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疟疾多抗原表位基因表达载体的构建及其在烟草中的表达 总被引:7,自引:0,他引:7
本文首次报道疟疾多表位抗原基因在转基因烟草中表达成功。疟 疾是当今最需要研究有效疫苗的主要染病之一。过去的研究表明,AWTE基因编码的疟 疾多种怕表位是有铲的抗疟表位,CTB基因编码的乱毒素B亚基是一种既能引起细胞免疫又能引起体液免疫的免疫载体和佐剂。本研究把AWTE-CTB融合基因构建到植物表达载体pBVG-nyl上,采用共转化的方法,通过基因枪导入转化烟草,经PCR扩增AWTE-CTB基因片段 相似文献
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以霍乱毒素B亚基(CTB)基因为载体,构建了含不同抗原表位的恶性疟原虫的融合基因CTB/ATE和CTB/AWTE。前者除含有恶性疟原虫裂殖子表面主要抗原表位杂合多肽基因SPf66外,还含有很强的T辅助细胞表位CST3和Tc细胞表位;后者在此基础上将我国发现的B细胞表位NKNDD基因经8次串联后融合其中。两种形式的融合基因经测序正确后转入大肠杆菌TK1046中,产量分别为10mg/L及5mg/L。表达产物CTB/AWTE经亲和层析纯化双抗夹心ELISA测定表明,该融合蛋白在保留了与抗CTB抗体结合的同时,与抗NKNDD单抗的结合效价达1:8000。 相似文献
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恶性疟原虫多抗原表位基因表达载体的构建及其在大豆幼胚中的表达 总被引:2,自引:0,他引:2
疟疾是当今最需要研究有效疫苗的主要传染病之一。AWTE基因编码恶性疟原虫多种抗原表位基因 ,CTB基因编码霍乱毒素 B亚基 ,是一种既能引起细胞免疫又能引起体液免疫的免疫载体和佐剂。把 AWTE- CTB融合基因构建到植物表达载体 p BVG- ny2上 ,通过基因枪导入法 ,转化大豆幼胚分生组织。 X- glu染色检测到 GUS基因的表达 ;抗原性分析实验结果表明 ,特异表达的融合蛋白可与 CTB和 AWTE抗体结合 ,具有 CTB抗原性。这个实验结果 ,表明疟疾多抗原表位基因首次在转基因大豆幼胚中得到瞬时表达 相似文献
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霍乱毒素B亚单位基因(CtxB)的克隆及其表达 总被引:7,自引:0,他引:7
从霍乱弧菌中抽提基因组DNA,用PCER方法获取霍乱毒素B亚单位基因(CtxB)。序列分析结果表明,CtxB基因编码124个氨基酸,其中编码62位Thr的密码子与文献报道有差异。将CtxB基因插入质粒pGEX-4T-2,构建pGEX-CTXB表达质粒,转化大肠相菌BL21(DE30,筛选表达菌株CTXB/BL21。工程株经IPTG诱导表达,可产生大量的表达蛋白,经SDS-PAGE分析,融合蛋白分子 相似文献
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以霍乱毒素B亚基(CTB)为载体,由其基因构建了含有不同时期不同抗原表位的恶性疟原虫的融合基因CTB~AWTE、CTB~NANP,前者除含有恶性疟原虫裂殖子表面主要抗原表位杂合多肽基因SPf66外,还含有很强的T辅助细胞表位CST3和Tc细胞表位,后者含有子孢子期的B、Th细胞表位。将纯化的质粒免疫Balb/c纯系小鼠,3次免疫后诱导机体产生了体液免疫和细胞免疫,免疫的小鼠进行疟原虫子孢子攻击实验,保护率为60%一80%。将纯化的质粒混合后免疫恒河猴,3次免疫后诱导机体产生了体液免疫和细胞免疫,免疫的恒河猴进行食蟹疟原虫攻击实验,显示了一定的保护作用。 相似文献
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用PCR法扩增出编码人FAS分子胞外区的cDNA片段,直接克隆到pGEM-T载体上,经DNA序列测定后,再插入到谷胱甘肽转硫酶(GST)融合蛋白表达载体pGEX-KG的EcoRⅠ和SalⅠ位点之间,构成重组质粒pKG-hFAS,将此质粒导入大肠杆菌,经IPTG诱导后获得GST-hFAS重组融合蛋白的表达,用谷胱甘肽偶联的Sepharose4B经亲合层析获得纯化的GST-hFAS蛋白,经凝血酶酶切和二次亲合层析去除GST部分,得到纯化的FAS蛋白.用纯化的FAS抗原免疫家兔制备了抗FAS抗体,经检测发现抗FAS抗体能诱导U937细胞发生细胞凋亡 相似文献
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合成了5对寡核苷酸片段,分别连接在两种恶性疟原虫杂合多肽(45肽和58肽)抗原基因片段(HPFGA和HPFGB)的头部和尾部,将这两种片段分别与霍乱毒素B亚单位(CT-B)基因末端融合。两种杂合多肽抗原分别含有数个红内期和红外期有代表性的、并能被T或B淋巴细胞识别的保护性抗原表位,CT-B基因前端具有促使分泌的信号肽序列。将这两种融合基因的不同重组质粒分别转化大肠杆菌,对转化菌的培养上清检测表明融合蛋白被分泌性表达,既具有恶性疟原虫和CT-B抗原性,又保持了CT-B与其受体神经节苷脂CM1结合的生物学活性。 相似文献
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A protocol has been developed to produce a cholera toxin B subunit (CTB) in tobacco tolerant to the herbicide phosphinothricin
(PPT) by means of in vitro selection. The synthetic CTB subunit gene was altered to modify the codon usage to that of tobacco
plant genes. The gene was then cloned into a plant expression vector and was under the control of the ubiquitin promoter and
transformed into tobacco plants by Agrobacterium-mediated transformation. Transgenic plantlets were selected in a medium supplemented with 5 mg/L PPT. Polymerase chain reaction
analysis confirmed stable integration of the synthetic CTB gene into a chromosomal DNA. A high level of CTB (1.8% of total
soluble protein) was expressed in transgenic plants, which was 18-fold higher than that under the control of the expressed
CaMV 35S promoter with native gene. The transgenic plants when transferred to a greenhouse proved to be resistant to 2% PPT. 相似文献
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Modification of the cholera toxin B subunit coding sequence to enhance expression in plants 总被引:4,自引:0,他引:4
Kang Tae-Jin Loc Nguyen-Hoang Jang Mi-Ok Yang Moon-Sik 《Molecular breeding : new strategies in plant improvement》2004,13(2):143-153
The cholera toxin B subunit (CTB) contains five identical polypeptides and targets glycosphingolipid receptors on eukaryotic cell surfaces. Increased expression of CTB in plants is critical for the development of edible vaccines. In this study, the coding sequence of the CTB gene was optimized, based on the modification of codon usage to that of tobacco plant genes and the removal of mRNA-destabilizing sequences. The synthetic CTB gene was cloned into a plant expression vector and expressed in tobacco plants under the control of the CaMV 35S promoter. The recombinant CTB protein constituted approximately 1.5% of the total soluble protein in transgenic tobacco leaves. This level of CTB production was approximately 15-fold higher than that in tobacco plants that were transformed with the bacterial CTB gene. The recombinant CTB produced by tobacco plants demonstrated strong affinity for GM1-ganglioside, which indicates that the sites required for binding and proper folding of the pentameric CTB structure were conserved. This is the first report on the optimization of the CTB-coding sequence to give a dramatic increase in CTB expression in plants. 相似文献
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Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts 总被引:31,自引:0,他引:31
The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens. Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copies per cell), resulted in the accumulation of up to 4.1 % of total soluble tobacco leaf protein as functional oligomers (410-fold higher expression levels than that of the unmodified LTB gene expressed via the nuclear genome). However, expression levels reported are an underestimation of actual accumulation of CTB in transgenic chloroplasts, due to aggregation of the oligomeric forms in unboiled samples similar to the aggregation observed for purified bacterial antigen. PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that the chloroplast- synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB. Also, binding assays confirmed that chloroplast-synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed in chloroplasts. Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses. Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant-based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible. Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants. Furthermore, since the quaternary structure of many proteins is essential for their function, this investigation demonstrates the potential for other foreign multimeric proteins to be properly expressed and assembled in transgenic chloroplasts. 相似文献
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Oszvald M Kang TJ Tomoskozi S Jenes B Kim TG Cha YS Tamas L Yang MS 《Molecular biotechnology》2008,40(3):261-268
The synthetic cholera toxin B subunit (CTB) gene, modified according to the optimized codon usage of plant genes, was introduced
into a plant expression vector and expressed under the control of the Bx17 HMW (high molecular weight) wheat endosperm-specific
promoter containing an intron of the rice act1. The recombinant vector was transformed into rice plants using a biolistic-mediated transformation method. Stable integration
of the synthetic CTB gene into the chromosomal DNA was confirmed by PCR amplification analysis. A high level of CTB (2.1%
of total soluble protein) was expressed in the endosperm tissue of the transgenic rice plants. The synthetic CTB produced
only in the rice endosperm demonstrated strong affinity for GM1-ganglioside, thereby suggesting that the CTB subunits formed an active pentamer. The successful expression of CTB genes in
transgenic plants makes it a powerful tool for the development of a plant-derived edible vaccine. 相似文献
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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
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Assessment of the utility of the tomato fruit-specific E8 promoter for driving vaccine antigen expression 总被引:4,自引:0,他引:4
To assess the utility of the tomato fruit-specific E8 gene's promoter for driving vaccine antigen expression in plant, the 2.2 kb and 1.1 kb E8 promoters were isolated and sequenced from Lycopersicon esculentum cv. Jinfeng #1. The 1.1 kb promoter was fused to vaccine antigen HBsAg M gene for the transfer to Nicotiana tabacum, and the CaMV 35S promoter was used for comparison. Cholera toxin B (ctb) gene under the control of the 1.1 kb promoter was transformed into both N. tabacum and L. esculentum. Southern blot hybridization confirmed the stable integration of the target genes into the tomato and tobacco genomes. ELISA assay showed that the expression product of HBsAg M gene under the control of the 1.1 kb E8 promoter could not be detected in transgenic tobacco tissues such as leaves, flowers, and seeds. In contrast, the expression of HBsAg M gene driven by CaMV 35S promoter could be detected in transgenic tobacco. ELISA assay for CTB proved that the 1.1 kb E8 promoter was able to direct the expression of exotic gene in ripe fruits of transgenic tomato, but expression was absent in leaf, flower, and unripe fruit of tomato, and CTB protein was not detected in transgenic tobacco tissues such as leaves, flowers, and seeds when the gene was under the control of the 1.1 kb E8 promoter. The results indicated that the E8 promoter acted not only in an organ-specific, but also in a species-specific fashion in plant transformation. 相似文献
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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
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