金黄葡萄球菌中毒休克综合征毒素1的研究

目的 应用PCR技术,检测金黄葡萄球菌的mecA基因和染色体上编码中毒休克综合征毒素1(TSST-1)的tst基因,了解tst基因的携带情况.方法 用PCR法对我院2006年8月-2007年5月临床分离的84株金黄葡萄球菌mecA基因和tst基因进行体外扩增,建立快速、特异、灵敏的检测产TSST-1耐甲氧西林金黄葡萄球菌(MRSA)的方法.结果 成功的对金黄葡萄球菌mecA基因和tst基因进行了检测,并进行基因测序.在我院84株受检金黄葡萄球菌中,41株金黄葡萄球菌的mecA基因扩增呈阳性,阳性株占48.81%.16株tst基因阳性,阳性株占19.05%.10株金黄葡萄球菌同时扩增出mecA基因和tst基因,阳性株占24.39%(10/41).结论 tst基因阳性株在临床分离的耐甲氧西林金黄葡萄球菌中占有较高的比例,应予以足够重视.     

热休克蛋白90α(HSP90α)在大肠杆菌中的表达

目的 :探讨热休克蛋白 90α的生物学功能 ,获得高质量及充足的HSP90α蛋白。方法 :采用PCR的方法扩增HSP90αcDNA5’端 42 4bp片段 ,在起始密码子位置制造一个NcoI酶切位点 ,将PCR产物克隆到中间载体 ,再通过合适的酶切位点将其余片段连接到此载体 ,形成一个带有NcoI突变位点的HSP90αcDNA全长克隆。最终将HSP90α全部编码cDNA插入到pET 16b表达载体中。结果 :经诱导表达 ,SDS PAGE凝胶电泳分离显示在分子量大约 83kD处有一诱导蛋白带。WesternBlot证明表达产物与抗HSP90α抗血清有特异的免疫反应。结论 :构建成了T7启动子控制下的HSP90α表达质粒。     

金黄色葡萄球菌 eap 基因的克隆与表达

 目的 构建携带 eap 基因的原核表达载体，诱导表达具有活性的重组 EAP 融合蛋白。 方法 PCR 法扩增金黄色葡萄球菌基因组 DNA，回收、 纯化的扩增产物与 pMD18-T 载体相连接得重组质粒 pMD18-T-EAP，转化 E.coli BL21（DE3）感受态细胞，酶切鉴定；未酶切组作为对照组重组质粒 pMD18-T-EAP 和 pET28a（+）表达载体分别用 Nde I 和 Xho I 限制性内切酶双酶切、连接，转化 E.coli BL21（DE3）感受态细胞，酶切鉴定；空载体作为对照组。用不同浓度（终浓度 1、2、4、8 mmol/L）和不同诱导时间（1、2、3、4、5、6 h）的异丙基-β-D-硫代半乳糖苷（IPTG）对阳性重组菌进行表达优化，分别取 E.coli上清液和沉淀做电泳分析。应用 MagneHis^TM 蛋白纯化系统纯化重组 EAP 融合蛋白，并通过薄层扫描测定蛋白质的浓度。 结果 所获 eap 基因与 GeneBank 的基因序列同源性 > 99%；氨基酸同源性达 100%。重组质粒经 IPTG 诱导，阳性重组菌转化子均有表达；当吸光度（A ）值等于 0.6 ~ 0.8 时，相对分子质量约 70 000 处出现目的蛋白条带。破碎的重组菌 pET28a-EAP上清液中目的蛋白条带较清楚，沉淀中几乎看不到。终浓度 1 mmol/L 为最佳蛋白表达工作浓度。IPTG 诱导 1 h 重组 EAP 融合蛋白有一定量的表达，随着时间的延长，表达量增加不明显，3 h 时的表达量达最高，之后，蛋白表达量变化不明显。表达的重组 EAP 融合蛋白含量占全菌体蛋白的 29.6%。 结论 成功地克隆和表达了金黄色葡萄球菌重组 EAP 融合蛋白，为进一步研究以 EAP 蛋白作为免疫原预防和治疗由金黄色葡萄球菌引起的疾病奠定基础。     

分泌抗金黄色葡萄球菌C1型肠毒素杂交瘤细胞系的建立及初步应用

应用常规方法建立了３株稳定分泌抗金黄色葡萄球菌Ｃ１型肠毒素（ＳＥＣ１）单克隆抗体（ＭｃＡｂ）的小鼠杂交瘤细胞系Ｂ３、Ｃ４和Ｇ８。其中Ｂ３和Ｃ４均为ＩｇＧ１（ｋ），Ｇ８为ＩｇＧ２ａ（ｋ）。Ｂ３和Ｇ８与ＳＥＡ，ＳＥＢ及ＳＥＤ均无交叉反应；Ｃ４虽与ＳＥＡ和ＳＥＤ无交叉反应，但与ＳＥＢ有交叉反应。间接ＥＬＩＳＡ测定小鼠腹水效价为１０＾－５～１０＾－８。应用识别不同表位的ＭｃＡｂ建立了双ＭｃＡｂ夹心ＥＬＩＳ     

金黄色葡萄球菌核酸酶在大肠杆菌中的表达及其活性分析

目的:构建金黄色葡萄球菌核酸酶(SN)基因的原核表达载体,研究其在大肠杆菌中的表达,并制备兔抗SN抗体。方法:从质粒pPLCSN中扩增SN基因片段,插入表达载体pLEX后转化大肠杆菌GI724,以色氨酸诱导表达。以表达的重组蛋白免疫日本大耳白兔制备抗SN抗体,用Westernblot分析兔抗SN抗体的特异性。结果:重组表达载体pLEXSN在大肠杆菌中获得表达,表达的可溶性蛋白占菌体蛋白总量的37%,具有良好的生物学活性。制备的兔抗SN抗体能够特异识别SN。结论:成功地在大肠杆菌中表达具有生物学活性的SN,并制备了兔抗SN抗体,为进一步探讨其作为抗病毒因子应用于病毒性疾病的治疗奠定了基础。     

金黄色葡萄球菌重组蛋白AtlM的体外抗菌效应初步探讨

目的通过基因工程技术获取金黄色葡萄球菌（ ATCC25923）自溶素片段AtlM重组蛋白（ rAtlM）,初步探讨其体外抗菌效应。方法根据GenBank 中金黄色葡萄球菌atlM基因序列（ AC：D17366）设计合成特异引物,采用PCR技术扩增相应的序列并构建重组表达质粒pET-32а（＋）/atlM,经由IPTG诱导后通过等电点洗脱技术获取纯化的rAtlM;采用微量肉汤稀释法测定rAtlM对ATCC25923及其苯唑西林诱导耐药株的最低抑菌浓度（ minimal inhibitory concentration ,MIC）;体外试验测定rAtlM对ATCC25923菌株及其耐药株的抗菌活性。结果成功构建了原核表达载体pET-32а（＋）/atlM,表达的重组蛋白AtlM对ATCC25923标准株和耐药株的MIC分别为8μg/ml和64μg/ml。体外抑菌试验显示rAtlM作用于ATCC25923标准株和耐药株1 h后即对细菌生长具有抑制作用（ P值分别为0．004和0．026）,对标准株作用持续到5 h测定点（P＝0．012）,对苯唑西林耐药株作用持续到3 h测定点（P＝0．001）,5 h后与未加药物的对照组相比差异无统计学意义（P＝0．102）。结论50μg/ml的rAtlM对金黄色葡萄球菌（ ATCC25923）及苯唑西林耐药株初步显示出一定的抑菌作用,具有作为抗菌药物的可能性。     

金黄色葡萄球菌膜蛋白nEBPS原核表达及抗体制备

目的：构建金黄色葡萄球菌弹性纤维结合蛋白N端蛋白（N-terminal elastin binding proteins of S.aureus,nEBPS）的原核表达系统,制备其多克隆抗体。方法：设计引物,PCR方法扩增nEBPS基因（nebps）,分别构建克隆载体pMD19-T（＋）/nebps和原核表达载体pQE30（＋）/nebps,经PCR、双酶切和测序鉴定后,阳性表达载体转化大肠杆菌表达宿主菌M15[pREP4],经1mM异丙基硫代-β-D-半乳糖苷（IPTG）诱导表达,表达产物用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质印迹法（Western Bloting,WB）分析鉴定,经镍螯合亲和层析（Ni-NTAAgrose）纯化后再用SDS-PAGE和WB法鉴定。制备抗原,免疫新英格兰白兔,得到抗血清并对其进行鉴定。结果：pQE30（＋）/nebps重组表达载体构建成功,目的蛋白在构建的原核表达系统中较高表达,纯化后得到高纯度的nEBPS,免疫动物后得到理想的多克隆抗体。结论：通过基因重组技术,金黄色葡萄球菌膜蛋白nEBPS在构建的原核表达系统M15[pREP4]中得以成功表达,并获得了高纯度的目的蛋白及其多克隆抗体。     

金黄色葡萄球菌外毒素在脓毒症发病中的作用与机制探讨

诸多研究证实，细菌、毒素、病毒感染等在机体的脓毒性反应中均可起触发作用，其中革兰阴性（G^-）菌及其内毒素在脓毒症发病中的作用与机制已进行了广泛、深入的研究。然而，长期以来，人们对于革兰阳性（G^＋）菌及其外毒素的致病意义认识不足。1．金葡菌及其外毒素的分布、产毒率与临床意义：流行病学资料显示，G^＋菌感染中金葡菌发病率位居首位，是烧伤创面感染、急性肝功能衰竭等疾病的重要病原菌。我们调查了近8年间从创面分离病原菌的分布情况，其中金葡菌分离率从1995年的17．7％（居第3位）上升为1999年的29．3％（居第1位）、2003年的44．4％，并呈现进一步升高的趋势。细菌学研究表明，可溶性外毒素的产生是G^＋菌感染的重要标志之一，其中金葡菌肠毒素因其“超抗原”（superantigen）特性以及在中毒性休克综合征（TSS）、多器官功能障碍综合征（MODS）发病中的特殊意义而倍受关注。细菌学调查显示，从我院烧伤患者及医务人员手和鼻腔中分离的127株金葡菌中，产毒率高达91．6％，而且以同时产生多种类型毒素者居多[同时产肠毒素B、C（SEB、SEC）者最为常见]，其中90％以上为耐药菌株。     

金黄色葡萄球菌肠毒素A基因的克隆及原核表达

目的从分离培养的金黄色葡萄球菌中提取肠毒素A（SEA）基因,亚克隆入表达载体pET-28a（＋）,诱导融合蛋白的表达,为肠毒素A应用研究奠定基础。方法根据GenBank中SEA的序列,设计一对特异性引物,以金黄色葡萄球菌DNA为模板PCR扩增SEA,纯化DNA进行BamHⅠ、XholⅠ双酶切鉴定及测序鉴定,并与做相应酶切的pET-28a（＋）连接,转化大肠杆菌BL21,并对质粒进行双酶切鉴定及基因序列分析,用IPTG诱导融合蛋白的表达,His标签单克隆抗体进行免疫印迹验证融合蛋白的表达。结果以金黄色葡萄球菌DNA为模板,成功扩增了SEA基因,基因大小为774bp,重组PET-28a（＋）-SEA双酶切鉴定可见目的片段,测序结果显示SEA在正确读框中,序列比对分析显示其与相关报道核苷酸序列一致性达99.9%。经IPTG诱导后,SDS-PAGE可见pET-28a（＋）-SEA/BL21在相应分子量（约30000Mr）条带大量表达,免疫印迹能检测到目的蛋白条带,说明其与His标签融合表达。结论克隆了金黄色葡萄球菌SEA基因,并成功在大肠杆菌BL21中融合表达,为肠毒素A应用研究奠定了基础。     

IgG-Antikörper gegen das Toxic shock Syndrom Toxin-1 (TSST-1) in Humanseren

Summary IgG antibodies against toxic shock syndrome toxin-1 (TSST-1) in 2002 human sera were determined using an enzyme linked immunosorbent assay. The results indicate a very early common exposure to TSST-1 in German males and females. The antibody titers are increasing up to the age of 20. They continue to increase further from the midthirties, reaching a peak level in the midfifties. Toxic shock syndrome is rarely described in Germany. It is possible, therefore, that most of the infections with TSST-1 producingS. aureus are of a subclinical nature. An atypical, less dramatic course than the commonly known toxic shock syndrome is also discussed.

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Abkürzung TSST-1 Toxic shock Syndrom Toxin-1 Herrn Prof. Dr. med. Wilhelm Wundt zu seiner Emeritierung gewidmet     

人白细胞介素4在大肠杆菌中的优化表达

目的：通过优化设计利用大肠杆菌表达人白细胞介素４（ＩＬ４） 并提高其表达量。方法：根据原核翻译起始序列的局部二级结构自由能,设计了ＡＵＧ 上下游序列,并在人ＩＬ４ 基因下游插入部分大肠杆菌ＬａｃＺ 序列以提高ｍＲＮＡ 的稳定性。结果：成功地构建了人ＩＬ４ 的高效表达克隆,命名为ｐＬＣＭ１８２ｈＩＬ４ 。ＳＤＳＰＡＧＥ 分析显示所表达的重组ＩＬ４ 蛋白质占细菌总蛋白的３０ ％ 。目的基因下游没有插入ＬａｃＺ序列所构建的表达克隆ｐＣＺＨｈＩＬ４ 在大肠杆菌中的表达量则占细菌总蛋白的２０ ％ 左右。结论：所设计的优化表达方法对人ＩＬ４ 基因的表达是成功的,在细胞因子的工程化表达中,优化设计对基因的表达量至关重要。     

Lack of muco-cutaneous signs of toxic shock syndrome when T cells are absent: S. aureus shock in immunodeficient adults with multiple myeloma

Staphylococcal toxic shock syndrome (TSS) is an acute life threatening disease. The diagnosis can be made clinically based on diagnostic criteria. The clinical manifestations are caused in large part by there lease of high levels of T-cell-derived cytokines as a result of potent toxins, also called superantigens (SAg), produced by Staphylococcus aureus, but it is not clear which clinical symptoms/signs are strictly T-cell dependent. Here, we report on three adults with multiple myeloma (MM) presenting with S.aureus sepsis/shock, and two patients with typical TSS. The MM patients had compromised humoral immunity because of depression of normal immunoglobulin (Ig) levels at the expense of the M protein.In addition, their T cells were absent due to high dose chemotherapy initiated for bone marrow trans-plantation. The MM cases lacked mucosal hyperemia, erythroderma and desquamation, but were otherwise indistinguishable from the TSS cases. All patients grew S. aureus and in each case, SAg genes were detected by PCR. In several cases, the plasma contained biological SAg activity resulting in VP specific proliferation of indicator T cells in vitro. The same specific activity was observed with the supernatant fluids of S. aureus broth cultures from the respective bacterial isolates. This confirms the presence of bio-active toxins in the plasma but did not lead to full blown TSS when T cells were lacking.Thus, S. aureus sepsis/shock can be clinically distinguished from typical TSS, and we suggest that mucocutaneous manifestations of TSS are the most telling signs of massive T-cell-dependent cytokine release.     

IgG Antikörper gegen das toxic shock Syndrom Toxin 1 (TSST-1) in Human-Immunglobulinen

Summary IgG antibodies against toxic shock syndrome toxin-1 in human immunoglobulins were determined using the ELISA technique. Of the drugs for intramuscular application, hemogamma and beriglobin contained the highest amount of antibodies. The highest concentration of antibodies in drugs for intravenous application was found inPseudomonas polyglobin and in Venimmun.

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Abkürzungen TSS toxic shock Syndrom - TSST-1 toxic shock Syndrom Toxin-1     

hIL-1Ra基因的克隆及在原核细胞中的表达

目的 :克隆hIL 1Ra基因并在大肠杆菌中高效表达。方法 :从人外周血中分离白细胞并提取其总RNA。用RT PCR获得hIL 1RacDNA ,克隆入pBV2 2 0表达载体进行诱导表达。对表达产物进行复性和纯化。结果 :成功地构建了pBV2 2 0 IL 1Ra表达载体 ,并在大肠杆菌中获得高效表达 ,表达量占全菌的 4 0 %。对表达产物进行分离纯化及活性分析表明 ,获得纯度大于98%的样品。该样品具有明显抑制IL 1刺激EL 4细胞分泌IL 2的作用。结论 :在大肠杆菌中成功地表达有活性的hIL 1Ra基因 ,为进一步的开发利用奠定了实验基础。     

Pathogenesis of the toxic shock syndrome: T cell mediated lethal shock caused by the superantigen TSST-1

The pathogenesis of the toxic shock syndrome (TSS) is only incompletely understood. We now present evidence that TSS toxin-1 (TSST-1), one of the superantigens produced by Staphylococcus aureus, induces lethal shock in D-galactosamine sensitized mice. In this model TSS is dependent on T cells, since cyclosporin A (CsA) completely blocked development of shock, and since T cell-deficient SCID mice did not show signs of disease upon injection with TSST-1. However, SCID mice repopulated with T cells succumbed to lethal shock. The disease is characterized by a burst of lymphokines like interleukin-2 (IL-2) and tumor necrosis factor (TNF) released into the sera of TSST-1-treated animals. Already 1–2 h after TSST-1 application TNF serum levels peaked and IL-2 levels peaked around 4 h after treatment. TNF appears as key mediator of TSS, because anti-TNF monoclonal antibodies protected TSST-1-challenged mice. Interestingly, the burst of TNF in serum was noted well in advance of detectable markers of T cell activation. Thus, about 5 % of all peripheral T cells started to express the IL-2 receptors as late as 4 h after treatment. Comparing TSST-1- and endotoxin-induced shock we conclude that TNF effects shock in both diseases. However, the type of cells involved appears distinct in that T cells cause TSS triggered by the exotosin TSST-1 while macrophages mediate the shock induced by endotoxins.     

Clonal expansion of T lymphocytes causes arthritis and mortality in mice infected with toxic shock syndrome toxin-1-producing staphylococci

Erosive arthritis is a common and feared complication of staphylococcal infection. The reason(s) for the progressive course of the arthritis is unknown. It has been recently established that enterotoxins produced by Staphylococcus aureus display superantigen properties leading to stimulation of T cells carrying distinct T cell receptor Vβ elements. This finding provides a potential connection between staphylococcal exoproteins and endogenous immune mechanisms participating in the infectious process. We have recently described successful induction of infectious arthritis in mice after intravenous inoculation of a toxic shock syndrome toxin-1 (TSST-1)-producing S. aureus LS-1 strain. Using this model we have now found a clonal expansion of T cells expressing Vβ11⁺ T cell receptor in the synovial tissue of arthritic mice. The role of TSST-1 as a superantigen inducing oligoclonal expansion was confirmed in an in vitro culture system. The expansion of Vβ11⁺ T cells proved to be of arthritogenic significance since mice genomically deleted of the Vβ11⁺ T cells did not develop arthritis and since pretreatment of healthy mice with anti-CD4 or anti-Vβ11 monoclonal antibodies inhibited arthritis. In addition, CD4⁺ and Vβ11⁺ T cells showed themselves to be of pathogenic significance in staphylococcal-induced mortality, since mice depleted of such populations showed increased survival. We propose that in hematogenously spread S. aureus-induced arthritis the TSST-1-dependent clonal expansion of CD4⁺ Vβ11⁺ T cells is a driving pathogenic force.