大肠杆菌热稳定肠毒素与霍乱毒素B亚单位融合蛋白免疫原性的评价

构建了霍乱毒素B亚单位(CTB)和大肠杆菌热稳定肠毒素(ST）的重组表达载体pMCST1、pMCST2。二的不同是,前为单拷贝ST,后为双拷贝ST。在大肠杆菌DH50α中融合蛋白高效表达。用这两种重组蛋白分别免疫小鼠,都诱导产生了高滴度的抗CTB和抗ST的血清。表明这两组融合蛋白具有良好的CTB和ST的免疫原性,为进一步构建抗CTB和抗ST的产毒性细菌腹泻疫苗打下了基础。     

CTB/CS3融合蛋白与GM1结合能力和免疫原性的分析

免疫霍乱毒素B亚单位（CTB）或肠毒素大肠杆菌（ETEC）定居因子CS3可使人体对ETEC的侵染有保护作用.为探索研制ETEC双组分亚单位疫苗的可行性,利用大肠杆菌诱导表达系统表达了CTB与CS3的融合蛋白（CTB/CS3）.蛋白质印迹结果表明,诱导表达的29 ku蛋白具有CTB和CS3蛋白双重抗原性.经Ni-NTA亲和层析纯化获得重组蛋白CTB/CS3,复性的重组蛋白可以部分形成五聚体并保留了与神经节苷脂GM1的结合能力.动物实验表明,融合蛋白CTB/CS3具有CTB和CS3蛋白的双重免疫原性,同时,CTB的免疫载体作用提高了CS3的免疫强度.     

霍乱毒素B亚单位与胰岛素原融合基因的克隆、表达及重组蛋白特性分析

构建了霍乱毒素B亚单位 (CholeratoxinBsubunit,CTB)与胰岛素原 (Proinsulin)的融合基因CTB -PROIN ,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pETCPI,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 ,其表达产物经过 15 %SDS PAGE分析表明该菌株可以表达融合蛋白 ,其分子量约为 21.6kD ,且主要以包涵体形式存在 ,约占全菌蛋白的 25%。含CTB-PROIN重组蛋白的包涵体经过变性和复性后 ,CTB-PROIN可以在体外自组装成五聚体结构。Westernblotting分析结果表明重组CTB PROIN蛋白可分别被霍乱毒素的抗体和胰岛素的抗体识别 ,说明该蛋白具有霍乱毒素和胰岛素的双重抗原性。同时在体外 ,CTB PROIN蛋白可与神经节苷脂GM1(monosialoganglioside)特异性结合 ,表明了该融合蛋白在体外具有生物活性。这些研究结果为利用原核生物表达系统研制廉价、高效的I型糖尿病口服疫苗奠定了基础。     

霍乱毒素B亚单位与胰岛素B链融合基因的克隆及原核表达分析

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。     

不相容双质粒共表达霍乱毒素类似嵌合蛋白

霍乱毒素由5个B亚基(CTB)和l个A亚基(CTA)(包括CTA1和CTA2)组成。该蛋白结构可帮助有毒的CTA1分子进入细胞。本研究拟利用原核不相容双质粒共表达系统获得霍乱毒素类似嵌合蛋白,用于大分子蛋白质黏膜给药的载体研究。将CTB基因片段克隆至载体p ET-28a中,获得重组质粒p ET-28a-CTB;以增强型绿色荧光蛋白(EGFP)代替有毒的CTA1,在CTA2序列N端融合穿膜肽(TAT),将EGFP-CTA2-TAT基因克隆至载体p ET-22b(+)中,获得重组质粒p ET-22b-EGFP-CTA2-TAT。利用p ET-28a-CTB和p ET-22b-EGFP-CTA2-TAT二者不同抗性,将双质粒分步转化到大肠杆菌BL21中。表达条件为0.75 mmol/L IPTG、20℃、200 r/min诱导20 h,重组嵌合蛋白能以可溶形式表达。经过Ni-NTA、Sephadex G-75纯化,Western blotting对蛋白质特异性进行鉴定,确定获得(CTB)5/EGFP-CTA2-TAT嵌合蛋白。     

乙肝PreS_2抗原决定簇和霍乱毒素B亚基基因的融合与表达

本研究通过全化学法按大肠杆菌密码偏性合成了HBV PreS_2抗原决定簇基因,与ctxB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CTB的基本高级结构和生物学功能;ELISA实验证明融合蛋白具有CTB和HBV PreS_2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,为研究融合蛋白的免疫原性并进一步构建基因工程肽苗奠定了基础。     

霍乱毒素B亚单位与谷氨酸脱羧酶抗原表位肽融合蛋白的表达及鉴定

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。     

CTB-FimA融合蛋白原核表达纯化及其免疫效果

[目的]构建牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)菌毛蛋白(FimA)与霍乱毒素B亚基(CTB)重组质粒pET-32a/CTB-FimA,表达纯化CTB-FimA融合蛋白并对其黏膜免疫效果进行研究。[方法]根据GenBank查找FimA基因和CTB基因,利用(Gly₄Ser)₃连接肽序列构建pET-32a/CTB-FimA原核表达质粒,转化至大肠杆菌BL21(DE3),确定IPTG诱导浓度及温度,SDS-PAGE分析目的蛋白的可溶性;采用亲和层析和凝胶过滤层析纯化目的蛋白CTB-FimA;用纯化的目的蛋白作为免疫原鼻腔滴注免疫BALB/c小鼠观察其黏膜免疫效果。[结果]经双酶切和测序鉴定pET-32a/CTB-FimA重组表达质粒构建成功;IPTG最适诱导浓度为0.5 mmol/L,最适诱导温度为23℃,重组蛋白CTB-FimA主要以可溶形式表达;Expasy软件ProtParam工具预测重组蛋白分子量约为72.08 kDa,亲水性总平均值为-0.313;每升发酵液表达CTB-FimA蛋白约100 mg;免疫...     

肠毒素性大肠杆菌CS6菌毛抗原与霍乱毒素B亚基在痢疾菌中的表达及其免疫学效果

构建了携带asd、霍乱毒素B亚基(CTB)基因的表达质粒pYX201,与福氏2a痢疾菌T32的△asd突变株FaD构成宿主质粒平衡致死系统,用于在没有抗生素选择压力的情况下,稳定表达CTB抗原基因。以此为基础,构建了单独表达肠毒素性大肠杆菌CS26菌毛抗原基因的重组质粒pYX202,以及同时表达CS6和CTB的共表达质粒pYX203。Western blotting和ELISA检测结果证实CS6及CTB在痢疾菌FaD中可以有效表达。重组菌免疫家兔后可诱生相应的血清抗体,特别是CTB的抗体效价较高,并持续较长时间。本研究为细菌性腹泻疫苗的研究提供了候选株。     

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

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。G_M1-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。     

霍乱弧菌CTB基因的克隆、表达及重组蛋白活性分析

霍乱弧菌CTB蛋白具有免疫佐剂活性。本研究根据已发表CTB基因的序列设计一对引物,从一株霍乱弧菌中扩增出CTB基因,测序后发现该基因全长375 bp,与国内分离的六株CTB基因的同源性达96.0%～99.2%。将该基因与pTWIN1连接构建了原核表达载体pTWIN1-CTB,重组表达载体转化BL21（DE3）表达菌株,0.8 mmol/L IPTG诱导4 h后,收获的细菌总蛋白SDS-PAGE电泳显示CTB在原核表达系统中得到表达,融合蛋白大小与理论值符合,蛋白产量占细菌总蛋白的20%左右,主要以包涵体形式存在,western杂交和GM1-ELISA结果表明重组蛋白具有免疫原性和粘膜佐剂活性。     

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.     

Synthesis of cholera toxin B subunit gene: cloning and expression of a functional 6XHis-tagged protein in Escherichia coli

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.     

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 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     

霍乱毒素B亚基融合蛋白表达系统的构建

霍乱毒素Ｂ亚基（ＣＴＢ）是良好的免疫佐剂和载体蛋白。本研究通过定点突变,在ＣＴＢ基因（ｃｔｘＢ）３′端终止密码前引入了限制性内切酶ＥｃｏＲＩ,构建了质粒ｐＭＣ０５。ｐＭＣ０５中ＣＴＢ与下游ｌａｃＺ′基因阅读框架相同,转化大肠杆菌后能够表达ＣＴＢ与β－半乳糖苷酶α肽的融合蛋白;所表达的融合蛋白能与ＧＭ１结合,说明融合蛋白保持ＣＴＢ的基本高级结构和生物学活性;融合蛋白能与抗－ＣＴＢ抗体结合,说明融合蛋白具有ＣＴＢ的抗原性。以上结果表明：通过将外源抗原决定簇基因融合至ｃｔｘＢ的３′端,在大肠杆菌中表达融合蛋白,构建基因工程肽苗是可行的。还探索了转录终止序列对融合基因蛋白表达水平的影响,构建了高效表达融合蛋白的载体－宿主系统。     

Expression of Cholera Toxin B Subunit in Transgenic Rice Endosperm

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 G_M1-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.     

High-level expression of codon optimized foot-and-mouth disease virus complex epitopes and cholera toxin B subunit chimera in Hansenula polymorpha

A codon optimized DNA sequence coding for foot-and-mouth disease virus (FMDV) capsid protein complex epitopes of VP1 amino acid residues 21-40, 135-160, and 200-213 was genetically fused to the N-terminal end of a 6x His-tagged cholera toxin B subunit (CTB) gene with the similar synonymous codons preferred by the methylotropic yeast Hansenula polymorpha. The fusion gene was synthesized based on a polymerase chain reaction (PCR) and subsequently overexpressed in H. polymorpha. The chimeric protein was successfully secreted into the culture medium (up to 100mg/L) and retained the antigenicity associated with CTB and FMDV antibodies by Western blot analysis. The chimera after purification through Co(2+)-charged resin column bound specifically to GM1 ganglioside receptor and thus retained the biological activity of CTB. This study has important implications in the construction of CTB chimera for mucosal vaccines against FMDV.     

Very efficient extracellular production of cholera toxin B subunit using Bacillus brevis

Abstract We have constructed a very efficient synthesis and secretion system for cholera toxin B subunit (CTB) of Vibrio cholerae 569B using Bacillus brevis . The constructed expression-secretion vector has the multiple promoters and the signal peptide coding region of the mwp gene, a structural gene for one of the major cell wall proteins of B. brevis strain 47, directly followed by the gene encoding the mature CTB. A large amount of mature CTB (1.4 g per liter of culture) was secreted into the medium. It had the same amino terminal amino acid sequence as that of authentic CTB and was fully active in GM1 ganglioside binding assay.