合成多肽P72对葡萄球菌肠毒素超抗原抑制效应的研究

目的 观察合成多肽P72对葡萄球菌肠毒素超抗原(SEs)促人外周血单个核细胞(PBMC)增殖活性的抑制作用.方法 3H-TdR掺入法检测人 PBMC的增殖;ELISA检测IL-2、IFN-γ、TNF-β分泌情况.结果 P72对 SEA、SEB、SEC的促PBMC增殖及分泌IL-2、IFN-γ、TNF-β具有抑制作用.结论 细胞试验显示合成多肽P72对超抗原SEA、SEB、SEC的活性具有抑制作用.     

重组葡萄球菌B型肠毒素超抗原的制备及抗肿瘤活性分析

目的 采用基因工程技术制备葡萄球菌B型肠毒素（SEB），并对其体内外抗肿瘤活性进行分析。方法 对SEB在大肠杆菌中进行高效表达和分离纯化，并对其超抗原活性和免疫学活性与天然SEB进行比较研究，观察重组SEB对人肝癌细胞的体外抑制作用，以及对小鼠肝癌和肉瘤的体内抑瘤效果。结果 SEB获得高效表达，并一步将其纯化至均质，与天然毒素相比较，重组SEB的免疫学活与超抗原活性基本相似，首次证明，即使SEB的浓度为10ng/L，仍然对人肝癌细胞有显著地抑制作用，其对小鼠体内的肝癌和肉瘤有一定的抑制作用。结论 重组SEB在体内外均有一定肿瘤抑制作用。是潜在的肿瘤免疫治疗药物。     

超抗原金黄色葡萄球菌肠毒素基因B的原核表达

目的构建重组pET-30a-SEB原核表达载体,转化感受态大肠杆菌BL-21(DE3),诱导表达超抗原金黄色葡萄球菌肠毒素B(staphylococcalenterotoxinB,SEB)。方法利用PCR技术从产SEB的金黄色葡萄球菌标准菌株CMCC-26075基因组DNA中克隆SEB全长序列,将其克隆到pGEM-TEasy载体中并进行测序。构建pET-30a-SEB原核表达质粒,转化感受态大肠杆菌BL21(DE3),异丙基硫代β-D半乳糖苷(isopropyl-beta-D-thiogalactopyranoside,IPTG)诱导表达,经镍离子螯合亲和层析纯化后免疫鉴定。结果PCR获得超抗原SEB基因片段,与克隆载体连接后经测序与文献报道的基本一致;成功构建了pET-30a-SEB原核表达质粒且成功诱导表达出相对分子质量(Mr)约31×103的蛋白。结论成功克隆了seb基因序列,并进行了原核表达和鉴定,获得了SEB蛋白,为后续对超抗原SEB的进一步研究奠定了基础。     

葡萄球菌肠毒素B突变体基因表达和功能的初步研究

目的 应用PCR致突变基因克隆技术,获得抗原性保留,但超原毒性明显下降的葡萄球菌毒素B（SEB）突变体,用作新型淋巴细胞激活分子和超抗原疫苗的研究。方法 用PCR和PCR致变技术,从SEB标准株扩增SEB（SEB－N）和SEB突变基因（SEB－M）,分别与原核表达质粒pTrc99A重组,转化大肠杆菌JM109,经筛选获得重组质粒pTrcNb和pTrcMb,用双脱氧链终止法作序列分析。表达的SEB－N和SEB－M蛋白,经双向免疫扩散试验作抗的性鉴定后,刺激小鼠脾细胞并由ELISA法检测培养上清中IL－2的水平。结果 SEB－N150位苏氨酸（密码子ACT）非定向突变为丙氨酸（密码GCT）,SEB－M23位天冬酰胺（密码子AAT）定向突变为丝氨酸（密码子AGT）。两种突变基因表达的蛋白均与抗SEB形成明显沉淀线,与天然SEB刺激小鼠脾细胞产生IL－2水平相比,SEB-N格SEB－M突变体蛋白分别下降12．5倍和40倍。结论 获得了能表达SEB－N和SEB－M突变体蛋白的2株工程菌。表达的突变体蛋白具有良好的免疫反应性,但刺激小鼠脾细胞产生白细胞介素2的超抗原生物学活性明显下降。     

葡萄球菌D型肠毒素超抗原TCR Vβ结合位点的研究

目的：研究金黄色葡萄球菌D型肠毒素(SED)超抗原与TCRVβ结合的关键位点.方法：利用定点突变技术，构建了6种SED的突变体.用^3H-TdR掺入法检测这些突变体促进T细胞增殖的活性。对促增殖活性降低的突变体，进一步用流式细胞仪检测他们与MHC-Ⅱ类分子结合的活性和其TCRVβ特异性。结果：发现N23位氨基酸是SED与人TCRVβ5结合的关键位点。H26位氨基酸可能是SED与人的其他TCRVβ除TCRVβ5、TCRVβ8和TCRVβ12．1)结合的关键位点，值得进一步研究。结论：本结果丰富了对超抗原免疫识别的认识，为葡萄球菌超抗原相关疾病的防治，以及基于超抗原设计新的抗肿瘤免疫制剂提供了理论依据.     

葡萄球菌肠毒素超抗原广谱抑制性多肽的设计及空间结构研究

目的:以SEs氨基酸高度保守序列作靶序列设计抑制性多肽,研究筛选出的多肽P72的空间结构。方法:用生物信息学软件"Vector NTI 10.3,Insight II 2000,Discovery Studio 1.7"等分析及预测多肽P72的空间结构。结果:P72在SEA、SEB和SEC的同源序列在空间结构具有高度的相似性,P72远离SEB的TCR和MHCⅡ结合位。结论:P72可能不是与MHCⅡ类分子及TCR结合而产生的抑制作用,其具体的抑制机制有待深入研究。     

超抗原-SE抗肿瘤作用的研究进展

葡萄球菌肠毒素(Staphylococcal entero-toxin，SE)是一种外源性超抗原，仅需微量即能高效地刺激T细胞增殖，促使其产生大量的细胞因子及细胞毒作用，提高机体总体细胞免疫水平，达到抗肿瘤的能力。体外细胞水平和动物体内的研究结果表明：SE能够产生强大的杀伤和抑制肿瘤细胞效应，是一种很有前途的新型肿瘤免疫治疗剂。目前已开始应用于肿瘤病人的治疗及对肿瘤病人在进行放化疗时保护机体白细胞降低以及防止其他毒副反应的发生。     

原核表达的葡萄球菌D型肠毒素超抗原的纯化及其促淋巴细胞增殖作用

目的　纯化原核表达的葡萄球菌D型肠毒素 (StaphylococcalenterotoxinD ,SED) ,检测其促淋巴细胞增殖活性 ,为SED免疫识别研究奠定基础。方法　用Ni2 + NTA金属螯合亲和层析法纯化 6×His SED融合蛋白 ,SDS PAGE和毛细管电泳检测蛋白纯度 ,BCA法进行定量 ,以3H TdR掺入法检测纯化的SED对人和小鼠淋巴细胞的促增殖活性。结果　获得可满足后续功能实验纯度较高的蛋白质 ;SED对人淋巴细胞的促增殖作用呈质量浓度依赖关系 ,0 .1μg mL时 ,细胞增殖最明显 ;淋巴细胞的增殖与IL 2分泌水平呈正相关。结论　原核表达的SED具有促人和小鼠淋巴细胞增殖活性     

超抗原葡萄球菌肠毒素A拮抗伊马替尼抑制T细胞活化作用的研究

 目的：探讨葡萄球菌肠毒素A(staphylococcal enterotoxin A, SEA)对甲磺酸伊马替尼（imatinib mesylate,IM）抑制T淋巴细胞活化的影响。方法：2 mg／L SEA 和50 nmol/L IM联合作用于Jurkat细胞24 h后,用实时荧光定量PCR技术检测CD3ε和ζ链mRNA表达水平变化;Western blotting技术检测CD3ε和ζ链蛋白水平变化。结果：单纯IM组CD3ε和ζ链mRNA及蛋白表达均表现下调;SEA与IM联合用药组可以逆转IM下调CD3ε和ζ链mRNA及蛋白表达水平作用。SEA拮抗IM抑制作用中,对CD3ε链mRNA表达上调的作用明显大于CD3ζ链。结论:SEA能拮抗IM对T细胞CD3ε和ζ链表达的抑制作用。     

超抗原-SE抗肿瘤作用的研究进展

葡萄球菌肠毒素(Staphylococcal entero-toxin,SE)是一种外源性超抗原,仅需微量即能高效地刺激T细胞增殖,促使其产生大量的细胞因子及细胞毒作用,提高机体总体细胞免疫水平,达到抗肿瘤的能力.体外细胞水平和动物体内的研究结果表明SE能够产生强大的杀伤和抑制肿瘤细胞效应,是一种很有前途的新型肿瘤免疫治疗剂.目前已开始应用于肿瘤病人的治疗及对肿瘤病人在进行放化疗时保护机体白细胞降低以及防止其他毒副反应的发生.     

Surface anchorage of superantigen SEA promotes induction of specific antitumor immune response by tumor-derived exosomes

Tumor-derived exosomes have been regarded as a new kind of cancer vaccine; however, their therapeutic efficacy needs to be further improved. Superantigen staphylococcal enterotoxin A (SEA)-coated tumor cells have been shown to potently induce tumor-specific T cell response. To increase efficacy of tumor-derived exosomes to induce antitumor immune response, we modified the exosomes by protein transfer of SEA tailed with a highly hydrophobic transmembrane domain (SEA-TM) and designated those SEA-TM-anchored exosomes as Exo/SEA-TM. We found the exosomes secreted from murine lymphoma E.G7-OVA cell line were round vesicles with the sizes of 40-100 nm limited by a bilayer membrane. Interestingly, the inner structure of the exosomes were visible under the transmission electron microscope; those "honeycomb-like" inner structure has not been described by other labs. Immunization with Exo/SEA-TM inhibited tumor growth and prolonged survival of the mice challenged with parental tumor cells more significantly than with exosomes (Exo) and even more than with the mixture of exosomes and SEA-TM. The results of mixed lymphocyte-tumor reaction (MLTR) showed that the increased IL-2, IFN-gamma secretion, and specific cytotoxic T lymphocyte (CTL) could be effectively induced from the splenic lymphocytes of the mice immunized with Exo/SEA-TM. In vivo depletion experiments showed that CD8(+) T cells are the main effector cells, and both CD4(+) T cells and NK cells are also involved in the antitumor effect of Exo/SEA-TM immunization. Therefore, tumor-derived exosomes surface anchored with SEA-TM can efficiently induce tumor-specific CTL thereby resulting in more potent inhibition of tumor growth. Our data provide an efficient and novel approach to tumor immunotherapy by protein modification of tumor-derived exosomes.     

Antibodies to highly conserved peptide sequence of staphylococcal and streptococcal superantigens in Kawasaki disease

Superantigen-mediated disease such as toxic shock syndrome is seen in patients who have a weak antibody response to the antigen toxic shock syndrome toxin 1 (TSST-1). We hypothesized that there may be deficiency in antibody production to staphylococcal and streptococcal toxins in Kawasaki disease (KD) children. A peptide was constructed from the homologous portion of the staphylococcal enterotoxins (SE) and streptococcal pyrogenic enterotoxins (SPE), and antibodies to the peptide were made. The anti-peptide antibody immunoblotted several of the SE toxins and SPE toxins. Presence of the peptide antibodies was investigated via ELISA in the sera of acute KD (n = 30), convalescent KD (n = 12), control adults (n = 10), and children (n = 19). The mean anti-peptide antibodies were indistinguishable between control children and KD before treatment with immunoglobulin (P = 0.7) but rose significantly after therapy (P < 0.01). The adults had significantly higher antibodies than the KD, both acute and late (P < 0.0001) and the control children (P < 0.0001). Thus, KD patients do not have a defective serological response against toxins such as SPE/SE/TSST-1. Normal children have significantly lower antitoxin antibody levels to the toxins compared to the adults.     

Anti-idiotypic antibody D12 and superantigen SPA both interact with human VH3-encoded antibodies on the external face of the heavy chain involving FR1, CDR2 and FR3

The mouse monoclonal antibody (mAb) D12 specifically binds in the variable region (idiotype) of human V_H3 encoded antibodies. We used mutational analysis to determine the subregions of a V_H3 encoded antibody which effect the interaction with mAb D12. Recombinant antibodies composed of mutant heavy chains were produced using the baculovirus expression system. The results of this topographical study indicate that the combined conformations of FR1, CDR2 and FR3 are critical for mAb D12 binding. MAb D12 binding was not effected either by the heavy chain CDR3 sequence nor by the light chain. We previously demonstrated that structures within the same three subregions are required for the B cell superantigen Staphylococcal protein A (SPA) binding to V_H3 encoded antibodies. Thus, some anti-idiotypic antibodies can interact with antibodies in a similar fashion to superantigens.     

中和抗体识别短肽对IFN-α生物学活性的影响

目的研究合成肽ＷＬＤＰＲＨ的免疫反应性和对干扰素（ＩＦＮ）诱导的生物学活性的影响。方法根据我们早先的研究，已用中和抗体从噬菌体展示６肽库中筛选出了两个与ＩＦＮ－α有同源性的肽片段，将其中之一进行了人工合成（ＷＬＤＰＲＨ），并进行了同源性比较分析，体外竞争酶联免疫吸附实验（ＥＬＩＳＡ），体外ＩＦＮ诱导抗病毒和抗增殖分析。结果计算机分析表明，人工合成肽ＷＬＤＰＲＨ与推理的Ｉ型ＩＦＮ受体结合区ＬｏｏｐＡＢ（２９－３５位）有相当的同源性。体外竞争ＥＬＩＳＡ实验表明，合成肽在２５μｇ／ｍｌ浓度时能特异性抑制ＩＦＮ－α与中和抗体４Ｃ１结合。体外抗病毒结果显示，干扰素在亚保护剂量时（２０～７０ｐｇ／ｍｌ）时，合成肽能提高ＩＦＮ－α作用细胞后的抗病毒活性，并且合成肽ＷＬＤＰＲＨ在１．４μｇ／ｍｌ时，ＩＦＮ－α抗病毒保护率从４０％提高到８６％，为最高保护率的最低剂量，但合成肽为１．４μｇ／ｍｌ，ＩＦＮ浓度为５～５５ｎｇ／ｍｌ时，对ＩＦＮ－α诱导的抗增殖作用不明显。结论用中和抗体筛选的短肽ＷＬＤＰＲＨ能影响ＩＦＮ分子作用模式，并且有可能模拟ＩＦＮ分子ＬｏｏｐＡＢ的结构和功能，这为免疫治疗及ＩＦＮ的小分子模拟设计奠定基础。