13 bacteriophage
vectors for the expression of foreign proteins in Escherichia coli: the rabies
glycoprotein, J. Mol. Appl .Genet. 2 (1984) 331342.
[24] E.S. Norton, J.F. Obijeski, D.V. Goeddel, Rabies virus glycoprotein analogs:
biosynthesis in Escherichia coli, Science 219 (1983) 614620.
[25] S.R. Klepfer, C. Debouck, J. Uffelman, P. Jacobs, A. Bollen, E.V. Jones,
Characterization of rabies glycoprotein expressed in yeast, Arch. Virol. 128
(1993) 269286.
[26] V. Yusibov, D.C. Hooper, S.V. Spitsin, N. Fleysh, R.B. Kean, T. Mikheeva, D. Deka,
A. Karasev, S. Cox, J. Randall, H. Koprowski, Expression in plants and
immunogenicity of plant virus-based experimental rabies vaccine, Vaccine
20 (2002) 31553164.
[27] H.S. Masson, D.M.K. Lam, C.J. Arntzen, Expression of hepatitis B surface antigen
in transgenic plants, Proc. Natl. Acad. Sci. USA 89 (1998) 11745
11749.
[28] J.A. Napier, G. Richard, P.R. Sherwry, Trafcking and stability of heterologous
proteins in transgenic plants, Meth. Biotechnol. 3 (1998) 189202.
[29] M.T. Dertzbaugh, C.O. Elson, Reduction in oral immunogenicity of cholera
toxin B subunit by N-terminal peptide addition, Infect. Immun. 61 (1993) 384
390.
[30] J. Sanchez, S. Johansson, B. Lowenadler, A. Svennerholm, J.M. Holmgren,
Recombinant cholera toxin B subunit and gene fusion proteins for oral
vaccination, Res. Microbiol. 141 (1990) 971979.
[31] R.A. Gonzalez, J. Sanchez, J. Holmgren, S. Lopez, C.F. Arias, Immunological
characterization of a rotavirus-neutralizing epitope fused to the cholera toxin
B subunit, Gene 133 (1993) 227232.
[32] C.H. Shi, C. Cao, J.S. Xhing, J. Li, Q.J. Ma, Gene fusion of cholera toxin B subunit
and HBV PreS2 epitope and the antigenicity of fusion protein, Vaccine 13
(1995) 933.
[33] T. Zhang, E. Li, S.L. Stanley Jr., Oral immunization with the dodecapeptide
repeat of the serine-rich Entamoeba histolytica protein (SREHP) fused to the
cholera toxin B subunit induces a mucosal and systemic anti-SREHP antibody
response, Infect. Immun. 63 (1995) 13491355.
[34] M. Backstrom, J. Holmgren, F. Schodel, M. Lebens, Characterization of an
internal permissive site in the cholera toxin B-subunit and insertion of
epitopes from human immunodeciency virus-1, hepatitis B virus and
enterotoxigenic Escherichia coli, Gene 165 (1995) 163168.
[35] M. Backstrom, M. Lebens, F. Schrodel, J. Holmgren, Insertion of a HIV-1neutralizing epitope in a surface-exposed internal region of the cholera toxin B
subunit, Gene 149 (1994) 211217.
[36] C. Peach, J. Velten, Expression variability (position effect) of CAT and GUS
reporter genes driven by linked divergent T-DNA promoters, Plant Mol. Biol.
17 (1991) 4960.
[37] A. Anilionis, W.H. Wunner, P.J. Curtis, Structure of the glycoprotein gene in
rabies virus, Nature 294 (1981) 275278.
[38] A.J. Parodi, Role of N-oligosaccharide endoplasmic reticulum processing
reactions in glycoprotein folding and degradation, Biochem. J. 348 (2000) 1
13.
[39] E.S. Trombetta, A. Helenius, Lectins as chaperones in glycoprotein folding, Curr.
Opin. Struct. Biol. 8 (1998) 587592.
[40] J. Holmgren, V. Elwing, P. Fredman, L. Svennerholm, Polystyrene adsorbed
gangliosides for investigation of the structure of the tetanus toxin receptor,
Eur. J. Biochem. 106 (1980) 371379.