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Sharma MK Jani D Thungapathra M Gautam JK Meena LS Singh Y Ghosh A Tyagi AK Sharma AK 《Journal of biotechnology》2008,135(1):22-27
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Matsumoto Y Suzuki S Nozoye T Yamakawa T Takashima Y Arakawa T Tsuji N Takaiwa F Hayashi Y 《Transgenic research》2009,18(2):185-192
Cereal crops such as maize and rice are considered attractive for vaccine production and oral delivery. Here, we evaluated
the rice Oryza sativa for production of As16—an antigen protective against the roundworm Ascaris suum. The antigen was produced as a chimeric protein fused with cholera toxin B subunit (CTB), and its expression level in the
endosperm reached 50 μg/g seed. Feeding the transgenic (Tg) rice seeds to mice elicited an As16-specific serum antibody response
when administered in combination with cholera toxin (CT) as the mucosal adjuvant. Although omitting the adjuvant from the
vaccine formulation resulted in failure to develop the specific immune response, subcutaneous booster immunization with bacterially
expressed As16 induced the antibody response, indicating priming capability of the Tg rice. Tg rice/CT-fed mice orally administered
A. suum eggs had a lower lung worm burden than control mice. This suggests that the rice-delivered antigen functions as a prophylactic
edible vaccine for controlling parasitic infection in animals. 相似文献
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Eno E. Ekong Daniel N. Okenu Jayanti Mania-Pramanik Qing He Joseph U. Igietseme Godwin A. Ananaba Deborah Lyn Carolyn Black & Francis O. Eko 《FEMS immunology and medical microbiology》2009,55(2):280-291
The Vibrio cholerae ghost (rVCG) platform is an effective carrier and delivery system for designing efficacious Chlamydia vaccines. We investigated whether CTA2B, the nontoxic derivative of cholera toxin, can augment protective immunity conferred by an rVCG-based chlamydial vaccine and enhance cross-protection against heterologous chlamydial strains. An rVCG vaccine coexpressing chlamydial major outer membrane protein and CTA2B was genetically constructed and antigens were targeted to the inner membrane of V. cholerae before ghost production by gene E -mediated lysis. Effective immunomodulation by CTA2B was demonstrated by the ability of the vaccine construct to enhance the activation and maturation of dendritic cells in vitro . Also, C57BL/6 mice immunized via mucosal and systemic routes showed increased specific mucosal and systemic antibody and T-helper type-1 (Th1) responses, irrespective of the route. The enhanced production of IFN-γ, but not IL-4 by genital mucosal and splenic T cells, indicated a predominantly Th1 response. Clearance of the Chlamydia muridarum vaginal infection was significantly enhanced by codelivery of the vaccine with CTA2B, with the intravaginal route showing a moderate advantage. These results indicate that the rVCG-based vaccine is capable of inducing cross-protection against heterologous chlamydial serovars and that incorporation of mucosal adjuvants, such as CTA2B in the rVCG delivery platform, may enhance protective immunity. 相似文献
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Nguyen-Xuan Huy Nguyen-Quang-Duc Tien Mi-Young Kim Tae-Geum Kim Yong-Suk Jang Moon-Sik Yang 《Plant Cell, Tissue and Organ Culture》2016,127(2):369-380
Porcine epidemic diarrhea virus (PEDV) belongs to the Coronaviridae family and causes acute enteritis in pigs. A fragment of the large spike glycoprotein, termed the S1D epitope (aa 636–789), alone and fused with cholera toxin B subunit, were independently cloned into plant expression vectors, yielding plasmids pMYV717 and pMYV719, respectively. Plant expression vectors were transformed into Agrobacterium tumefaciens and subsequently infiltrated into Nicotiana benthamiana leaves. The highest expression level of S1D was found at 2?days post infiltration (dpi), reached 0.04?% of total soluble protein, and rapidly decreased thereafter. The expression and assembly of CTB–S1D fusion protein were confirmed by Western blot and GM1-ELISA. The highest expression level of CTB–S1D fusion protein was 0.07?% of TSP at 4 dpi, with a rapid decrease thereafter. In the presence of p19 protein from tomato bushy stunt virus, the S1D and CTB–S1D protein levels peaked at 6 dpi and were fourfold to sevenfold higher than in the absence of p19, respectively. After oral administration of transiently expressed CTB–S1D fusion protein, or with bacterial cholera toxin or rice callus expressing mutant cholera toxin 61F, mice exhibited significantly greater serum IgG and sIgA levels against bacterial CTB and S1D antigen, peaking at week 6. Transiently expressed CTB–S1D fusion protein will be administered orally to pigs to assess the immune response against PEDV. 相似文献
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Cholera toxin B protein in transgenic tomato fruit induces systemic immune response in mice 总被引:8,自引:0,他引:8
Cholera toxin B (CTB) subunit is a well-characterized antigen against cholera. Transgenic plants can offer an inexpensive
and safe source of edible CTB vaccine and may be one of the best candidates for the production of plant vaccines. The present
study aimed to develop transgenic tomato expressing CTB protein, especially in the ripening tomato fruit under the control
of the tomato fruit-specific E8 promoter by using Agrobacterium-mediated transformation. Transgenic plants were selected using PCR and Southern blot analysis. Exogenous protein extracted
from leaf, stem, and fruit tissues of transgenic plants was detected by ELISA and Western blot analysis, showing specific
expression in the ripening fruit, with the highest amount of CTB protein being 0.081% of total soluble protein. Gavage of
mice with ripe transgenic tomato fruits induced both serum and mucosal CTB specific antibodies. These results demonstrate
the immunogenicity of the CTB protein in transgenic tomato and provide a considerable basis for exploring the utilization
of CTB in the development of tomato-based edible vaccine against cholera. The rCTB antigen resulted in much lower antibody
titers than an equal amount of exgenous CTB in trangenic fruits, suggesting the protective effect of the fibrous tissue of
the fruit to the exogenous CTB protein against the degradation of protease in the digestive tracts of mice.
Xiao-Ling Jiang and Zhu-Mei He contributed equally to this work. 相似文献
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Juthika Kundu Rupa Mazumder Ranjana Srivastava & Brahm S. Srivastava 《FEMS immunology and medical microbiology》2009,56(2):179-184
Intranasal immunization, a noninvasive method of vaccination, has been found to be effective in inducing systemic and mucosal immune responses. The present study was aimed at investigating the efficacy of intranasal immunization in inducing mucosal immunity in experimental cholera by subunit recombinant protein vaccines from Vibrio cholerae O1. The structural genes encoding toxin-coregulated pilus A (TcpA) and B subunit of cholera toxin (CtxB) from V. cholerae O1 were cloned and expressed in Escherichia coli . Rabbits were immunized intranasally with purified TcpA and CtxB alone or a mixture of TcpA and CtxB. Immunization with TcpA and CtxB alone conferred, respectively, 41.1% and 70.5% protection against V. cholerae challenge, whereas immunization with a mixture of both antigens conferred complete (100%) protection, as assayed in the rabbit ileal loop model. Serum titers of immunoglobulin G (IgG) antibodies to TcpA and CtxB, and anti-TcpA- and anti-CtxB-specific sIgA in intestinal lavage of vaccinated animals were found to be significantly elevated compared with unimmunized controls. Vibriocidal antibodies were detected at remarkable levels in rabbits receiving TcpA antigen and their titers correlated with protection. Thus, mucosal codelivery of pertinent cholera toxoids provides enhanced protection against experimental cholera. 相似文献
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Priming immunization against cholera toxin and E. coli heat-labile toxin by a cholera toxin short peptide-beta-galactosidase hybrid synthesized in E. coli.
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A synthetic oligodeoxynucleotide encoding for a small peptide was employed for the expression of this peptide in a form suitable for immunization. The encoded peptide, namely, the region 50-64 of the B subunit of cholera toxin (CTP3), had previously been identified as a relevant epitope of cholera toxin. Thus, multiple immunizations with its conjugate to a protein carrier led to an efficient neutralizing response against native cholera toxin. Immunization with the resulting fusion protein of CTP3 and beta-galactosidase, followed by a booster injection of a sub-immunizing amount (1 microgram) of cholera toxin, led to a substantial level of neutralizing antibodies against both cholera toxin and the heat-labile toxin of Escherichia coli. 相似文献
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通过全化学法按大肠杆菌密码偏性合成了乙肝炎病毒(HBV)前S2抗原(PreS2)抗原决定簇基因,与霍乱毒素B亚基基因的3’端融合。重组质粒转化大肠杆菌后融合基因得到高效表达,表达量达30μg/mL,表达产物95%以上分泌到胞外。表达的融合蛋白能与神经节苷脂GM1结合,说明融合蛋白保持了霍乱毒素B亚基(CTB)的基本高级结构和生物学功能;酶联免疫吸附实验证明融合蛋白具有CTB和HBVPreS2的抗原性;应用亲和层析纯化后得到了电泳纯融合蛋白制品,为研究融合蛋白免疫原性并进一步构建基因工程肽苗奠定了基础。 相似文献
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Neutralization of heat-labile toxin of E. coli by antibodies to synthetic peptides derived from the B subunit of cholera toxin. 总被引:5,自引:2,他引:3
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Antibodies elicited by six synthetic peptides corresponding to various fragments of B subunit of cholera toxin (CT) were evaluated for their cross-reactivity with heat-labile toxin (LT) of Escherichia coli. The antiserum directed towards the peptide CTP3 (residues 50-64) was found highly cross-reactive with the LT, in radioimmunoassay and immunoblotting. This peptide was also the most cross-reactive with intact CT. The antiserum against CTP1 (residues 8-20) was also cross-reactive with the two toxins, although to a much lower extent. Antisera to both CTP1 and CTP3, which are inhibitory towards CT, were found equally effective in neutralizing the biological activity of the E. coli LT. This was manifested by inhibition of both adenylate cyclase activity and fluid secretion into ligated ileal loops of rats. These results might indicate the potential of such synthetic peptides as the basis for a general vaccine against several types of infectious diarrhea. 相似文献
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A组轮状病毒VP6与霍乱毒素B亚基融合蛋白在大肠杆菌中的表达及生物活性分析 总被引:4,自引:0,他引:4
利用霍乱毒素B亚基 (CholeratoxinBsubunit,CTB)的免疫载体作用 ,将轮状病毒相关抗原引入口服免疫体系 ,可激起有效的粘膜免疫反应 ,这里报道了CTB基因与A组轮状病毒地方株T114VP6全基因的融合 ,并在大肠杆菌BL21(DE3 )中进行了融合蛋白的表达。在IPTG诱导下得到分子量为 5 6kD的融合蛋白 ,表达量占菌体蛋白的15 %。分别用抗CT的抗体和抗A组轮状病毒的高价免疫血清进行WesternBlot检测 ,结果证明融合蛋白CTB VP6保留了天然霍乱毒素B亚基及轮状病毒VP6的抗原性。GM1-ELISA检测表明 ,复性后的融合蛋白具有与神经节苷脂GM1 结合的能力。 相似文献
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纯化重组霍乱毒素B亚单位与天然霍乱毒素B亚单位性质的对比研究 总被引:1,自引:0,他引:1
霍乱毒素B亚单位(BS)已用于新型口服霍乱疫苗、佐剂及蛋白质载体,但成本高,来源困难.用重组霍乱毒素B亚单位(rBS)代替BS可克服上述缺点.rBS用于上述目的前必须证实其在物理、化学及免疫学性质方面与天然同类产品的一致性.用亲和层析法从各批次大罐发酵所获工程菌E.coliMM2(pMM-CTB)培养物上清中制备得到了小批量rBS纯品,在同等条件下与BS(Sig-ma公司产品)进行理化、免疫学性质的对比研究,证实二者在SDS-PAGE中电泳带位置一致、分子量相同,纯度达99%;在反相HPLC中出峰行为一致,纯度达100%;在半干式聚焦电泳分析中电泳带分布相同,等电点为7.91.rBSN端起的20个氨基酸序列为TPQNITDLCAEYHNTQIHTL,与克隆基因来源株的毒素B亚单位同一段序列完全一致.氨基酸组成分析证实rBS与BS相近.在免疫学性质分析中,rBS与BS在免疫双扩散试验中与抗CT均出一条沉淀线且相互吻合;在免疫电泳试验中二者与抗CT在相应位置上产生一条沉淀弧;二者均能与神经节苷脂GM1结合且这种结合均可通过二者与抗CT的预保温处理而被阻断.对比研究结果揭示rBS与BS性质完全一致,可代替BS用于 相似文献
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Expression of Escherichia coli heat-labile enterotoxin B subunit (LTB) in Saccharomyces cerevisiae 总被引:1,自引:0,他引:1
Rezaee MA Rezaee A Moazzeni SM Salmanian AH Yasuda Y Tochikubo K Pirayeh SN Arzanlou M 《Journal of microbiology (Seoul, Korea)》2005,43(4):354-360
Heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli (ETEC) is both a strong mucosal adjuvant and immunogen. It is a subunit vaccine candidate to be used against ETEC-induced diarrhea. It has already been expressed in several bacterial and plant systems. In order to construct yeast expressing vector for the LTB protein, the eltB gene encoding LTB was amplified from a human origin enterotoxigenic E. coli DNA by PCR. The expression plasmid pLTB83 was constructed by inserting the eltB gene into the pYES2 shuttle vector immediately downstream of the GAL1 promoter. The recombinant vector was transformed into S. cerevisiae and was then induced by galactose. The LTB protein was detected in the total soluble protein of the yeast by SDS-PAGE analysis. Quantitative ELISA showed that the maximum amount of LTB protein expressed in the yeast was approximately 1.9% of the total soluble protein. Immunoblotting analysis showed the yeast-derived LTB protein was antigenically indistinguishable from bacterial LTB protein. Since the whole-recombinant yeast has been introduced as a new vaccine formulation the expression of LTB in S. cerevisiae can offer an inexpensive yet effective strategy to protect against ETEC, especially in developing countries where it is needed most. 相似文献
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目的表达并纯化产肠毒素大肠埃希菌(Enterotoxigenic Escherichia coli,ETEC)CFA/I定居因子的CfaB亚单位,分析其免疫原性。方法将不含信号肽序列的cfaB基因克隆到pQE一30上,构建重组质粒pQE-30-cfaB并转化EcoliM15,表达融合蛋白6xHis—CfaB,将表达的蛋白纯化和复性后免疫BALB/c小鼠,制备抗CfaB的抗血清,用ELISA法检测抗血清效价。结果6×His—CfaB融合蛋白高效表达,相对分子质量为15.5kD,纯化复性后免疫小鼠所得抗血清效价为1:125000。结论成功构建了高效表达6×His-CfaB融合蛋白的重组质粒,表达的融合蛋白免疫小鼠后获得了高效价的抗血清,为进一步研制以双歧杆菌为表达系统的新型ETEC亚单位口服疫苗奠定了基础。 相似文献
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Arêas AP Oliveira ML Ramos CR Sbrogio-Almeida ME Raw I Ho PL 《Protein expression and purification》2002,25(3):481-487
Cholera toxin B subunit (CTB) has been extensively studied as immunogen, adjuvant, and oral tolerance inductor depending on the antigen conjugated or coadministered. It has been already expressed in several bacterial and yeast systems. In this study, we synthesized a versatile gene coding a 6XHis-tagged CTB (359bp). The sequence was designed according to codon usage of Escherichia coli, Lactobacillus casei, and Salmonella typhimurium. The gene assembly was based on a polymerase chain reaction, in which the polymerase extends DNA fragments from a pool of overlapping oligonucleotides. The synthetic gene was amplified, cloned, and expressed in E. coli in an insoluble form, reaching levels about 13 mg of purified active pentameric rCTB per liter of induced culture. Western blot and ELISA analyses showed that recombinant CTB is strongly and specifically recognized by polyclonal antibodies against the cholera toxin. The ability to form the functional pentamers was observed in cell culture by the inhibition of cholera toxin activity on Y1 adrenal cells in the presence of recombinant CTB. The 6XHis-tagged CTB provides a simple way to obtain functional CTB through Ni(2+)-charged resin after refolding and also free of possible CTA contaminants as in the case of CTB obtained from Vibrio cholerae cultures. 相似文献
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Structural characterization of pertussis toxin A subunit 总被引:14,自引:0,他引:14
D L Burns S Z Hausman W Lindner F A Robey C R Manclark 《The Journal of biological chemistry》1987,262(36):17677-17682
The relationship between the structure of the A subunit of pertussis toxin and its function was analyzed. Limited tryptic digestion of the A subunit converted the protein to two stable fragments (Mr = 20,000 and 18,000). Antibodies raised to synthetic peptides homologous to regions in the A subunit were used to map these fragments. Both fragments were shown to contain the NH2-terminal portion but not the COOH-terminal portion of the A subunit. While these fragments exhibited NAD glycohydrolase activity, they were unable to reassociate with the B oligomer of the toxin. Thus the COOH-terminal portion of the A subunit does not contain the residues which are required for the NAD glycohydrolase activity of the toxin. However, this region of the molecule may be important for maintaining the oligomeric structure of the toxin. These results suggest that the A subunit of pertussis toxin is similar in structure to the A subunit of cholera toxin. In addition, antibodies raised to a synthetic peptide identical to residues 6-17 of the A subunit of pertussis toxin will bind to the A subunit of cholera toxin. 相似文献