志贺氏毒素B亚单位的分离纯化及其多克隆抗体的制备

从高效表达志贺氏毒素Ｂ亚单位（ＳｔｘＢ）的工程菌株ＤＨ５α／ｐＳＵ１０８分离纯化了ＳｔｘＢ,并用它制备了多克隆抗体。ＥＬＩＳＡ试验表明抗ＳｔｘＢ抗血清的滴度达１×１０４。Ｗｅｓｔｅｒｎｂｌｏｔ结果显示该抗血清能与ＳｔｘＢ发生特异反应。这为研究志贺氏毒素Ｂ亚单位的免疫保护作用和痢疾志贺氏Ⅰ型菌苗的研制打下了基础     

StxB／HlyA（CT）融合蛋白的分泌表达和StxB在小鼠内的特异免疫反应

本文将志贺氏毒Ｂ亚单位（ＳｔｘＢ）与大肠杆菌溶血素Ａ的Ｃ末端（ＨｌｙＡ（ＣＴ））相融合,蛋白ＳｔｘＢ／ＨｌｙＡ（ＣＴ）不仅能在大肠杆菌中分泌到胞外,而且也能从ａｒｏＡ突变的减毒鼠伤寒沙门氏菌中分泌出去。当用高拷贝质粒ｐＵＣ１８作载体时,上述融合蛋白对宿主细胞有毒性,但以低拷贝质粒ｐＢＲ３２２取代之后,该融合蛋白不再对窠主细胞产生任何不利影响。融合蛋白ＳｔｘＢ／ＨｌｙＡ（ＣＴ）无论是在ａｅｒｏｂａｃ     

志贺氏毒素

<正>志贺氏菌引起的严重腹泻和痢疾综合征(志贺氏菌病)系由具蛋白质的外毒素介导。在志贺氏培养物或细菌溶解产物中有细胞毒素、神经毒素和肠毒素的证据。显然,所有这些毒素的活性系由同一蛋白质毒素引起。许多研究者对培养细胞系中部分纯化的志贺氏毒素的作用进行了研究,表明在某些     

霍乱毒素B亚基基因上游序列对B亚基表达水平的影响

本研究通过缺失突变和移码突变研究了ｃｔｘ Ｂ基因上游Ａ基因部分序列对ｃｔｘＢ表达水平的影响,结果表明：（１）将霍乱毒素操纵子ＸｂａＩ－ＥｃｏＲｉ片段克隆至ｐＵＣ１９,构建的质粒ｐＵＣ１９ＣＴＢ中Ａ亚基的部分序列不能翻译,该质粒转化大肠菌后的ＣＴＢ的表达产量为３０μｇ／μｌ;（２）在质粒ｐＵＣ１９ＣＴＢ的ＸｂａＩ位点引入移码突变,构建质粒ｐＭＣ０２Ｃ,使Ａ亚基基因部分序列能够翻译至自然的终止密码,Ｂ     

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     

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

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。     

从噬菌体表面展示肽库中筛选志贺毒素受体结合抑制剂

利用抗体捕获法 ,从表面展示随机肽序列的噬菌体文库中筛选到与志贺毒素B亚基 (StxB)结合 ,并能抑制志贺毒素细胞毒效应的噬菌体克隆 ;依据其中 1个克隆序列 (A12 )合成的肽可以与志贺毒素的受体Gb3竞争结合StxB ,并抑制志贺毒素(Stx)的细胞毒和肠毒活性 ;抑制 5×CD₅₀ 剂量的Stx细胞毒效应需 2 2 .7μmol的A12合成肽 .筛选得到的 2个噬菌体克隆 (A3 ,A12 )编码的氨基酸序列不同 ,但能竞争结合StxB ,推测它们形成相同或相似的空间结构 .为志贺毒素抑制剂进一步研究打下基础 ,对其他相关药物的研制亦有参考价值 .     

Leu122对Hsp16.3组装过程中亚基相互作用的影响

小分子热休克蛋白是种类最多的热休克蛋白家族 ,它们均以寡聚体的形式存在 .研究表明 ,来自结核杆菌的小分子热休克蛋白Hsp16 3是以 3个三聚体的形式存在的九聚体 .为了探讨Hsp16 3体外组装过程中的亚基相互作用和识别 ,利用野生型Hsp16 3及其L12 2A突变体蛋白为模型 ,采用高效液相分子筛层析、非变性聚丙烯酰胺凝胶电泳和脲梯度凝胶电泳等方法进行研究 .结果表明 ,Hsp16 3在体外能自发地再组装成九聚体 .12 2位的亮氨酸残基对Hsp16 3体外再组装过程中的亚基相互作用有重要的影响 ,并且在Hsp16 3的组装过程中 ,亚基之间的相互识别是高度灵敏和特异的 ,野生型蛋白的亚基和L12 2A突变体蛋白的亚基并不能形成杂合体 ,只有完全相同的亚基才能组装成九聚体     

霍乱毒素B亚基基因具有自己的启动子

本研究发现并证实霍乱毒素Ｂ亚基基因上游ＸｂａＩ～ＣｌａＩ限制性片段内存在具有启动子活性的序列;在该启动子作用下,霍乱毒素Ｂ亚基表达水平可达２００ｍｇ／Ｌ,氯霉素乙酰基转移酶基因表达水平随培养条件不同在０．３～１０ｍｇ／Ｌ之间,大肠杆菌β－半乳糖苷酶基因的表达量达４１００单位／ｍｌ。在该启动子的控制下霍乱毒素Ｂ亚基基因可以高效表达,该启动子的存在可能是霍乱毒素操纵子中霍乱毒素Ｂ亚基表达量是Ａ亚基的６倍的原因。     

大瓶螺凝集素的分离纯化及部分性质研究

大瓶螺蛋白腺经磷酸盐缓冲液抽提、硫酸铵分级沉淀、ＳｅｐｈａｄｅｘＧ－１００和Ｓｅｐｈａｒｏｓｅ４Ｂ凝胶过滤,可获得在不连续ＰＡＧＥ（ｐＨ４．３和ｐＨ８．９）上显示单一蛋白质染色带的大瓶螺凝集素（ＡＧＬ）．该凝集素对人血红细胞无血型专一性,但对Ａ型血红细胞的凝集作用最强．ＡＧＬ的血凝活力可被乳糖或半乳糖所抑制．ＡＧＬ分子中的中性糖含量为０．２４ｍｇ／ｍｇ蛋白质．用ＳＤＳ－ＰＡＧＥ法测得其亚基分子量为１５０００,且只有一种亚基．ＡＧＬ中Ｃｙｓ和Ｐｈｅ的含量较高,并较耐热．     

Screening and identification of receptor antagonist for shiga toxin from random peptides displayed on filamentous bacteriophages

The bacteriophage clones which can bind with shiga toxin B subunit (StxB) and inhibit cytotoxicity of shiga toxin were obtained by using antibody capturing method from a 15-mer random peptide library displayed on the surface of bacteriophage fd. Among them, one peptide encoded by the random DNA region of a selected bacteriophage (A12) was synthesized and tested in vitro and in vivo, where the peptide competed with the receptor of shiga toxin to bind StxB, and inhibited the cytotoxicity and enterotoxicity of shiga toxin. The peptide can also block other apparently unrelated StxB binding bacteriophage (A3), which suggests that there are overlapping StxB interaction sites for those ligands with different sequences. The results provide a demonstration of bacteriophage display to screen peptide ligands for a small and/or unable biotinylated molecule by antibodies-capturing strategy, and take the lead for the development of receptor antagonists for shiga toxin.     

Screening and identification of receptor antagonist for shiga toxin from random peptides displayed on filamentous bacteriophages

The bacteriophage clones which can bind with shiga toxin B subunit (StxB) and inhibit cytotoxicity of shiga toxin were obtained by using antibody capturing method from a 15-mer random peptide library displayed on the surface of bacteriophage fd. Among them, one peptide encoded by the random DNA region of a selected bacteriophage (A12) was synthesized and testedin vitro andin vim, where the peptide competed with the receptor of shiga toxin to bind StxB, and inhibited the cytotoxicity and enterotoxicity of shiga toxin. The peptide can also block other apparently unrelated StxB binding bacteriophage (A3), which suggests that there are overlapping StxB interaction sites for those ligands with different sequences. The results provide a demonstration of bacteriophage display to screen peptide ligands for a small and/or unable biotinylated molecule by antibodies-capturing strategy, and take the lead for the development of receptor antagonists for shiga toxin.     

Single-step method for purification of Shiga toxin-1 B subunit using receptor-mediated affinity chromatography by globotriaosylceramide-conjugated octyl sepharose CL-4B.

A new single-step purification method for Shiga toxin (Stx) was developed using receptor-mediated affinity chromatography, in which Gb3Cer (globotriaosylceramide) was conjugated to octyl Sepharose CL-4B as a carrier. This method achieves high yield and high purity in a small column on which Gb3Cer has been immobilized at high density. Using this affinity column, the Stx1 B subunit was purified with homogeneity by a one-step procedure from a crude extract of recombinant Stx1 B subunit-producing Escherichia coli. The purified Stx1 B subunit conserved a natural pentamer structure confirmed by gel filtration and sedimentation equilibrium analysis. Furthermore, the purified Stx1 B subunit was able to bind specifically to Gb3Cer expressed on Burkitt's lymphoma cells. This versatile purification method can be used to isolate various types of natural as well as recombinant Stx, facilitating fundamental studies of human diseases caused by this toxin.     

Analyzing of expression of novel polypeptide complexes consisting of Shiga toxin B subunit and Adherence Fimbriae of Escherichia coli based on in silico modeling

Enterohemorrhagic (EHEC) and enteroaggregative (EAEC) are two pathotypes of diarrheagenic Escherichia coli. EAEC strains express adhesins called aggregate adherence fimbriae (AAFs) which the bacteria use to adhere to intestinal mucosa. EHEC virulence factor is Shiga toxin which belongs to the AB5 toxin family. B subunit, the nontoxic part of Shiga toxin (StxB), forms a homo pentamer and is responsible for binding to target cells. StxB has recently been proven to have adjuvant activity. In the current study we fused StxB encoding gene to 3' end of genes encoding two variants of AAFs, i.e., AAF/I and AAF/II. The in silico studies on tertiary structure and biochemical characteristics of Shiga toxin A subunit (StxA) revealed more resemblance to AAF/II than AAF/I. The constructs were prepared in a way that StxB could imitate its natural structure (pentamer formation) and its position (C-terminus) in the native toxin complex. The expression of these constructs showed the formation of AAF/II-B as a protein complex but with lower molecular mass than its expected size. In contrast, the AAF/I-B complex was not formed. Overall, the results of in silico studies and expression experiments together revealed that despite AAF/II-B expression, StxB failed to form pentamer. Therefore the observed protein complex has lower molecular mass. Since StxB is bound to AAF/II through disulfide bond, this bond prevents pentamer formation of StxB. However, due to the lack of disulfide bond between AAF/I and StxB, no protein complex is formed, thus StxB maintains its pentamer structure.     

Tumor delivery of ultrasound contrast agents using Shiga toxin B subunit

The present study demonstrates the targeting of ultrasound contrast agents to human xenograft tumors by exploiting the overexpression of the glycolipid Gb3 in neovasculature. To this end, microbubbles were functionalized with a natural Gb3 ligand, the B subunit of the Shiga toxin (STxB). The targeting of Gb3-expressing tumor cells by STxB microbubbles was first shown by flow cytometry and fluorescence microscopy. A significantly higher proportion of STxB microbubbles were associated with Gb3-expressing tumor cells compared to cells in which Gb3 expression was inhibited. Moreover, ultrasonic imaging of culture plates showed a 12 dB contrast enhancement in average backscattered acoustic intensity on the surface of Gb3-expressing cells compared to Gb3-negative cells. Also, a 18 dB contrast enhancement was found in favor of STxB microbubbles compared to unspecific microbubbles. Microbubble signal intensity in subcutaneous tumors in mice was more than twice as high after the injection of STxB-functionalized microbubbles compared to the injection of unspecific microbubbles. These in vitro and in vivo experiments demonstrated that STxB-functionalized microbubbles bind specifically to cells expressing the Gb3 glycolipid. The cell-binding moieties of toxins thus appear as a new group of ligands for angiogenesis imaging with ultrasound.     

Development and Characterization of Recombinant Antibody Fragments That Recognize and Neutralize In Vitro Stx2 Toxin from Shiga Toxin-Producing Escherichia coli

Background

Stx toxin is a member of the AB₅ family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes.

Methods and Findings

In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA.

Conclusion

In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro.     

Regulated expression of the Shiga toxin B gene induces apoptosis in mammalian fibroblastic cells

Shiga toxins (Stxs) produced by enterohaemorrhagic Escherichia coli may induce colonic ulceration, bloody diarrhoea and acute renal failure. The A subunit (StxA) is known to inhibit protein synthesis, whereas the B subunits (StxB) bind to Gb3 on the cell surface. However, the mechanisms by which Stxs kill target cells remain unclear. Stx1A or Stx1B genes were introduced into pcDNA3.1 vectors and transfected into NIH3T3 and HeLa cells. The Stx1B gene-transfected cells became apoptotic with accompanying DNA fragmentation, whereas the Stx1A gene-transfected cells were found to be necrotic and no DNA fragmentation occurred. The HeLa/C4 cells integrated with the Stx1B gene with a tetracycline-inducible promoter eventually produced cytoplasmic Stx1B, leading to DNA fragmentation on the addition of doxycycline. These apoptotic changes were abrogated by pretreatment with Z-VAD-fmk. These results suggest that the transfected Stx1B gene induces apoptosis by activating the caspase cascade after Stx1B expression in the cytoplasm.     

Cloning a Truncated Fragment (stx2a<Subscript>1</Subscript>) of the Shiga-Like Toxin 2A<Subscript>1</Subscript> Subunit of EHEC O157:H7: Candidate Immunogen for a Subunit Vaccine

Shiga toxins consist of enzymatically active A and B subunit multimers. The A subunit of shiga-like toxins can be proteolytically cleaved into two parts, A₁ and A₂, with A₁ being responsible for toxic activity. Antibody neutralizing the A₁ subunit of shiga toxin may protect against infection of the enterohemorrhagic Escherichia coli (EHEC O157:H7). It was difficult to express the full-length A₁ subunit of shiga toxin 2 (stx2A₁) in a previous study. We have now analyzed the full-length of stx2A₁ using bioinformatics software. The data show that the carboxyl terminal (of ~15 amino-acid residues) has strong hydrophobicity and low antigenicity. We cloned and expressed a truncated fragment of stx2A₁ (15 amino-acid residues of the carboxyl terminal being removed), designated stx2a₁, which can evoke a humoral immune response. Anti-Stx2a₁ antibodies can neutralize the native shiga toxin 2 both in vivo and in vitro, which suggests that Stx2a₁ serves as a candidate immunogen for a subunit vaccine that can also be used as the antigen to screen phage anti-shiga toxin antibody libraries. L. Liu and H. Zeng contributed equally to this study.