炭疽毒素保护性抗原第四结构域在大肠杆菌中的可溶性表达

人工优化设计并合成炭疽毒素保护性抗原第四结构域基因,并与噬菌体gⅢ蛋白N端结构域基因融合,在大肠杆菌中可溶性表达融合蛋白。结果表明合成了炭疽毒素保护性抗原第四结构域基因,并在大肠杆菌中获得了高效可溶性融合表达,可溶性表达产物占细菌总蛋白量的36％左右;经亲和层析纯化获得了重组蛋白;Western印迹分析表明,表达产物能与His单抗（重组蛋白羧基端带有6xHis）发生特异性结合反应。以上结果表明获得了炭疽毒素保护性抗原第四结构域,为利用人抗体库进行筛选抗炭疽毒素的人源性中和抗体奠定了基础。     

针对炭疽保护性抗原不同结构域的中和性单克隆抗体的筛选和鉴定

炭疽保护性抗原（PA）是炭疽毒素的重要组分,同时也是现有炭疽疫苗的主要有效成分,在炭疽杆菌的致病与免疫中发挥关键作用。以重组PA为免疫原,采用B淋巴细胞杂交瘤技术,结合炭疽毒素敏感细胞的毒性中和试验,大量筛选抗PA单克隆抗体,获得了9株炭疽毒素中和性单抗。进一步分析表明这些单抗以IgG1亚类为主,分别识别PA 3个结构域的4个不同中和表位区。针对结构域2的4株单抗识别同一表位区,其中3株单抗的中和活性强于抗PA多抗;针对结构域4的4株单抗识别两个不同表位区;另有1株单抗识别位于结构域3的表位。实验结果提示PA具有多个中和表位,分别位于其不同结构域,其中结构域2、4包含主要中和表位。实验中获得的针对不同表位的中和性单抗为深入研究PA的免疫保护机理提供了工具,也为研制针对炭疽毒素的被动免疫制剂和治疗药物打下基础。     

重组炭疽保护性抗原的表达、纯化与生物活性分析

构建分泌型表达质粒 ,在大肠杆菌中实现了重组炭疽保护性抗原 (rPA)的分泌型表达。重组蛋白位于细菌外周质 ,表达量约占菌体总蛋白的 10 %。以离子交换、疏水层析和凝胶过滤为基础 ,建立了rPA的纯化工艺 ,每升培养物可获得约 15mgrPA ,纯度可达 95 %以上。体外细胞毒性试验显示rPA具有较好的生物学活性。用rPA免疫家兔产生的抗血清在体外可抑制炭疽致死毒素的活性 ,表明rPA可诱导机体产生保护性免疫。以上结果为今后发展新一代炭疽疫苗打下基础     

炭疽芽孢杆菌保护性抗原在大肠杆菌中的表达及纯化

按照炭疽芽孢杆菌保护性抗原（PA）基因成熟肽编码序列设计引物,从炭疽杆菌pOX1质粒中扩增出PA基因片段,将该片段定向插入到原核表达载体pET-28a中,获得了pET-PA原核表达重组质粒,限制性酶切分析和DNA序列测定均证实该克隆插入片段为PA基因的成熟呔编码序列。将该重组质粒转化大肠杆菌BL21（DE3）,经IPTG诱导,重组蛋白在大肠杆菌表达系统中获得了高效表达;Western印迹分析表明表达产物具有良好的免疫学活性。     

炭疽杆菌保护性抗原在大肠杆菌中的表达及其纯化

利用基因重组技术获取炭疽杆菌保护性抗原(PA)。将炭疽杆菌保护性抗原编码基因pag与pET载体连接构建重组质粒,转化大肠杆菌DE3株,诱导表达炭疽杆菌保护性抗原,并经亲和层析及凝胶过滤纯化此抗原。实验成功构建了表达炭疽杆菌保护性抗原的重组菌株,纯化后PA纯度达90%,且经检测纯化产物具有天然PA的生物学活性。同时表明从大肠杆菌中纯化PA较以往从炭疽杆菌中获取PA简便易行。     

炭疽毒素的细胞受体

炭疽杆菌外毒素是三组分蛋白质,构成两种毒素。水肿因子(EF)和致死因子(LF)分别是腺苷环化酶和金属蛋白酶,保护性抗原(PA)与细胞表面受体结合并将水肿因子或致死因子转移进细胞内发挥毒性作用。在哺乳动物细胞上己发现有两种受体,分别是肿瘤内皮标志8基因编码的细胞表面蛋白ATR／TEM8和毛细血管形态发生基因2编码的细胞表面蛋白CMG2。这两种受体蛋白的生理功能都不十分清楚,它们之间的氨基酸序列有很高的同源性(40％～60％)。氨基酸序列主要分信号肽、细胞外主基、跨膜区、脑浆尾四个区,细胞外主基内含von WIlle-brand因子A主基或称整合素插入主基(VWA／I主基),VWA／I主基内有金属离子依赖性粘连位点(MIDAS),是主基与PA蛋白质相互作用所必不可少的。这两种受体都有几种异构体,主要差异在于胞浆尾区的氨基酸长度不同。两种VWA／I主基都有封闭PA功能,阻止细胞中毒的作用,有望作为抗毒素治疗炭疽。     

破伤风毒素保护性抗原在毕氏酵母中的分泌表达

采用PCR方法．从C.Tetani 64008菌株中扩增大小为1353bp的破伤风毒素与靶细胞起结合作用的重链C端基因(Tetc),直接连接pGEM-T栽体进行测序,并以pPIC9K为表达栽体构建重组表达质粒,经线形化的重组质粒电转化毕氏酵母细胞GS1l5和KM71．甲醇诱导获得了分泌表达,表达产物存在于培养上清中,占分泌蛋白的10％,通过免疫印迹可以检测到重组表达产物,活性测定表明,重组蛋白具特异结合活性,本研究通过实现破伤风毒素保护性抗原在酵母系统中的分泌表达、研究其影响因素,为其它细菌毒素蛋白高效可溶性表达,及进一步抗原片段介导保护性免疫研究及抗毒素制备奠定了基础。     

炭疽毒素及其细胞受体的研究进展

炭疽毒素由 3种蛋白组成 :保护性抗原 (protectiveantigen ,PA)、致死因子 (lethalfactor,LF)和水肿因子 (edemafactor ,EF) .综述炭疽毒素研究的最新进展 .主要介绍炭疽毒素的关键致病因子———LF的结构与功能 ,炭疽毒素膜转运成分PA的结构及其受体 (anthraxtoxinreceptor ,ATR)和其cDNA克隆的结构 ,并讨论了在炭疽的治疗、预防和毒素在肿瘤治疗中的可能应用 .     

B型肉毒毒素保护性抗原Hc在大肠杆菌中的可溶性高表达

目的：通过序列优化及表达条件改进,在大肠杆菌中高效可溶性表达B型肉毒毒素保护性抗原He（Bont／B-He）。方法：对Bont／B-He基因片段优化,用大肠杆菌常用密码子替换稀有密码子,并将（C＋C）含量由76．2％降至56-3％;人工合成多条具有重叠互补序列的寡核苷酸片段,采用重叠延伸PCR方法获得了Bont／B-He的全长基因,并构建了原核可溶性表达载体;将经酶切和测序鉴定正确的重组质粒转化大肠杆菌B121（DE3）感受态细胞,用IPTC诱导Bont／B-He的表达并进行纯化及Western印迹鉴定。结果和结论：目的蛋白在大肠杆菌B121（DE3）中获得了可溶性高表达,占菌体裂解液上清总蛋白的26．7％,表达量达到30mg／L,是目前国内外已知表达的最高水平;经Ni柱一步纯化后,目的蛋白纯度可达到80.3％,为肉毒毒素中和抗体的制备及亚单位疫苗的研究奠定了基础。     

The Protective Antigen Component of Anthrax Toxin Forms Functional Octameric Complexes

The assembly of bacterial toxins and virulence factors is critical to their function, but the regulation of assembly during infection has not been studied. We begin to address this question using anthrax toxin as a model. The protective antigen (PA) component of the toxin assembles into ring-shaped homooligomers that bind the two other enzyme components of the toxin, lethal factor (LF) and edema factor (EF), to form toxic complexes. To disrupt the host, these toxic complexes are endocytosed, such that the PA oligomer forms a membrane-spanning channel that LF and EF translocate through to enter the cytosol. Using single-channel electrophysiology, we show that PA channels contain two populations of conductance states, which correspond to two different PA pre-channel oligomers observed by electron microscopy—the well-described heptamer and a novel octamer. Mass spectrometry demonstrates that the PA octamer binds four LFs, and assembly routes leading to the octamer are populated with even-numbered, dimeric and tetrameric, PA intermediates. Both heptameric and octameric PA complexes can translocate LF and EF with similar rates and efficiencies. Here, we report a 3.2-Å crystal structure of the PA octamer. The octamer comprises ∼ 20-30% of the oligomers on cells, but outside of the cell, the octamer is more stable than the heptamer under physiological pH. Thus, the PA octamer is a physiological, stable, and active assembly state capable of forming lethal toxins that may withstand the hostile conditions encountered in the bloodstream. This assembly mechanism may provide a novel means to control cytotoxicity.     

Molecular Dynamics Simulations of Complexes Between Wild-Type and Mutant Anthrax Protective Antigen Variants and a Model Anthrax Toxin Receptor

Abstract

Bacillus anthracis, a spore-forming infectious bacterium, produces a toxin consisting of three proteins: lethal factor (LF), edema factor (EF), and protective antigen (PA). LF and EF possess intracellular enzymatic functions, the net effect of which is to severely compromise host innate immunity. During an anthrax infection PA plays the critical role of facilitating entry of both EF and LF toxins into host cell cytoplasm. Crystal structures of all three of the anthrax toxins have been determined, as well as the crystal structure of the (human) von Willebrand factor A (integrin VWA/I domain)—an anthrax toxin receptor. A theoretical structure of the complex between VWA/I and PA has also been reported. Here we report on the results of 1,000 psec molecular dynamics (MD) simulations carried out on complexes between the Anthrax Protective Antigen Domain 4 (PA-D4) and the von Willebrand Factor A (VWA/I). MD simulations (using Insight II software) were carried out for complexes containing wildtype (WT) PA-D4, as well as for complexes containing three different mutants of PA-D4, one containing three substitutions in the PA-D4 “small loop” (residues 679–693) (D683A/L685E/Y688C), one containing a single substitution at a key site at the PA-D4—receptor interface (K679A) and another containing a deletion of eleven residues at the C-terminus of PA (A724–735). All three sets of PA mutations have been shown experimentally to result in serious deficiencies in PA function. Our MD results are consistent with these findings. Major disruptions in interactions were observed between the mutant PA-D4 domains and the anthrax receptor during the MD simulations. Many secondary structural features in PA-D4 are also severely compromised when VWA complexes with mutant variants of PA-D4 are subjected to MD simulations. These MD simulation results clearly indicate the importance of the mutated PA-D4 residues in both the “small loop” and at the carboxyl terminus in maintaining a PA conformation that is capable of effective interaction with the anthrax toxin receptor.     

Role of the Protective Antigen Octamer in the Molecular Mechanism of Anthrax Lethal Toxin Stabilization in Plasma

Anthrax is caused by strains of Bacillus anthracis that produce two key virulence factors, anthrax toxin (Atx) and a poly-γ-D-glutamic acid capsule. Atx is comprised of three proteins: protective antigen (PA) and two enzymes, lethal factor (LF) and edema factor (EF). To disrupt cell function, these components must assemble into holotoxin complexes, which contain either a ring-shaped homooctameric or homoheptameric PA oligomer bound to multiple copies of LF and/or EF, producing lethal toxin (LT), edema toxin, or mixtures thereof. Once a host cell endocytoses these complexes, PA converts into a membrane-inserted channel that translocates LF and EF into the cytosol. LT can assemble on host cell surfaces or extracellularly in plasma. We show that, under physiological conditions in bovine plasma, LT complexes containing heptameric PA aggregate and inactivate more readily than LT complexes containing octameric PA. LT complexes containing octameric PA possess enhanced stability, channel-forming activity, and macrophage cytotoxicity relative to those containing heptameric PA. Under physiological conditions, multiple biophysical probes reveal that heptameric PA can prematurely adopt the channel conformation, but octameric PA complexes remain in their soluble prechannel configuration, which allows them to resist aggregation and inactivation. We conclude that PA may form an octameric oligomeric state as a means to produce a more stable and active LT complex that could circulate freely in the blood.     

炭疽毒素受体胞外区在毕赤酵母中分泌表达、纯化与活性鉴定

使用分泌型表达载体,实现了重组炭疽毒素受体胞外区 (rATR(CMG2)-EXCELL) 在毕赤酵母 KM71H 培养物上清中的分泌表达 . 表达量约占培养物上清总蛋白质的 20%. 经过螯合柱初步纯化,每升诱导培养物可获得约 1 mg 电泳纯的 rATR(CMG2)-EXCELL. 体外与配基 PA 结合试验和细胞保护试验显示, rATR(CMG2)-EXCELL 具有很好的生物活性 . rATR(CMG2)-EXCELL 的成功表达为今后研究炭疽毒素受体的作用机理、发展新型炭疽治疗药物打下基础 .     

Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process

The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm. Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts). We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm. Importantly, altering raft integrity using drugs prevented LF delivery and cleavage of cytosolic MAPK kinases, suggesting that lipid rafts could be therapeutic targets for drugs against anthrax. Moreover, we show that internalization of PA is dynamin and Eps15 dependent, indicating that the clathrin-dependent pathway is the major route of anthrax toxin entry into the cell. The present work illustrates that although the physiological role of the ATR is unknown, its trafficking properties, i.e., slow endocytosis as a monomer and rapid clathrin-mediated uptake on clustering, make it an ideal anthrax toxin receptor.     

CHO细胞表达的抗炭疽保护性抗原人源化抗体的纯化及质量控制分析

目的:建立CHO细胞表达的抗炭疽保护性抗原人源化单抗纯化工艺和质量控制方法。方法:收获50 L生物反应器中无血清悬浮培养的CHO工程细胞培养液,通过高速离心去除细胞及碎片后超滤浓缩上清液,经亲和层析、SPFF阳离子交换层析后,将所得目的蛋白质经G25凝胶柱更换缓冲液以完成纯化;对纯化的产品进行单抗鉴别(Western印迹)、相对分子质量(SDS-PAGE和MOLDI-TOF)、纯度(SEC-HPLC)、生物学活性(毒素中和试验)、产品相关杂质(SEC-HPLC检测聚集体、降解产物)、工艺相关杂质(ELISA分析残余宿主蛋白、残余蛋白A)、安全性(凝胶法检测内毒素、薄膜过滤法考察无菌)分析。结果:样品回收率达61.7%;单抗鉴别实验阳性;MOLDI-TOF测定完整分子的相对分子质量为147 995,与预期相符;单体比例为99.25%,二聚体比例为0.75%;EC50值为0.1516μg/m L。残余宿主蛋白、残余蛋白A、内毒素、无菌检查结果符合药典要求。结论:初步建立了纯化工艺和质量控制方法,为抗炭疽人源化单抗药物的研制奠定了基础。