共查询到20条相似文献,搜索用时 359 毫秒
1.
Nguyen Hoang Loc Nguyen Van Song Nguyen Quang Duc Tien Tang Thuy Minh Phan Thi Quynh Nga Tae-Geum Kim Moon-Sik Yang 《Plant Cell, Tissue and Organ Culture》2011,105(1):39-45
A gene encoding the B subunit of the enterotoxigenic Escherichia coli heat-labile enterotoxin (LTB) was adapted to the optimized plant coding sequence, and fused to the endoplasmic reticulum
retention signal SEKDEL in order to enhance its expression level and protein assembly in plants. The synthetic LTB (sLTB)
gene was placed into a plant expression vector under the control of the CaMV 35S promoter, and subsequently introduced into
the watercress (Nasturtium officinale L.) plant by the Agrobacterium-mediated transformation method. The integration of the sLTB gene into the genomic DNA of transgenic plants was confirmed
by genomic DNA PCR amplification. The assembly of plant-produced LTB protein was detected by western blot analysis. The highest
amount of LTB protein produced in transgenic watercress leaf tissue was approximately 1.3% of the total soluble plant protein.
GM1-ganglioside enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to GM1-ganglioside, which is the receptor for biologically active LTB on the cell surface, suggesting that the plant-synthesized
LTB subunits formed biologically active pentamers. 相似文献
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3.
Nguyen-Xuan Huy Young-Sook Kim Sang-Chel Jun Zhewu Jin Seung-Moon Park Moon-Sik Yang Tae-Geum Kim 《Biotechnology and Bioprocess Engineering》2009,14(6):731-737
Plant-based vaccines have been produced in transgenic plants including tobacco, potatoes, corn, and rice. However, these plants
are not suitable for administration without cooking. To overcome this obstacle, a fusion gene encoding the synthetic enterotoxigenic
Escherichia coli heat-labile enterotoxin B subunit genetically fused with a synthetic neutralizing epitope of porcine epidemic diarrhea virus
(sLTB-sCOE) was introduced into lettuce cells (Lactuca sativa) by Agrobacterium-mediated transformation methods. The integration and expression of the sLTB-sCOE fusion gene was confirmed in transgenic
lettuce by genomic DNA PCR amplification and Northern blot analysis, respectively. Synthesis and assembly of the LTB-COE fusion
protein into oligomeric structures with pentamer size were observed in transgenic plant extracts by Western blot analysis
with anti-LTB or anti-COE antibodies. The binding of plantproduced LTB-COE to intestinal epithelial cell membrane glycolipid
receptors was confirmed by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). Based on the ELISA results, LTB-COE fusion protein made up about 0.026∼0.048% of the total soluble protein in the
transgenic lettuce leaf tissues. The synthesis and assembly of LTB-COE monomers into biologically active oligomers in transgenic
lettuce leaf tissues demonstrates the feasibility of using uncooked edible plant-based vaccines for mucosal immunization. 相似文献
4.
Tae-Geum Kim Bang-Geul Kim Mi-Young Kim Jae-Kwon Choi Eun-Sun Jung Moon-Sik Yang 《Molecular biotechnology》2010,44(1):14-21
Enterotoxigenic Escherichia coli is one of the leading causes of diarrhea in developing countries, and the disease may be fatal in the absence of treatment.
Enterotoxigenic E. coli heat-labile toxin B subunit (LTB) can be used as an adjuvant, as a carrier of fused antigens, or as an antigen itself. The
synthetic LTB (sLTB) gene, optimized for plant codon usage, has been introduced into rice cells by particle bombardment-mediated
transformation. The integration and expression of the sLTB gene were observed via genomic DNA PCR and western blot analysis,
respectively. The binding activity of LTB protein expressed in transgenic rice callus to GM1-ganglioside, a receptor for biologically active LTB, was confirmed by GM1-ELISA. Oral inoculation of mice with lyophilized transgenic rice calli containing LTB generated significant IgG antibody
titers against bacterial LTB, and the sera of immunized mice inhibited the binding of bacterial LTB to GM1-ganglioside. Mice orally immunized with non-transgenic rice calli failed to generate detectable anti-LTB IgG antibody titers.
Mice immunized with plant-produced LTB generated higher IgG1 antibody titers than IgG2a, indicating a Th2-type immune response.
Mice orally immunized with lyophilized transgenic rice calli containing LTB elicited higher fecal IgA antibody titers than
mice immunized with non-transgenic rice calli. These experimental results demonstrate that LTB proteins produced in transgenic
rice callus and given to mice by oral administration induce humoral and secreted antibody immune responses. We suggest that
transgenic rice callus may be suitable as a plant-based edible vaccine to provide effective protection against enterotoxigenic
E. coli heat-labile toxin. 相似文献
5.
6.
Expression of the B Subunit of E. coli Heat-labile Enterotoxin in the Chloroplasts of Plants and its Characterization 总被引:6,自引:0,他引:6
Transgenic chloroplasts have become attractive systems for heterologous gene expressions because of unique advantages. Here, we report a feasibility study for producing the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (LTB) via chloroplast transformation of tobacco. Stable site-specific integration of the LTB gene into chloroplast genome was confirmed by PCR and genomic Southern blot analysis in transformed plants. Immunoblot analysis indicated that plant-derived LTB protein was oligomeric, and dissociated after boiling. Pentameric LTB molecules were the dominant molecular species in LTB isolated from transgenic tobacco leaf tissues. The amount of LTB protein detected in transplastomic tobacco leaf was approximately 2.5% of the total soluble plant protein, approximately 250-fold higher than in plants generated via nuclear transformation. The GM1-ELISA binding assay indicated that chloroplast-synthesized LTB protein bound to GM1-ganglioside receptors. LTB protein with biochemical properties identical to native LTB protein in the chloroplast of edible plants opens the way for inexpensive, safe, and effective plant-based edible vaccines for humans and animals. 相似文献
7.
A cDNA encoding the simian-human immunodeficiency virus (SHIV 89.6p) Tat regulatory element protein was fused to the c-terminus of the cholera toxin B subunit gene (ctxB-tat) and introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods. The fusion gene was detected in the genomic DNA of transformed potato leaf cells by PCR DNA amplification. Synthesis and assembly of the CTB-Tat fusion protein into oligomeric structures of pentamer size was detected in transformed tuber extracts by immunoblot analysis. The binding of CTB-Tat fusion protein pentamers to intestinal epithelial cell membrane glycolipid receptors was quantified by G(M1)-ganglioside enzyme-linked immunosorbent assay (G(M1)-ELISA). Based on the ELISA results, CTB-Tat fusion protein made up about 0.005-0.007% of total soluble tuber protein or approximately 4.6mg per 100g potato tuber tissue. The synthesis and assembly of CTB-Tat monomers into biologically active oligomers in transformed potato tuber tissues demonstrates the feasibility of using viral pathogen antigens synthesized in edible plants for mucosal immunization against HIV-1 infection. 相似文献
8.
9.
Mi-Young Kim Tae-Geum Kim Han-Sang Yoo Moon-Sik Yang 《Plant Cell, Tissue and Organ Culture》2011,105(3):375-382
10.
Luzmila Martínez-González Sergio Rosales-Mendoza Ruth Elena Soria-Guerra Leticia Moreno-Fierros Rubén López-Revilla Schuyler S. Korban Juan C. Guevara-Arauza Ángel Gabriel Alpuche-Solís 《Plant Cell, Tissue and Organ Culture》2011,107(3):441-449
Transgenic plants serve as attractive systems for the production and delivery of subunit vaccines, thus expression of an enterotoxigenic
Escherichia coli (ETEC) antigen in an edible plant may lead to the development of a viable oral vaccine against cholera and ETEC diarrhea.
In this study, expression of the heat labile toxin B subunit (LTB) from ETEC was performed in lettuce, and its immunological
characterization was investigated. A total of 27 independent transgenic lines were established following Agrobacterium-mediated transformation. Selected lettuce lines were subjected to GM1-ELISA to confirm the proper quaternary structure of
the LTB protein. Levels of accumulation of the pentameric LTB reached up to 0.05% of the total soluble protein (TSP) in T1
and T2 progenies of these lines. Oral immunization of Balb/c mice was conducted using three weekly doses of lettuce-derived
LTB. This elicited specific and significant antibody responses in both serum and intestinal tissues. Moreover, mice immunized
with lettuce-derived LTB showed diminished intestinal fluid accumulation following challenge with the cholera toxin. This
study demonstrated that this plant-based vaccine may contribute to immunization practices against diarrheal diseases. 相似文献
11.
Synthesis and assembly of SIVmac Gag p27 capsid protein cholera toxin B subunit fusion protein in transgenic potato 总被引:4,自引:0,他引:4
A deoxyribonucleic acid (DNA) fragment encoding the cholera toxin B subunit (CTB) was linked 5′ to the simian immunodeficiency
virus (SIVmac) Gag p27 capsid gene (CTB-Gag). The fusion gene was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods and transformed plants regenerated. The CTB-Gag gene fusion was detected in transformed potato
leaf genomic DNA by polymerase chain reaction-mediated DNA amplification. The results of immunoblot analysis with anti-CTB
and anti-Gag antibodies verified the synthesis of biologically active CTB-Gag fusion protein in transformed leaf and tuber
tissues. Synthesis and assembly of the CTB-Gag fusion protein into oligomeric structures of pentamer size was confirmed by
GM1-ganglioside-enzyme-linked immunosorbent assay (GM1-ELISA) of transformed potato tuber tissue extracts. The binding of CTB-Gag fusion protein oligomers to intestinal epithelial
cell membrane receptors quantified by GM1-ELISA showed that CTB-Gag fusion protein made up approx 0.016–0.022% of the total soluble tuber protein. The synthesis of
CTB-Gag monomers and their assembly into biologically active CTB-Gag fusion protein oligomers in potato tuber tissues provides
the opportunity for employment of the carrier and adjuvant properties of CTB for the development of edible plant-based subunit
mucosal vaccines for enhanced mucosal immunity against SIV in macaques. 相似文献
12.
Synthesis and assembly of anthrax lethal factor-cholera toxin B-subunit fusion protein in transgenic potato 总被引:3,自引:0,他引:3
A DNA encoding the 27-kDa domain I of anthrax lethal factor protein (LF), was linked to the carboxyl terminus of the cholera toxin B-subunit (CTB-LF). The CTB-LF fusion gene was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated in vivo transformation methods and antibiotic-resistant plants were regenerated. The CTB-LF fusion gene was detected in transformed potato leaf genomic DNA by polymerase chain reaction (PCR)-mediated DNA amplification. Immunoblot analysis with anti-CTB and anti-LF primary antibodies verified the synthesis and assembly of biologically active CTB-LF fusion protein oligomers in transformed plant tuber tissues. Furthermore, the binding of CTB-LF fusion protein pentamers to intestinal epithelial cell membrane receptors measured by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA) indicated that the CTB-LF fusion protein made up approx 0.002% of the total soluble tuber protein. Synthesis of CTB-LF monomers and their assembly into biologically active CTB-LF fusion protein pentamers in potato tuber tissues demonstrates the feasibility of using edible plants for production and delivery of adjuvanted LF protein for CTB-mediated immunostimulation of mucosal immune responses against anthrax toxin. 相似文献
13.
Nguyen Hoang Loc Le Thi Thinh Moon-Sik Yang Tae-Geum Kim 《Biotechnology and Bioprocess Engineering》2011,16(3):576-580
The cholera toxin B subunit (CTB), a nontoxic molecule with potent biological properties, is a powerful mucosal and parenteral
adjuvant that induces a strong immune response against co-administered or coupled antigens. A gene encoding CTB, which was
modified based on the optimized codon usage in the plant, was synthesized and fused to the endoplasmic reticulum retention
signal KDEL to enhance its expression level in plants. The synthetic CTB (sCTB) gene was introduced into a plant expression
vector adjacent to the CaMV 35S promoter, and was transformed into tomato using an Agrobacterium-mediated transformation method. The integration of the sCTB gene into the genomic DNA of transgenic plants was confirmed
by genomic DNA PCR amplification. The synthesis and assembly of CTB protein in transgenic plants was demonstrated through
immunoblot analysis and GM1-ELISA. The highest amount of CTB protein produced in transgenic tomatoes was approximately 0.9% of total soluble fruit protein
which was 10-fold greater than the previously 0.081%. GM1-ELISA indicated that plant-synthesized CTB protein bound specifically to GM1-gangliosides, suggesting that the CTB subunits formed active pentamers. 相似文献
14.
Young-Sook Kim Bang-Geul Kim Tae-Geum Kim Tae-Jin Kang Moon-Sik Yang 《Plant Cell, Tissue and Organ Culture》2006,87(2):203-210
To increase expression level of cholera toxin B subunit (CTB) in lettuce plants, synthetic CTB (sCTB) gene based on the optimized codon usage was fused with an endoplasmic reticulum retention signal, KDEL. The sCTB gene was introduced into a plant expression vector and transformed to lettuce plants using Agrobacterium-mediated transformation system. As a selection marker, a bialaphos resistance (bar) gene that encodes phosphinothricin acetyltransferase (PAT), conferring tolerance to the herbicide phosphinothricin (PPT), was used. PCR amplification of genomic DNA confirmed the presence of the sCTB gene in the transgenic lettuce plants. Expressions of mRNA and protein of sCTB were observed by Northern and Western blot analyses, respectively. The sCTB synthesized in the transgenic lettuce showed strong affinity for GM1-ganglioside suggesting that the sCTB conserved the antigenic sites for binding and proper folding of pentameric CTB structure. The expression level of CTB was relatively high, reaching total soluble protein (TSP) levels of 0.24% in transgenic lettuce. 相似文献
15.
A DNA construct containing the cholera toxin B subunit (CTB) gene genetically fused to a nucleotide sequence encoding three
copies of tandemly repeated diabetes-associated autoantigen, the B chain of human insulin, was produced and transferred into
low-nicotine tobaccos by Agrobacterium. Integration of the fusion gene into the plant genome was confirmed by polymerase chain reaction (PCR). The results of immunoblot
analysis verified the synthesis and assembly of the fusion protein into pentamers in transgenic tobacco. GM1–ELISA showed
that the plant-derived fusion protein retained GM1–ganglioside receptor binding specificity. The fusion protein accounted
for 0.11% of the total leaf protein. The production of transgenic plants expressing CTB–InsB3 offers a new opportunity to test plant-based oral antigen therapy against autoimmune diabetes by inducing oral tolerance. 相似文献
16.
Assembly of cholera toxin B subunit full-length rotavirus NSP4 fusion protein oligomers in transgenic potato 总被引:4,自引:0,他引:4
A CTB-NSP4(175) fusion gene encoding the entire 175-aa murine rotavirus NSP4 enterotoxin protein was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB-NSP4(175) enterotoxin fusion gene was detected in the genomic DNA of transformed leaves by PCR DNA amplification. Synthesis and assembly of the full-length CTB-NSP4(175) fusion protein into oligomeric structures of pentamer size was detected in transformed tuber extracts by immunoblot analysis. The binding of CTB-NSP4(175 )fusion protein pentamers to intestinal epithelial cell membrane receptors was quantified by G(M1)-ganglioside enzyme-linked immunosorbent assay (G(M1)-ELISA). The ELISA results showed that CTB-NSP4(175) fusion protein was 0.006-0.026% of the total soluble tuber protein. The synthesis of CTB-NSP4(175) monomers and their assembly into biologically active oligomers in transformed potato tubers demonstrates the feasibility of using edible plants for the synthesis of enterocyte-targeted full-length rotavirus enterotoxin antigens that retain all of their pathogenic epitopes for initiation of a maximum mucosal immune response. 相似文献
17.
ARAKAWA TAKESHI CHONG DANIEL K.X. LAWRENCE MERRITT J. LANGRIDGE WILLIAM H.R. 《Transgenic research》1997,6(6):403-413
A gene encoding the cholera toxin B subunit protein (CTB), fused to an endoplasmic reticulum (ER) retention signal (SEKDEL) was inserted adjacent to the bi-directional mannopine synthase P2 promoter in a plant expression vector containing a bacterial luciferase AB fusion gene (luxF) linked to the P1 promoter. Potato leaf explants were transformed by Agrobacterium tumefaciens carrying the vector and kanamycin-resistant plants were regenerated. The CTB-SEKDEL fusion gene was identified in the genomic DNA of bioluminescent plants by polymerase chain reaction amplification. Immunoblot analysis indicated that plant-derived CTB protein was antigenically indistinguishable from bacterial CTB protein, and that oligomeric CTB molecules (Mr 50 kDa) were the dominant molecular species isolated from transgenic potato leaf and tuber tissues. Similar to bacterial CTB, plant-synthesized CTB dissociated into monomers (Mr 15 kDa) during heat or acid treatment. The maximum amount of CTB protein detected in auxin-induced transgenic potato leaf and tuber tissues was approximately 0.3% of total soluble plant protein. Enzyme-linked immunosorbent assay methods indicated that plant-synthesized CTB protein bound specifically to GM1-ganglioside, the natural membrane receptor of cholera toxin. In the presence of the SEKDEL signal, CTB protein accumulates in potato tissues and is assembled into an oligomeric form that retains native biochemical and immunological properties. The expression of oligomeric CTB protein with immunological and biochemical properties identical to native CTB protein in edible plants opens the way for preparation of inexpensive food plant-based oral vaccines for protection against cholera and other pathogens in endemic areas throughout the world 相似文献
18.
SIVmac Gag p27 capsid protein gene expression in potato 总被引:3,自引:0,他引:3
A cDNA encoding the Simian immunodeficiency virus type (SIV(mac)) Gag capsid protein was introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods. The gag gene was detected in the genomic DNA of transformed leaf tissues by PCR DNA amplification. Immunoblot analysis of transformed potato plant extracts with anti-Gag monoclonal antibody showed that biologically active Gag protein was synthesized in transformed tuber tissues. Based on ELISA results, recombinant Gag protein made up 0.006-0.014% of total soluble tuber protein. The synthesis of SIV Gag in transformed potato tubers opens the way for development of Gag-based edible plant vaccines for protection against SIV and potentially HIV-1 infection. 相似文献
19.
Eun-Ah Shin Yong Keun Park Kang Oh Lee William H. R. Langridge Jin-Yong Lee 《Molecular biotechnology》2009,43(2):138-147
Periodontal disease caused by the gram-negative oral anaerobic bacterium Porphyromonas gingivalis is thought to be initiated by the binding of P. gingivalis fimbrial protein to saliva-coated oral surfaces. To assess whether biologically active fimbrial antigen can be synthesized
in edible plants, a cDNA fragment encoding the C-terminal binding portion of P. gingivalis fimbrial protein, fimA (amino acids 266–337), was cloned behind the mannopine synthase promoter in plant expression vector pPCV701. The plasmid
was transferred into potato (Solanum tuberosum) leaf cells by Agrobacterium tumefaciens in vivo transformation methods. The fimA cDNA fragment was detected in transformed potato leaf genomic DNA by PCR amplification methods. Further, a novel immunoreactive
protein band of ~6.5 kDa was detected in boiled transformed potato tuber extracts by acrylamide gel electrophoresis and immunoblot
analysis methods using primary antibodies to fimbrillin, a monomeric P. gingivalis fimbrial subunit. Antibodies generated against native P. gingivalis fimbriae detected a dimeric form of bacterial-synthesized recombinant FimA(266–337) protein. Further, a protein band of ~160 kDa
was recognized by anti-FimA antibodies in undenatured transformed tuber extracts, suggesting that oligomeric assembly of plant-synthesized
FimA may occur in transformed plant cells. Based on immunoblot analysis, the maximum amount of FimA protein synthesized in
transformed potato tuber tissues was approximately 0.03% of total soluble tuber protein. Biosynthesis of immunologically detectable
FimA protein and assembly of fimbrial antigen subunits into oligomers in transformed potato tuber tissues demonstrate the
feasibility of producing native FimA protein in edible plant cells for construction of plant-based oral subunit vaccines against
periodontal disease caused by P. gingivalis. 相似文献
20.
Toc159, a protein located in the outer envelope membrane and the cytosol, is an important component of the receptor complex for nuclear-encoded chloroplast proteins. We investigated the molecular mechanism of protein import into chloroplasts by atToc159 using the ppi2 mutant, which has a T-DNA insertion at atToc159, shows an albino phenotype, and does not survive beyond the seedling stage due to a defect in protein import into chloroplasts. First we established that transiently expressing atToc159 in protoplasts obtained from the white leaf tissues of ppi2 plants complements the protein import defect into chloroplasts. Using this transient expression approach and a series of deletion mutants, we demonstrated that the C-terminal membrane-anchored (M) domain is targeted to the chloroplast envelope membrane in ppi2 protoplasts, and is sufficient to complement the defect in protein import. The middle GTPase (G) domain plays an additional critical role in protein import: the atToc159[S/N] and atToc159[D/L] mutants, which have a mutation at the first and second GTP-binding motifs, respectively, do not support protein import into chloroplasts. Leaf cells of transgenic plants expressing the M domain in a ppi2 background contained nearly fully developed chloroplasts with respect to size and density of thylakoid membranes, and displayed about half as much chlorophyll as wild-type cells. In transgenic plants, the isolated M domain localized to the envelope membrane of chloroplasts but not the cytosol. Based on these results, we propose that the M domain is the minimal structure required to support protein import into chloroplasts, while the G domain plays a regulatory role. 相似文献