细胞分裂素结合蛋白的研究进展

迄今为止,已从多种植物中分离到细胞分裂素结合蛋白（ＣＢＰｓ）,它们可能在细胞分裂素的信号转导、体内运输及代谢中起作用。根据现有研究结果认为,大多数ＣＴＫｓ受体可能位于膜上,通过与Ｇ－蛋白耦联的信号转导系统或双组分信号转导系统完成ＣＴＫｓ信号的跨膜转导。少数ＣＴＫｓ受体可能位于细胞质中,与胞内ＣＴＫｓ结合后进入细胞核,直接调节基因的表达。本文综述了近年来对ＣＢＰｓ的研究进展,分析了ＣＴＫｓ受体的可能     

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

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。     

黄地老虎颗粒体病毒DNA片段的克隆与颗粒体蛋白基因的定位

用ｐＵＣ１９质粒作载体,克隆了黄地老虎颗粒体病毒（Ａｇｒｏｌｉｓｓｅｇｅｔｕｍｇｒａｎｕｌｏｓｉｓｖｉｒｕｓ,简称ＡｓＧＶ）ＤＮＡＰｓｔＩ－Ｄ．Ｅ．Ｆ．Ｇ．Ｈ．Ｊ．Ｋ．等７个片段。以［￣（３２）Ｐ］－ｄＣＴＰ标记的油桐尺蠖核型多角体病毒（Ｂｕｚｕｒａｓｕｐｐｒｅｓｓａｒｉａｎｕｃｌｃａｒｐｏｌｙｈｅｄｒｏｓｉｓｖｉｒｕｓ简称ＢｓＮＰＶ）多角体蛋白基因为探针,在３７℃条件下对ＡｓＧＶ）颗粒体蛋白基因进行了定位,将其分别定位于ＢｓｌⅡ－Ｓ或ＴＰｓＴＩ－Ａ或Ｂ和ＥｃｉＲＩ－Ａ片段上。     

霍乱毒素B亚单位基因（CtxB）的克隆及其表达

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     

鲑鱼降钙素基因在大肠杆菌中的克隆与表达

以鲑鱼基因组ＤＮＡ为模板,采用ＰＣＲ获得ｓＣＴ基因,并为ＤＮＡ序列分析所证实．以ｐＧＥＸ－３Ｘ为表达载体,利用体外定点突变技术成功地构建了融合蛋白ＧＳＴ－ｓＣＴ的重组表达质粒ｐＧＥＸ－３Ｘ－ｓＣＴ,在大肠杆菌中得到高效表达,其表达量约为菌体总蛋白的３０％;利用亲合层析法对融合蛋白ＧＳＴ－ｓＣＴ进行纯化,再经Ｆａｃ－ｔｏｒΧａ酶切后获得了重组ｓＣＴ,并对其进行活性检测．初步实验证明,重组ｓＣＴ具有较强的生物活性     

鲑鱼降钙素基因在大肠杆菌中的克隆与表达

以鲑鱼基因组ＤＮＡ为模板,采用ＰＣＲ获得ｓＣＴ基因,并为ＤＮＡ序列分析所证实。以ｐＧＥＸ－３Ｘ为表达载体,利用体外定点突变技术成功地构建了融合蛋白ＧＳＴ－ｓＣＴ的重组表达质粒ｐＧＥＸ－３Ｘ－ｓＣＴ,在大肠杆菌中得到高效表达,其表达量约为菌体总蛋白的３０％;利用亲合层析法对融合蛋白ＧＳＴ－ｓＣＴ进行纯化,再经Ｆａｃｔｏｒ Ｘａ酶切后获得了重组ｓＣＴ,并对其进行活性检测。初步实验证明,重组ｓＣＴ具有较     

人肿瘤坏死因子可溶性受体I与人IgG：Fc的融合基因的构建及其在大？…

采用ＰＣＲ方法引入编码氨基酸残基序列为ＧＧＧＧＳＧＧＧＧＳ的镜头,将ｓＴＮＦＲ１ ｃＤＮＡ与人ＩｇＧ：Ｆｃ ｃＤＮＡ片段连接,构成融合基因ｆｕｓｉｏｎ １。将ｆｕｓｉｏｎ １克隆在ｐＢＳⅡ ＳＫ＾＋载体上。测定序列后,转入ｐＲＳＥＴ－Ｂ表达载体,在大肠杆菌中表达,得到分子量为４５ｋＤａ的蛋白,超声破碎后电泳证明表达的蛋白为包涵体,经免疫蛋白印迹证实为我们构建的融合蛋白。     

大鼠基底动脉和尾动脉平滑肌G蛋白的异质性

用离体血管收缩功能实验法分析了大鼠基底动脉（ＢＡ）和尾动脉（ＣＡ）平滑肌细胞Ｇ蛋白的异质性。结果显示,当ＢＡ和ＣＡ平滑肌被精氨酸血管加压素（ＡＶＰ）激动后,该两种血管对Ｇ蛋白非选择性激活剂ＧＴＰγＳ的收缩反应发生了相反的变化：ＢＡ对ＧＴＰγＳ的收缩增强,并且这种收缩增强不受百日咳毒素（ＰＴ,Ｇｉ和Ｇｏ抑制剂）的影响。而霍乱毒素（ＣＴ,Ｇｓ激活剂）则完全抑制这种收缩,呈单相反应。与ＢＡ相反,ＣＡ对ＧＴＰγＳ的收缩减弱,后者可被ＰＴ预温育反转为收缩增强,而且ＣＴ对这种收缩的抑制作用短暂,呈双相反应。结果提示,ＢＡ和ＣＡ平滑肌细胞介导激动剂收缩反应的Ｇ蚕白存在异质性。     

日本血吸虫26kD抗原基因在BCG中的表达

研究了外源基因日本血吸虫２６ｋＤ抗原（Ｓｊ２６ＧＳＴ）在卡介苗（ｂａｃｉｌｕｓＣａｌｍｅｔｅ－Ｇｕｅｒｉｎ,ＢＣＧ）、耻垢分枝杆菌（Ｍ．ｓｍｅｇｍａｔｉｓ）和大肠杆菌（Ｅ．ｃｏｌｉ）中的表达．运用重组ＤＮＡ和聚合酶链反应（ＰＣＲ）等分子生物学技术,以表达Ｓｊ２６ＧＳＴ的Ｅ．ｃｏｌｉｐＧＥＸ衍生质粒为模板,经ＰＣＲ得到编码Ｓｊ２６ＧＳＴ的全长ｃＤＮＡ片段．将其按正确的阅读框顺序,克隆到人结核杆菌热休克蛋白（ｈｅａｔｓｈｏｃｋｐｒｏｔｅｉｎ,ＨＳＰ）７０的启动子下游,再将ＨＳＰ７０启动子和Ｓｊ２６ＧＳＴ基因一起亚克隆到Ｅ．ｃｏｌｉ－分枝杆菌穿梭质粒ｐＢＣＧ－２０００中,得到Ｅ．ｃｏｌｉ－分枝杆菌穿梭表达质粒ｐＢＣＧ－Ｓｊ２６．ｐＢＣＧ－Ｓｊ２６电转化入ＢＣＧ和Ｍ．ｓｍｅｇｍａｔｉｓｍｃ２１５５中表达Ｓｊ２６ＧＳＴ抗原,所表达的天然重组Ｓｊ２６ＧＳＴ（ｒＳｊ２６ＧＳＴ）为可溶性蛋白,在ＳＤＳ－ＰＡＧＥ上分子量为２６ｋＤ处可见明显的表达蛋白带．其表达量分别占ＢＣＧ和Ｍ．ｓｍｅｇｍａｔｉｓ菌体总蛋白的１５％和１０％．可见,Ｓｊ２６ＧＳＴ基因能在ＢＣＧ中高效表达．     

p16抑癌基因定点突变及其在大肠杆菌中的表达与纯化

为了研究错义突变对ｐ１６功能的影响,应用ＰＣＲ体外定点突变方法对ｐ１６ｃＤＮＡ进行体外定点突变,并将野生型和突变型ｐ１６ｃＤＮＡ克隆于ｐＧＥＸ－５Ｔ载体,在大肠杆菌中经ＩＰＴＧ诱导表达,Ｗｅｓｔｅｒｎ印迹鉴定确证表达．而后用谷胱甘肽－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析纯化野生型和突变型ｐ１６融合蛋白．得到了第４８位密码子ＣＣＧ（Ｐｒｏ）→ＣＴＧ（Ｌｅｕ）突变的ｐ１６－Ｐ４８突变体,并在大肠杆菌中表达了４２ｋＤ的ＧＳＴ－ｐ１６和ＧＳＴ－ｐ１６Ｐ４８Ｌ融合蛋白．最后经纯化得到了野生和Ｐ４８Ｌ突变的ｐ１６融合蛋白     

Purified cholera toxin B subunit from transgenic tobacco plants possesses authentic antigenicity

Cholera toxin B subunit (CTB) mature protein was stably expressed in transgenic tobacco plants under the control of the CaMV 35S promoter and TMV Omega fragment. Fusion of the PR1b signal peptide coding sequence to the CTB mature protein gene increased the expression level by 24-fold. The tobacco-synthesized CTB (tCTB) was purified to homogeneity by a single step of immunoaffinity chromatography. The purified tCTB is predominantly in the form of pentamers with molecular weight identical to the native pentameric CTB, indicating that the PR1b-CTB fusion protein has been properly processed in tobacco cells. Furthermore, by immunodiffusion and immunoelectrophoresis, we have shown that the antigenicity of the purified tCTB is indistinguishable from that of the native CTB protein.     

Modification of the cholera toxin B subunit coding sequence to enhance expression in plants

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.     

Expression of a synthetic cholera toxin B subunit in tobacco using ubiquitin promoter and bar gene as a selectable marker

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.     

Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts

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.     

Assessment of the utility of the tomato fruit-specific E8 promoter for driving vaccine antigen expression

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.     

Cholera toxin B protein in transgenic tomato fruit induces systemic immune response in mice

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.     

Expression of cholera toxin B subunit oligomers in transgenic potato plants

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     

Highly expressed cholera toxin B subunit in the fruit of a transgenic tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> L.)

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 G_M1-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%. G_M1-ELISA indicated that plant-synthesized CTB protein bound specifically to G_M1-gangliosides, suggesting that the CTB subunits formed active pentamers.     

Expression of cholera toxin B subunit and the B chain of human insulin as a fusion protein in transgenic tobacco plants

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.