野生型金黄色葡萄球菌肠毒素C2在E.coli中的表达和纯化研究

目的克隆金黄色葡萄球菌肠毒素C2(SEC2)基因在E.coli重组表达,纯化重组SEC2(rSEC2)并进行生物学活性分析。方法PCR获得正确编码SEC2的基因片断,构建表达质粒pET-28a-sec2在E.coliBL21中表达。利用离子交换和分子筛色谱纯化rSEC2,四甲基偶氮唑盐(MTT)法对rSEC2和天然SEC2(nSEC2)生物学活性进行分析和比较。结果可溶性rSEC2占菌体总蛋白质的40%,两步纯化后蛋白质纯度约达95%,rSEC2和nSEC2均具有显著的超抗原活性。结论成功获得与nSEC2有着类似活性的高纯度rSEC2,为进一步研究该蛋白质的抗肿瘤机理奠定物质基础。     

重组金黄色葡萄球菌肠毒素A突变体D227A蛋白的表达、纯化和鉴定

建立从金黄色葡萄球菌肠毒素A突变体D227A原核表达系统pET32a-SEAD227A-E.coliBL21DE3中,表达、纯化和鉴定重组金黄色葡萄球菌肠毒素A突变体D227A(rSEA-D227A)蛋白的方法。采用不同浓度的IPTG(0.1、0.5和1.0mmol.L-1)诱导原核表达系统表达rSEA-D227A,10%SDS-PAGE检测表达产量,Ni-NTA亲和层析法纯化rSEA-D227A,采用HPLC检测其纯度,Western-blotting进行免疫学鉴定。从已构建的金黄色葡萄球菌肠毒素A突变体D227A原核表达系统中成功表达和纯化了rSEA-D227A蛋白,产量约占细菌总蛋白的38.2%,纯度为88.7%。为后续进一步研究该蛋白的生物活性奠定基础。     

超抗原金黄色葡萄球菌肠毒素A对人外质血T细胞活化作用的观察

目的 观察超抗原对外周血T细胞的活化作用。方法 用不同浓度的金黄色葡萄球菌肠毒素A(SEA)刺激人外周血T细胞，观察细胞增殖、IL-2的产生及细胞表型情况。结果 当SEA为lμg／ml时，T细胞表现了最大的增殖活性；SEA对CD4 、CD8 T细胞有同等活化作用；IL-2在培养后的48～72小时产生的量最大。结论 SEA对外周血T细胞有明显的促增殖作用。     

重组表达葡萄球菌肠毒素B及其突变体M177的免疫原性

目的 研究重组表达的葡萄球菌肠毒素B（rSEB）及其突变体M177的免疫学活性,探讨rSEB及其突变体M177作为超抗原疫苗的可行性。方法 通过小鼠急性毒性实验、抗原抗体结合实验、主动免疫和被动免疫保护实验来研究rSEB及其突变体M177的免疫学活性。结果 rSEB对BALB/c小鼠的致死作用与野生型SEB的致死作用基本相同,而突变体M177对小鼠的致死作用下降了21倍,M177具有与rSEB和SEB相同的免疫活性。结论 突变体M177有望发展为SEB超抗原疫苗。     

食源性金黄色葡萄球菌分离株肠毒素及耐药性分析

目的 ：对92株食源性金黄色葡萄球菌分离株进行肠毒素及耐药性检测,以了解其致病性和耐药性。方法 ：采用酶联免疫吸附试验测定食源性金黄色葡萄球菌分离株肠毒素携带情况,利用VITEK^?2 Compact全自动微生物分析系统对菌株进行药敏试验,采用聚合酶链式反应对菌株mecA耐药基因进行检测。结果 ：92株食源性金黄色葡萄球菌分离株肠毒素检出率为5.43%(5株),对青霉素、四环素、红霉素、克林霉素4种抗生素的耐药率较高,分别为100%、 30.43%、19.57%、14.13%,检出3株耐甲氧西林金黄色葡萄球菌(MRSA)。结论 ：食源性金黄色葡萄球菌分离株产生的肠毒素不容忽视,多重耐药及MRSA的检出提示耐药形势比较严峻,加强对食源性金黄色葡萄球菌致病性和耐药性的监测,对降低食品安全风险有重要意义。     

重组金葡菌肠毒素O的克隆表达及生物学活性分析

克隆金葡菌肠毒素O(SEO)的全长基因，实现其可溶性表达,并对纯化的表达产物进行生物学活性分析。从金葡菌FRI 100菌株基因组中得到SEO基因，克隆至谷胱甘肽S-转移酶(GST)融合表达载体pGEX-4T-1，转化大肠杆菌，获高效表达。融合蛋白GST-SEO经Glutathione Sepharose 4B亲和纯化和凝血酶消化获重组SEO(rSEO)后，MTT法检测脾淋巴细胞的增殖作用，分析纯化后rSEO的生物学活性。测序结果表明，得到正确的肠毒素SEO基因序列，并获高效表达的融合蛋白；MTT结果表明，rSEO具有与SEC相当的显著的促淋巴细胞增殖以及抑制肿瘤细胞生长的能力。本研究成功克隆、表达、纯化了具有抗肿瘤生物学活性的rSEO蛋白，为进一步研究该蛋白的抗肿瘤机制奠定了基础，并有望成为一种新的超抗原制剂用于肿瘤的临床治疗。     

金黄色葡萄球菌食品和患者分离株肠毒素耐药性比较

目的：本文对金黄色葡萄球菌食品分离株和患者分离株的肠毒素进行分型和耐药性检测,比较二者的差异性。方法：用国标方法分离金黄色葡萄球菌,测定分离菌株对于抗生素的耐药性。结果：患者分离株中金葡菌类型较多,占82%,同时含有2种及以上肠毒素基因的菌占73%。食品中检测到肠毒素A~E类型的比率为65%,同时携带2种及以上毒素类型的占19%。A型和B型菌株数最多,各占25%。结论：金葡菌患者分离株和食品分离株的肠毒素类型不同,其耐药性也有较大差异。     

超抗原SEC抗胶质瘤的研究

目的　观察超抗原SEC活化淋巴细胞对胶质瘤的杀伤作用。方法　采用APAAP法对SEC活化的淋巴细胞进行亚群分析 ;按MTT法测定经SEC活化的淋巴细胞对胶质瘤的体外杀伤作用 ;通过建立荷瘤动物模型及人外周血淋巴细胞———SCID小鼠免疫嵌合模型 ,观察肿瘤生长曲线。结果　经SEC活化的淋巴细胞主要为CD4 + 细胞 ;经SEC活化的淋巴细胞对 8株胶质瘤细胞均产生强大的杀伤作用 ,其中以 1× 10 4U·L-1剂量组作用最明显。生长曲线显示 :A组、C组和B组对胶质瘤生长抑制率分别为 5 8 5 %、4 6 2 %及 36 3%。结论　经超抗原SEC活化的淋巴细胞对胶质瘤有杀伤作用。     

体温与抵御B型葡萄球菌肠毒素攻击能力的关系及在确定疫苗剂量程序中的应用

B型葡萄球菌肠毒素(SEB)是一种被称为细菌超抗原(BSAg)的外毒素，能引起发热症状，严重时可危及生命。作者通过观察恒河猴在使用重组(r)SEB疫苗前后及遭受SEB攻击前后的体温变化来检测疫苗的安全性和有效性，同时明确体温能否作为预测有效疫苗及确定疫苗剂量程序的一个相关生物学标志。     

金黄色葡萄球菌肠毒素A基因全序列的克隆、鉴定与扩增

目的 克隆SEA基因作为后续基因治疗的目的基因。方法 以金黄色葡萄球茵基因组DNA(ATCC-13565)为模板,以金黄色葡萄球菌肠毒素A(SEA)基因特异性引物为介导，采用普通PCR和降落PCR方法分别克隆SEA基因全序列，克隆入T载体后进行DNA测序。结果 2％琼脂糖凝脂电泳和DNA测序证实该基因克隆成功。结论 通过PCR方法，可以获得后续实验所需的SEA基因。     

Assessment of the Functional Regions of the Superantigen Staphylococcal Enterotoxin B

The functional activity of superantigens is based on capacity of these microbial proteins to bind to both the β-chain of the T cell receptor (TcR) and the major histocompatibility complex (MHC) class II dimer. We have previously shown that a subset of the bacterial superantigens also binds to a membrane protein, designated p85, which is expressed by renal epithelial cells. This binding activity is a property of SEB, SEC1, 2 and 3, but not SEA, SED, SEE or TSST. The crystal structure of the tri-molecular complex of the superantigen staphylococcal enterotoxin B (SEB) with both the TcR and class II has previously been reported. However, the relative contributions of regions of the superantigen to the overall functional activity of this superantigen remain undefined. In an effort to better define the molecular basis for the interaction of SEB with the TcR β-chain, we report studies here which show the comparative contributions of amino- and carboxy-terminal regions in the superantigen activity of SEB. Recombinant fusion proteins composed of bacterial maltose-binding protein linked to either full-length or truncated toxins in which the 81 N-terminal, or 19 or 34 C-terminal amino acids were deleted, were generated for these studies. This approach provides a determination of the relative strength of the functional activity of the various regions of the superantigen protein.     

Sulfasalazine Attenuates Staphylococcal Enterotoxin B-Induced Immune Responses

Staphylococcal enterotoxin B (SEB) and related exotoxins are important virulence factors produced by Staphylococcus aureus as they cause human diseases such as food poisoning and toxic shock. These toxins bind directly to cells of the immune system resulting in hyperactivation of both T lymphocytes and monocytes/macrophages. The excessive release of proinflammatory cytokines from these cells mediates the toxic effects of SEB. This study examined the inhibitory activities of an anti-inflammatory drug, sulfasalazine, on SEB-stimulated human peripheral blood mononuclear cells (PBMC). Sulfasalazine dose-dependently inhibited tumor necrosis factor α, interleukin 1 (IL-1) β, IL-2, IL-6, interferon γ (IFNγ), and various chemotactic cytokines from SEB-stimulated human PBMC. Sulfasalazine also potently blocked SEB-induced T cell proliferation and NFκB activation. These results suggest that sulfasalazine might be useful in mitigating the toxic effects of SEB by blocking SEB-induced host inflammatory cascade and signaling pathways.     

Intranasal Rapamycin Rescues Mice from Staphylococcal Enterotoxin B-Induced Shock

Staphylococcal enterotoxin B (SEB) and related exotoxins produced by Staphylococcus aureus are potent activators of the immune system and cause toxic shock in humans. Currently there is no effective treatment except for the use of intravenous immunoglobulins administered shortly after SEB exposure. Intranasal SEB induces long-lasting lung injury which requires prolonged drug treatment. We investigated the effects of rapamycin, an immunosuppressive drug used to prevent graft rejection, by intranasal administration in a lethal mouse model of SEB-induced shock. The results show that intranasal rapamycin alone delivered as late as 17 h after SEB protected 100% of mice from lethal shock. Additionally, rapamycin diminished the weight loss and temperature fluctuations elicited by SEB. Intranasal rapamycin attenuated lung MCP-1, IL-2, IL-6, and IFNγ by 70%, 30%, 64%, and 68% respectively. Furthermore, short courses (three doses) of rapamycin were sufficient to block SEB-induced shock. Intranasal rapamycin represents a novel use of an immunosuppressant targeting directly to site of toxin exposure, reducing dosages needed and allowing a wider therapeutic window.     

Uncoupling of T Cell Receptor Zeta Chain Function during the Induction of Anergy by the Superantigen, Staphylococcal Enterotoxin A

Staphylococcus aureus enterotoxins have immunomodulatory properties. In this study, we show that Staphylococcal enterotoxin A (SEA) induces a strong proliferative response in a murine T cell clone independent of MHC class II bearing cells. SEA stimulation also induces a state of hypo-responsiveness (anergy). We characterized the components of the T cell receptor (TCR) during induction of anergy by SEA. Most interestingly, TCR zeta chain phosphorylation was absent under SEA anergizing conditions, which suggests an uncoupling of zeta chain function. We characterize here a model system for studying anergy in the absence of confounding costimulatory signals.     

Staphylococcal Enterotoxin H Induced Apoptosis of Bovine Mammary Epithelial Cells in Vitro

Staphylococcal enterotoxins (SEs) are powerful superantigenic toxins produced by Staphylococcus aureus (S. aureus). They can cause food poisoning and toxic shock. However, their impact on bovine mammary epithelial cells (bMECs) is still unknown. In this study, the distribution of SE genes was evaluated in 116 S. aureus isolates from bovine mastitis, and the most prevalent genes were seh (36.2%), followed by sei (12.1%), seg (11.2%), ser (4.3%), sec (3.4%), sea (2.6%) and sed (1.7%). To better understand the effect of staphylococcal enterotoxin H (SEH) on bMECs, the seh gene was cloned and inserted into the prokaryotic expression vector, pET28a, and transformed into Escherichia coli BL21 (DE3). The recombinant staphylococcal enterotoxin H (rSEH) was expressed and purified as soluble protein. Bioactivity analysis showed that rSEH possessed the activity of stimulating lymphocytes proliferation. The XTT assay showed that 100 μg/mL of rSEH produced the cytotoxic effect on bMECs, and fluorescence microscopy and flow cytometry analysis revealed that a certain dose of rSEH is effective at inducing bMECs apoptosis in vitro. This indicates that SEs can directly lead to cellular apoptosis of bMECs in bovine mastitis associated with S. aureus.     

超抗原高聚金葡素对恶性体腔液的免疫学治疗作用

目的 :了解超抗原高聚金葡素对恶性体腔积液的治疗效果并探讨其免疫作用机理。方法 :对 36例恶性体腔积液患者体腔内注射高聚金葡素治疗 ,采用流式细胞仪监测治疗前后患者外周血及体腔积液中T淋巴细胞亚群、NK细胞及B细胞活性的变化。结果 :体腔内注射高聚金葡素 ,体腔积液中CD+ 4 、CD16及CD+ 4 /CD+ 8比值进行性增高 ,CD+ 3 、CD+ 8、CD19治疗前及治疗后各组均无显著性差异。患者外周血中CD+ 3 、CD+ 4 、CD+ 4 /CD+ 8比值、CD16均明显低于正常对照 ,CD+ 8与正常对照比较无显著差异 ,CD19则明显高于正常对照。高聚金葡素体腔内注射治疗后 ,外周血中CD+ 3 、CD+ 4 、CD+ 4 /CD+ 8比值、CD16及CD19均明显增高 ,但CD+ 8无明显改变。高聚金葡素对恶性体腔积液治疗有效率达到 78 6 %。结论 :恶性体腔积液患者细胞免疫功能处于抑制状态。高聚金葡素对恶性体腔积液具有良好的治疗作用 ,其作用是通过刺激CD+ 4 细胞和NK细胞增殖分化 ,对肿瘤细胞产生非特异性杀伤效应而实现的。体腔内注射高聚金葡素 ,同样能增强机体的免疫功能     

Human Leukemia T-Cell Lines as Alternatives to Animal Use for Detecting Biologically Active Staphylococcal Enterotoxin Type B

Staphylococcal enterotoxin type B (SEB) is associated with food poisoning. Current methods for the detection of biologically active SEB rely upon its ability to cause emesis when administered to live kittens or monkeys. This technique suffers from poor reproducibility and low sensitivity and is ethically disfavored over concerns for the welfare of laboratory animals. The data presented here show the first successful implementation of an alternative method to live animal testing that utilizes SEB super-antigenic activity to induce cytokine production for specific novel cell-based assays for quantifiable detection of active SEB. Rather than using or sacrificing live animals, we found that SEB can bind to the major histocompatibility complex (MHC) class II molecules on Raji B-cells. We presented this SEB–MHC class II complex to specific Vβ5.3 regions of the human T-cell line HPB-ALL, which led to a dose-dependent secretion of IL-2 that is capable of being quantified and can further detect 10 pg/mL of SEB. This new assay is 100,000 times more sensitive than the ex vivo murine splenocyte method that achieved a detection limit of 1 µg/mL. The data presented here also demonstrate that SEB induced proliferation in a dose-dependent manner for cells obtained by three different selection methods: by splenocyte cells containing 22% of CD4⁺ T-cells, by CD4⁺ T-cells enriched to >90% purity by negative selection methods, and by CD4⁺ T-cells enriched to >95% purity by positive selection methods. The highly enriched and positively isolated CD4⁺ T-cells with the lowest concentration of antigen-presenting cells (APC) (below 5%) provided higher cell proliferation than the splenocyte cells containing the highest concentration of APC cells.     

Superantigen staphylococcal enterotoxin C1 mutant can reduce paraquat pulmonary fibrosis

Abstract

A network of inflammation factors is related to pulmonary fibrosis induced by paraquat (PQ) poisoning. At high doses, the superantigen staphylococcal enterotoxin C1 (SEC1) can induce immunological unresponsiveness and inhibit release of inflammation factors. In this study, site-directed mutagenesis was performed at the H118 and H122 amino acid residues of SEC1 to reduce SEC1 toxicity. The SEC1 mutant showed significantly decreased pyrogenic toxicity, but retained clonal anergy at high dosages in vitro. Pretreatment with the SEC1 mutant prior to PQ poisoning in mice reduced symptom duration and severity, prolonged survival time, and decreased the splenocyte response to ConA induction. The SEC1 mutant also down-regulated several important cytokines related to fibrosis in the plasma after PQ poisoning. SEC1 decreased the expression of genes related to pulmonary fibrosis, including α-SMA, COL1a1, COL3 and TGF-β1, in PQ poisoned mice. Histomorphological observation indicated alleviation of pathological changes in the lungs after SEC1 pretreatment compared to mice in the PQ group. In conclusion, the SEC1 mutant reduced pulmonary interstitial fibrosis induced by PQ poisoning.     

Expression and bioactivity analysis of staphylococcal enterotoxin G and staphylococcal enterotoxin I

Context: The filtrate of Staphylococcus aureus culture, named staphylococcal enterotoxin C injection, has been used for 10 years in China. SEC2 has been claimed to be the only staphylococcal enterotoxins (SEs) without certifiable evidence.

Objectives: To present an efficient procedure for the expression and purification of recombinant proteins SEG and SEI, from S. aureus.

Materials and methods: In present work, we extracted total DNA from S. aureus (FRI 1230) and the recombinant proteins of SEG and SEI were then cloned, expressed and purified using E. coli. Splenic lymphocytes were used as effector cells and K562 and B16 cells were used as target cells to evaluate the inhibitory and stimulatory abilities of purified rSEG and rSEI on in vitro proliferation.

Results: The size of amplified products of SEG and SEI genes were found to be about 400 and 467?bp, respectively. pGEX-SEG and pGEX-SEI were constructed successfully. SEG and SEI were demonstrated to be active stimulators of T-cell proliferation; moreover, they inhibited the proliferation of K562 cells and B16 cells.

Discussion: The current findings suggest that SEC2 might not be the only active component of staphylococcal enterotoxin C injection and may involve other essential proteins like SEG and SEI in its clinical efficacy.

Conclusion: This efficient procedure for the expression and purification of SEG and SEI and may be useful for mass production of therapeutically important proteins. In the future, proteins acting as active stimulators of T-cell proliferation may help in developing effective cancer therapy.     

Cure and Curse: E. coli Heat-Stable Enterotoxin and Its Receptor Guanylyl Cyclase C

Enterotoxigenic Escherichia coli (ETEC) associated diarrhea is responsible for roughly half a million deaths per year, the majority taking place in developing countries. The main agent responsible for these diseases is the bacterial heat-stable enterotoxin STa. STa is secreted by ETEC and after secretion binds to the intestinal receptor guanylyl cyclase C (GC-C), thus triggering a signaling cascade that eventually leads to the release of electrolytes and water in the intestine. Additionally, GC-C is a specific marker for colorectal carcinoma and STa is suggested to have an inhibitory effect on intestinal carcinogenesis. To understand the conformational events involved in ligand binding to GC-C and to devise therapeutic strategies to treat both diarrheal diseases and colorectal cancer, it is paramount to obtain structural information on the receptor ligand system. Here we summarize the currently available structural data and report on physiological consequences of STa binding to GC-C in intestinal epithelia and colorectal carcinoma cells.