金黄色葡萄球菌肠毒素中毒的免疫治疗策略

金黄色葡萄球菌肠毒素（staphylococcal enterotoxins,SEs）是由金黄色葡萄球菌（Staphylococcus aureus）分泌的一类具有呕吐活性的细菌外毒素，可引起严重的炎症反应和食物中毒。SEs具有超抗原活性，可与T细胞受体的可变区和MHC II类分子形成三元复合物（TCR-SEs-MHC II），直接刺激T淋巴细胞大量产生肿瘤坏死因子-α (tumor necrosis factor α,TNF-α)、白介素（interleukin,IL）-2、IL-6和γ干扰素（interferon γ,IFN-γ）等细胞因子，从而导致中毒性休克综合征（toxicshocksyndrome, TSS）。在临床常见的SEs中，肠毒素A (staphylococcalenterotoxin A, SEA)和肠毒素B(staphylococcal enterotoxin B,SEB)是出现频率最高、毒性最大、危害最严重的两种。目前尚没有针对SEs中毒治疗策略的综述性研究。本文首先概述了SEs的分类、结构及毒性作用，重点围绕SEA和SEB，分析了TCR-SEs-MHC II类...     

金黄色葡萄球菌肠毒素

金黄色葡萄球菌是一种重要的病原体,它产生多种类型的毒素,从而引起各种类型的疾病。金黄色葡萄球菌肠毒素(Staphylococcal enterotoxins,SEs),是一组血清学上互不相同的热稳定肠毒素,有10个血清型。由于食入了被SEs污染的食品而主要引起肠胃炎,此外,SEs还是一种强的超抗原,它可以刺激非特异性T细胞增殖。SEs各型之间有着相似的结构和功能。     

IL-2与金黄色葡萄球菌肠毒素A和B融合基因的克隆及表达

目的：IL-2与金黄色葡萄球菌肠毒素A和B融合基因的克隆及表达。方法：分别在金黄色葡萄球菌肠毒素A227Ala、B基因的两端克隆上两个酶切点Hind Ⅲ,Kpn Ⅰ。将IL-2基因突变,设计一段linker使之分别与SEA227Ala和SEB相连并克隆到PET表达载体中,在大肠杆菌DH5a（DE3）-Pass中表达。结果：表达的蛋白占总蛋白15%。结论：IL-2与金黄色葡萄球菌肠毒素A和B融合蛋白能在大肠杆菌中有效表达。     

金黄色葡萄球菌肠毒素A Asp227Ala基因的克隆及表达

目的：金黄色葡萄球菌肠毒素A Asp227ala基因的克隆及表达。方法：利用错配PCR方法,从含有金黄色葡萄球菌肠毒素A（Staphylococcal enterotoxn A,SEA)基因的质粒中扩增出约720bp的DNA片段,将其克隆到表达载体7ZTS中,并转化于JM109（DE3）。结果：重组质粒的测序结果表明,它含有702bp（不包括N端72bp的信号肽编码区）,其核苷酸序列与文献报道完全一致,推导的氨基酸序列显示227位的天冬氨酸已突变为丙氨酸。结论：该基因所表达的蛋白为可溶性蛋白,表达量占总蛋白51.5%。表达的蛋白与天然肠毒素A产生的抗体能发生凝集作用,具有与天然SEA类同的抗原活性。     

金黄色葡萄球菌肠毒素C的原核表达、纯化及活性鉴定

目的：在大肠杆菌中表达金黄色葡萄球菌肠毒素C（SEC）,并对其活性进行检测。方法：以金黄色葡萄球菌基因组为模板扩增SEC基因,经测序正确后插入原核表达载体pBV220中,转化大肠杆菌DH5α后温度热诱导重组SEC表达;表达产物经阳离子柱纯化后,用ELISA鉴定其抗原性;通过观测表达产物对鼠脾淋巴细胞的刺激增殖情况,检测其超抗原活性。结果：构建了SEC-pBV220原核表达载体,在大肠杆菌中可快速、高效表达SEC蛋白,表达产物具有超抗原活性。结论：实现了SEC的原核表达。     

金黄色葡萄球菌肠毒素A的基因克隆、表达及活性试验

利用PCR从金黄色葡萄球菌标准株（Staphylococcus aureus, ATCC13565）中克隆了金黄色葡萄球菌肠毒素A(SEA)的基因,序列测定结果与报道完全一致。构建了表达载体pETSEA并获得高效表达,重组蛋白（rSEA）在37℃诱导时以包涵体形式存在,降低温度则出现可溶表达,可溶性rSEA占总rSEA的55%。可溶性rSEA经Ni²⁺亲和层析纯化,达电泳纯。通过同源模建对rSEA对SEA进行结构比较,结果表明尽管rSEA比野生型SEA多了9个氨基酸但其结构并没有明显的变化。单核细胞增殖试验进一步证明了该结论：将rSEA与SEA同外周血单个核细胞共同培养,两者均能有效地促进其增殖。将rSEA与体内激活的脾细胞共培养,则能增强脾细胞的体外抑瘤活性。     

抗金黄色葡萄球菌单链抗体(scFv)的原核表达及蛋白纯化

旨在将来源于鸡的金黄色葡萄球菌单链抗体(scFv)进行原核诱导表达,获得有抗体活性的目的蛋白。构建含有目的抗体基因的重组质粒,将此质粒进行原核诱导表达并鉴定所获得蛋白的生物活性。结果显示,(1)成功构建了含有金黄色葡萄球菌单链抗体(scFv)的重组质粒p Cold I-scFv,质粒成功转化到大肠杆菌表达菌株中;(2)经过诱导表达后,目的蛋白主要以包涵体的形式存在于沉淀中;(3)使用4 mol/L尿素成功地将包涵体变性溶出;(4)通过柱层析法及透析法获得了纯化及复性效果较好的目的蛋白;(5)间接ELISA鉴定证实所获蛋白具有金黄色葡糖球菌抗体活性。通过质粒构建及原核诱导表达、包涵体溶出和复性等步骤,最终获得了有金黄色葡萄球菌抗体活性的目的蛋白。     

金黄葡萄球菌fnbB基因的克隆及在大肠杆菌中的表达

金黄色葡萄球菌（Staphylococcus aureus）是引起奶牛乳房炎主要致病菌之一,主要通过其菌体表面的黏附素侵入寄主细胞引起疾病,为奶牛业造成巨大损失。金黄色葡萄球菌表面蛋白纤连蛋白结合蛋白(fibronectin-binding protein,FnBP)是其关键的黏附因子,在研制抗金黄色葡萄球菌的新型疫苗中占有重要地位.本文根据GenBank中纤连蛋白结合蛋白B基因（fnbB）序列设计特异性引物,以金黄葡萄球菌基因组DNA为模板,进行PCR扩增,获得3 458 bp 的DNA片段。使用T-A克隆技术,将PCR产物克隆至pGEM T easy Vector中,成功构建出了克隆质粒pGEM-fnbB。以 BamHI和XhoI 双酶切pGEM-fnbB和pET28a(+),并将纯化的基因fnbB 亚克隆至pET28a(+)中,构建出原核表达质粒pET28a-fnbB,并将其转化至E.coli BL21(DE3)感受态细胞中,经1 mmol/L的IPTG诱导和SDS-PAGE分析,在约165 ku 处出现了与预期目的蛋白相一致的外源蛋白带,Western blot分析结果表明该蛋白具有金黄葡萄球菌的抗原性。金黄葡萄球菌pET28a-fnbB成功表达为金黄葡萄球菌引起的奶牛乳房炎的诊断和研究新型疫苗奠定基础。     

产毒金黄色葡萄球菌鉴定及在百叶中生长与产毒特性分析

从生鲜食品中分离鉴定得到产肠毒素SEA的金黄色葡萄球菌（Staphyloccocus aureus）菌株,研究金黄色葡萄球菌在百叶中的生长变化及产肠毒素特性。将不同浓度的金黄色葡萄球菌同时接种到减菌（样品组）和未减菌（对照组）百叶中,高、低初始接种量分别控制在4.0 lg(cfu/g)和2.0 lg(cfu/g)左右,并定时追踪不同储存温度条件下百叶中金黄色葡萄球菌的活菌数及产毒情况。金黄色葡萄球菌肠毒素的检测采用商业化的ELISA试剂盒。在37 ℃储存过程中,金黄色葡萄球菌在样品组中不但能生长良好并在18 h后菌落数可达到7.0 lg(cfu/g)并被检测出肠毒素,对照组中金黄色葡萄球菌生长良好菌落数可达到7.0 lg(cfu/g)却未检测出产肠毒素。在25、15和5 ℃储存过程中,样品组和对照组百叶都未检测出肠毒素。温度对金黄色葡萄球菌在百叶中是否产肠毒素具有决定性的作用,且百叶中本身存在的杂菌可能可以抑制金黄色葡萄球菌产肠毒素。     

细菌及其毒素诱发小鼠胸腺细胞凋亡的机制初探

给ＢＡＬＢ／ｃ小鼠腹腔注射产肠毒素Ｂ金黄色葡萄球菌（ＳＥＢＳ）、绿脓杆菌（ＰＡ）或葡萄球菌肠毒素Ｂ（ＳＥＢ）,均可引起小鼠胸腺萎缩,呈现胸腺重量减轻、胸腺细胞数减少、胸腺细胞存活率降低。实验发现,在这些细菌或毒素作用下,胸腺细胞发生了具有细胞凋亡的特征性形态学和生化学变化。进一步研究表明,小鼠胸腺细胞凋亡的机制可能与这些细菌或毒素诱导宿主产生ＴＮＦ－α、ＩＦＮｒ和ＩＬ－６等细胞因子有关。     

Expression and bioactivity analysis of Staphylococcal enterotoxin M and N

Staphylococcal enterotoxins (SEs) are powerful superantigens that stimulate non-specific T-cell proliferation produced by Staphylococcus aureus and draw considerable attention as ideal drugs for cancer therapy. The filtrate of S. aureus culture has been used as ampul named Staphylococcal enterotoxin C injection in clinic for 10 years in China and proved to be effective. The superantigen SEC claimed to be the only active component without certifiable evidences. For further investigations of the active components of this injection and establishment of foundations for the development of novel anti-cancer drugs, in this research we extracted total DNA from S. aureus (FRI 1230), cloned, expressed and purified recombinant proteins of Staphylococcal enterotoxin M and N (rSEM and rSEN). The MTT assay of the purified rSEM and rSEN demonstrated that their abilities of stimulating T cells and inhibiting the proliferation of K562-ADM cells and B16 cells were equivalent to that of purified SEC2 in vitro. These findings suggested that SEC was not the only active component of Staphylococcal enterotoxin C injection and the effective procedure of expression and purification may be useful for mass productions of these therapeutically important proteins.     

Molecular docking analysis of Enterotoxin I from Staphylococcus aureus with Nafcillin analogues

Staphylococcal enterotoxins (SEs) are liked with food poisoning and other related infections. Nafcillin is an antibiotic used to treat S. aureus. Therefore, it is of interest to study the molecular interactions of 25 nafcillin analogues with enterotoxin I using molecular docking analysis. The analysis shows optimal interaction features of Nafcillin analogues with Enterotoxin I from Staphylococcus aureus for further consideration.     

A single-stranded DNA aptamer that selectively binds to Staphylococcus aureus enterotoxin B

The bacterium Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs). Staphylococcal food poisoning (SFP) results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify several SEs within a food or clinical sample. However, the success of these assays depends upon the availability of a monoclonal antibody, the development of which is non-trivial and costly. The current scope of the available immuno-affinity based methods is limited to the classical SEs and does not encompass all of the known or emergent SEs. In contrast to antibodies, aptamers are short nucleic acids that exhibit high affinity and specificity for their targets without the high-costs and ethical concerns of animal husbandry. Further, researchers may choose to freely distribute aptamers and develop assays without the proprietary issues that increase the per-sample cost of immuno-affinity assays. This study describes a novel aptamer, selected in vitro, with affinity to staphylococcal enterotoxin B (SEB) that may be used in lieu of antibodies in SE detection assays. The aptamer, designated APT(SEB1), successfully isolates SEB from a complex mixture of SEs with extremely high discrimination. This work sets the foundation for future aptamer and assay development towards the entire family of SEs. The rapid, robust, and low-cost identification and quantification of all of the SEs in S. aureus contaminated food is essential for food safety and epidemiological efforts. An in vitro generated library of SE aptamers could potentially allow for the comprehensive and cost-effective analysis of food samples that immuno-affinity assays currently cannot provide.     

Highly Expressed Recombinant SEB for Antibody Production and Development of Immunodetection System

Staphylococcal enterotoxins (SEs) are the second most common causal agents of food poisoning throughout the world. Staphylococcal enterotoxin B (SEB) is one of the most potent and a listed biological warfare agent. Therefore, its quick, accurate and sensitive detection is of paramount importance. But availability of sensitive and specific antibodies against SEB is the major bottleneck in the development of an immunodetection system. Therefore, in the present study seb gene was cloned and expressed in a heterologous host resulting in a yield of 92 mg pure toxin per litre of culture broth after Ni–NTA affinity purification. Antibodies raised against the recombinant toxin did not cross react with related enterotoxins and organisms that can gain access in the food. Further, a sandwich ELISA was developed to detect SEB after extraction from artificially spiked food samples like milk, orange juice, skim milk and khoya. The sandwich ELISA was able to detect SEB in the range of 0.25 to 0.49 ng/ml or g of food. The detection system developed in the present study is at least as specific and sensitive as other commercially available kits which use monoclonal antibodies.     

In vitro cell-based assay for activity analysis of staphylococcal enterotoxin A in food

Staphylococcal enterotoxins (SEs) are a leading cause of food poisoning and have two separate biological activities; it causes gastroenteritis and functions as a superantigen that activates large numbers of T cells. In vivo monkey or kitten bioassays were developed for analysis of SEs emetic activity. To overcome the inherent limitations of such bioassays, this study describes an in vitro splenocyte proliferation assay based on SEs superantigen activity as an alternative method for measuring the activity of staphylococcal enterotoxin A (SEA). After incubation of splenocytes with SEA, cell proliferation was measured by labeling the proliferating cells' DNA with bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) and quantifying the incorporated BrdU by immunohistochemistry. BrdU labeling is shown to be highly correlated with SEA concentration ( R ²=0.99) and can detect 20 pg mL⁻¹ of SEA, which is far more sensitive than most enzyme-linked immunosorbent assays. Our assay can also distinguish between active toxin and inactive forms of the toxin in milk. By applying immunomagnetic beads that capture and concentrate the toxin, our assay was able to overcome matrix interference. These results suggest that our in vitro cell-based assay is an advantageous practical alternative to the in vivo monkey or kitten bioassays for measuring SEA and possibly other SEs activity in food.     

The emetic activity of staphylococcal enterotoxins,SEK, SEL,SEM, SEN and SEO in a small emetic animal model,the house musk shrew

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are the most recognizable causative agents of emetic food poisoning in humans. New types of SEs and SE‐like (SEl) toxins have been reported. Several epidemiological investigations have shown that the SEs and SEl genes, particularly, SEK, SEL, SEM, SEN and SEO genes, are frequently detected in strains isolated from patients with food poisoning. The purpose of the present study was to evaluate the emetic activity of recently identified SEs using a small emetic animal model, the house musk shrew. The emetic activity of these SEs in house musk shrews was evaluated by intraperitoneal administration and emetic responses, including the number of shrews that vomited, emetic frequency and latency of vomiting were documented. It was found that SEs induce emetic responses in these animals. This is the first time to demonstrate that SEK, SEL, SEM, SEN and SEO possess emetic activity in the house musk shrew.     

A Novel ELISA Format for the Rapid and Sensitive Detection of Staphylococcal Enterotoxin A

Staphylococcal food poisoning is one of the leading causes of bacterial food poisoning each year. Detection kits for staphylococcal enterotoxins are commercially available and the assays can require from one and a half to twenty-four hours to complete with detection limits ranging from 0.5 to 2 ng enterotoxin per gram of food. We have successfully demonstrated a microsphere-packed capillary (MPC) ELISA for the detection of staphylococcal enterotoxin A (SEA) and have compared it to two commercially available kits. The MPC assay detected a lower amount of SEA in ham, chicken, cheese, and bean sprouts than either of the two commercially available kits. In addition, the novel MPC assay was completed in less than ten minutes, as compared to three and twenty-four hours for the two commercially available kits. This research also demonstrated that the MPC ELISA can contain integrated positive and negative controls and has the potential to simultaneously detect and identify multiple enterotoxins.     

Establishment of highly specific and quantitative immunoassay systems for staphylococcal enterotoxin A, B, and C using newly-developed monoclonal antibodies

Staphylococcal enterotoxin (SE) activities remain after boiling or treating with proteases. The main symptoms such as vomiting and diarrhea, are caused by the ingestion of SEs. Among SEs, SEA has been reported to be the major and most toxic protein. A highly specific and simple assay system is required to diagnose staphylococcal food poisoning. Therefore, the development of a suitable assay system is strongly anticipated. In this study, we have established a highly specific and sensitive avidin-biotin sandwich ELISA (ABS-ELISA) system for SEA, SEB, and SEC1 using newly-developed monoclonal antibodies. The linearity of these systems obtained was in the range of 0.78-25 ng/ml for each SE, and furthermore, the lower concentrations of SEs could also be detected. The recoveries of SEs from murine serum, skim milk solution, and raw milk were found to be over 90%, suggesting that our systems could detect SEs without any interventions, such as these from milk or serum proteins. We were also able to quantify SEs in 22 specimens of culture supernatants of S. aureus isolated in past occurrences. Our established system should be very useful not only in the clinical field but also in various fields of investigation because of its quantifi-cation and simplicity in detecting SEs.     

Molecular epidemiological characterization of Staphylococcus aureus isolates originating from food poisoning outbreaks that occurred in Tokyo,Japan

Staphylococcal food poisoning (SFP), one of the commonest food‐borne diseases, results from the ingestion of one or more staphylococcal enterotoxins (SEs) produced in foods by Staphylococcus aureus. In the present study, 203 S. aureus strains originating from 83 outbreaks that had occurred in Tokyo were examined for their coagulase type and genotype of SEs to analyze their molecular epidemiological characteristics. The representative subsets of the 83 S. aureus isolates were analyzed by multilocus sequence typing (MLST) and S. aureus pathogenicity island (SaPI) scanning. The isolates were integrated into eight specific clonal complexes (CC) s; CC81, CC8, CC6, CC5, CC508, CC59, CC20 and CC30. The profiles of the coagulase type, SE/SEl genotype and the suspected type of enterotoxin‐encoding mobile genetic element (MGE) indicated a correlation with each CC. SaPI scanning showed fixed regularity between the distributions of genomic islands, including SaPIs, and the phylogenetic lineage based on MLST. These results indicate that the S. aureus isolates, which classified into eight CCs, have distinguishable properties concerning specific coagulase type, enterotoxin genotype and MGE type. Strains of S. aureus harboring these particular elements possess the potential to cause SFP.     

Identification of a Fourth Staphylococcal Enterotoxin, Enterotoxin D

A fourth staphylococcal enterotoxin was identified serologically with antiserum to the very crude enterotoxic products of growth of a strain which also produces enterotoxin C, and then with antiserum to the considerably purified enterotoxic antigen of a strain which produces only the new enterotoxin. The identification of this antigen as enterotoxin D was based on the following observations. It was produced by strains which do not produce enterotoxins A, B, or C; it was absent in the growth products of nonenterotoxigenic strains; when appreciably purified, it was associated with emetic activity in the cat, and its biological activity was neutralized only by antisera containing its specific antibody and not by antibodies to enterotoxins A, B, and C. Staphylococcal strain 494 (ATCC 23235) was selected as the prototype strain. The production of this enterotoxin alone and together with enterotoxin A by strains of food-poisoning origin indicates that its role in food poisoning is second in frequency only to that of enterotoxin A. The incidence of production of enterotoxins A, B, C, and D, and of unidentified cat emetic substances by strains from several source categories, is presented.