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本实验室使霍乱肠毒素(CT)B亚单位基因在大肠杆菌中获得表达,并能分泌到胞外。将该菌株培养物上清经过超滤浓缩后,用偶联上霍乱毒素IgG的CNBr-Sepharose 4B进行亲和层析,得到表达产物霍乱毒素B亚单位纯蛋白。经PA-GE、HPLC及琼脂免疫扩散等方法鉴定证明该蛋白与天然霍乱肠毒素B亚单位完全相同。 相似文献
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霍乱毒素B亚单位是预防霍乱重要的保护性抗原,由霍乱毒素B亚单位组成的新的口服疫苗已经大量人体试验证明安全有效,此外霍乱毒素B亚单位是很强的粘膜免疫原,当与无关抗原结合在一起口服时,也是一个很好的佐剂。为获得大量霍乱毒素B亚单位,采用DNA重组技术,将霍乱毒素B亚单位基因与阿拉伯糖操纵子的表达载体pMPM-A4Ω质粒进行重组,pMPM-A4Ω表达载体是由阿拉伯糖(L-ara)所调控,具有高拷贝表达的特点。我们成功地构建了重组质粒和工程菌菌株,对其 相似文献
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构建了霍乱毒素B亚单位(choleratoxinBsubunit,CTB)与胰岛素(insulin)B链的融合基因CTB-INSB,将该融合基因克隆到大肠杆菌表达载体pET-30a(+)中,获得重组质粒pETCIB;并将该质粒转入大肠杆菌菌株BL21(DE3)中;重组菌株经IPTG诱导后的表达产物经15%SDS-PAGE分析表明可以表达融合蛋白,其分子量约为15.4kDa,且主要以包涵体形式存在,约占全菌蛋白的30%。含CTB-INSB重组蛋白的包涵体经变性和复性后,可在体外自组装成五聚体结构。Westernblotting分析结果显示CTB-INSB可分别被霍乱毒素的抗体和胰岛素的抗体识别,表明该蛋白具有霍乱毒素B亚单位与胰岛素的双重抗原性。同时GM1-ELISA分析结果表明CTB-INSB在体外可与神经节苷脂GM1(monosialoganglioside)特异结合,进一步证实了它能够形成类似CTB五聚体的高级结构,具有生物活性。 相似文献
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北非蝎昆虫毒素AaHIT1基因的原核表达和转基因分析 总被引:3,自引:0,他引:3
将北非蝎昆虫毒素 (AndroctonusaustralisHectorinsecttoxin ,AaHIT1)基因克隆入温度诱导表达载体pBV2 2 0 ,并在宿主菌E .coliDH5α中进行诱导表达 .表达定位分析发现 ,AaHIT1大部分存在于包涵体中 .蛋白质N 端测序证实了体外表达的AaHIT1蛋白的正确性 .将AaHIT1基因转化入烟草中并获得了转基因植株 ,基因组PCR、RT PCR及Northern印迹分别证实AaHIT1基因已正确地插入到烟草基因组中并已得到表达 .抗虫实验表明 ,该转基因烟草对白粉虱有明显的抗性 .该研究为蝎毒AaHIT1的生物学毒性实验以及获得抗虫转基因作物奠定了基础 相似文献
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霍乱肠毒素B亚单位在转基因番茄中表达的研究 总被引:7,自引:0,他引:7
将霍乱肠毒素B亚单位(CT-B)基因及内质网引导序列(SEKDEL)克隆到质粒pRTL2和pBI121中,分别构建植物双元表达载体pBI-CTB和pBI-CTBK,CT-B基因由Ca35S启动子控制表达。采用叶盘法经根癌农杆菌介导转化番茄(金丰1号,Jinfeng1)各表达载体得到一批转基因植株。经PCR和Southern blot分析表明CT-B基因整合到了番茄基因组中;ELISA和Western blot分析表明pBI-CTB和pBI-CTBK的转基因植株能够有效表达CT-B多肽,分别占番茄叶片可溶性蛋白的0.055%和0.084%。 相似文献
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目的:在大肠杆菌中表达霍乱毒素B单位(CTB)与谷氨酸脱羧酶(GAD)抗原表位肽段(531~545)的融合蛋白。方法:通过PCR技术将CTB基因与GAD基因融合在一起,插入pET22b载体,转化大肠杆菌BL21(DE3)后经诱导获得融合基因的表达;对表达产物进行包涵体复性后,纯化得到融合蛋白分子;对该融合蛋白分子进行了Western印迹和GM1-ELISA分析。结果:表达的融合蛋白的相对分子质量约为14000,Western印迹表明该融合蛋白具有霍乱毒素抗原性;GM1-ELISA实验表明该融合蛋白能够特异性地结合神经节苷酯GM1,表明该蛋白具有与CTB相似的五聚体结构。结论:融合蛋白CTB-GAD的成功表达,为后续动物实验提供了充足的抗原。 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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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传染病之一。过去的研究表明,AWTE基因编码的疟疾多种抗原表位是有效的抗疟表位,CTB基因编码的霍乱毒素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。本研究把AWTE-CTB融合基因构建到植物表达载体pBVG-ny1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基因片段检测,证实了疟疾多表位抗原基因在转基因烟草中的整合。SDS-PAGE蛋白电泳结果显示转基因烟草中表达了AWTE-CTB融合基因分子量相同的特异蛋白。经抗原性分析实验和Western免疫印迹实验结果表明,特异表达的融合蛋白可与CTB和AWTE抗体结合,具有CTB和AWTE抗原性。 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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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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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
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Bacterial and plant enterotoxin B subunit-autoantigen fusion proteins suppress diabetes insulitis 总被引:2,自引:0,他引:2
Carter JE Yu J Choi NW Hough J Henderson D He D Langridge WH 《Molecular biotechnology》2006,32(1):1-15
Several bacterial and plant enterotoxin B subunit-islet autoantigen fusion proteins were compared for their ability to serve
as islet autoantigen carriers and adjuvants for reduction of pancreatic islet inflammation associated with type 1 diabetes.
The cholera toxin B subunit (CTB), the heat-labile toxin B subunit from enterotoxigenic Escherichia coli (LTB), the Shigella toxin B subunit (STB), and the plant toxin ricin B subunit (RTB) were genetically linked to the islet autoantigens proinsulin
(INS) and glutamic acid decarboxylase (GAD). The adjuvant-autoantigen gene fusions were transferred to a bacterial expression
vector and the corresponding fusion proteins synthesized in E. coli. The purified adjuvant-autoantigen proteins were fed to 5-wk-old nonobese diabetic (NOD) mice once a week for 4 wk. Histological
examination of pancreatic islets isolated from inoculated mice showed significant levels of insulitis reduction in comparison
with uninoculated mice. The ratio of serum anti-INS and anti-GAD IgG2c to IgG1 antibody isotype titers increased in all ligand-autoantigen inoculated animal groups, suggesting an increase in effector
Th2 lymphocytes in B subunit-mediated insulitis suppression. The results of these experiments indicate that bacterial and
plant enterotoxin B subunit ligand-autoantigens enhance insulitis reduction in NOD mice. This research prompts further exploration
of a multiadjuvant/autoantigen co-delivery strategy that may facilitate type 1 diabetes prevention and suppression in animals
and humans. 相似文献