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蛋白的B细胞表位的预测

目的预测金黄色葡萄球菌肠毒素A蛋白（SEA）的B细胞表位。方法以金黄色葡萄球菌合肥乳源分离株M3基因组DNA为模板,PCR扩增SEA基因并进行序列测定与分析。应用DNAstar protean软件对SEA蛋白的二级结构、柔性、亲水性、表面可能性和抗原指数等多参数进行综合分析,预测其B细胞表位。结果M3分离株的SEA基因全长774bp,编码由257个氨基酸组成的相对分子量为29．67kDa的SEA蛋白,M3分离株SEA基因与标准株的核苷酸序列与氨基酸序列同源性分别为98．7％和98．4％。SEA蛋白的优势B细胞表位位于肽链的第64—68、100～107、138—141、156—160、166～173、213～217和237～244区段。结论预测出SEA蛋白的7个优势B细胞表位,为进而克隆表达表位蛋白,制备针对SEA表位的单克隆抗体奠定了基础。     

葡萄球菌肠毒素A全长基因的克隆和序列测定

分析和克隆超抗原（SAg）葡萄球菌肠毒素A（SEA）全长基因,为进行SAg基因应用于肿瘤导向治疗和基因治疗的研究奠定基础。设计并合成一对针对SEA全长基因的特异性引物,用PCR反应从产SEA的标准葡萄球菌菌株的基因组中扩增出SEA全长基因。PCR产物与克隆载体pGEM-T连接后进行基因序列测定。成功地从标准葡萄球菌菌株的基因组中扩增出一条约770bp的条带。基因序列测定表明,与巳发表的SEA全长基国序列完全一致。     

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。     

金黄色葡萄球菌肠毒素B基因的克隆和在大肠杆菌中高效表达

利用PCR方法从金黄色葡萄球菌TSTw基因组DNA中扩增出约700bp的DNA片段,将之克隆到pGEM7Zf(+)载体上并转化大肠杆菌 DH5α菌株。重组质粒的测序结果表明克隆到了seb基因,它含有717bp（不包括N端81bp的信号肽编码区）,其核苷酸序列与文献报道完全一致。将其连接于表达载体7ZTS上,转化到大肠杆菌JM109（DE3）内。表达的SEB占总蛋白33.5%。      

金黄色葡萄球菌肠毒素A、B、C、D、E重组载体的构建及表达

金黄色葡萄球菌肠毒素（Staphylococcal enterotoxins，SEs）是一组结构毒力相似、血清型不同的可溶性小分子蛋白质，平均分子质量为26~30 kD，是引起细菌性食物中毒及肠胃炎的主要因素之一。为了制备金黄色葡萄球菌肠毒素的纯品，首先合成了SEA、SEB、SEC、SED和SEE的基因序列，然后构建了5种肠毒素的原核表达载体，分别转入BL21（DE3）细胞中进行诱导表达。通过SDS-PAGE和Western blot验证，5种肠毒素蛋白均被成功表达，并且在较低的诱导温度（16 ℃）获得一定量的可溶性蛋白。成功制备了5种金黄色葡萄球菌肠毒素的可溶性蛋白，为今后更好地解决因SEs引起的食品安全问题奠定了基础。     

快速检测金黄色葡萄球菌肠毒素A基因方法的建立与应用

目的建立一种快速准确定量检测金黄色葡萄球菌肠毒素A的方法。方法以femB、SEA基因分别作为金黄色葡萄球菌菌株、肠毒素A的靶序列,设计合成引物和TaqM an探针;收集腹泻患者大便标本分离的金黄色葡萄球菌68株,并定量检测其肠毒素A。结果TaqM an探针荧光聚合酶链反应检测金黄色葡萄球和肠毒素A的灵敏度均为1.0×102拷贝。68株金黄色葡萄球菌中检出产肠毒素A菌株11例(16.2%,11/68),CT值为13.5~20.6。结论TaqM an探针荧光聚合酶链反应能够准确快速检测金黄色葡萄球菌肠毒素A。     

EGF-SEA融合蛋白在大肠杆菌中的表达和纯化

根据基因库中查到的金黄色葡萄球菌肠毒素A(SEA)基因序列和人体表皮生长因子(EGF)基因序列进行密码子优化,以适于大肠杆菌表达.人工合成SEA基因与EGF基因.将两目的基因克隆至原核表达栽体pFT22b中,经测序验证表明成功构建了重组表达质粒pET22b-EGF-SEA.将构建好的pET22b-EGF-SEA质粒转化大肠杆菌BL21(DE3),经IPTG诱导进行表达;SDS-PAGE分析表明融合基因EGF-SEA在大肠杆菌BL21(DE3)中以包涵体的形式得到了高效表达,产物相对分子质量约为44kDa,与理论值大小一致.包涵体经洗涤,变性、复性后用His Bind Kit进行分离纯化,所得蛋白纯度≥95%.高纯度EGF-SEA融合蛋白的获得为进一步研究其生物学活性及肿瘤治疗奠定了基础.     

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。     

葡萄球菌A型肠毒素的高效表达和分离纯化

根据已知葡萄球菌A型肠毒素 (SEA)的基因序列 ,用PCR从产毒标准株S .aureusFRI 10 0中扩增得到约70 0的SEA基因片段 ,并将该片段克隆至表达载体 pBV2 2 0中 ,实现了高效表达。表达产物以可溶性形式存在 ,表达的毒素用CM SephroseFF离子交换层析进行纯化 ,获得了高纯度的重组SEA ,SDS PAGE显示单一条带。ELISA试验证明所获重组SEA具有与天然SEA相似的免疫学性质。     

Crystal structure of the superantigen staphylococcal enterotoxin type A.

Staphylococcal enterotoxins are prototype superantigens characterized by their ability to bind to major histocompatibility complex (MHC) class II molecules and subsequently activate a large fraction of T-lymphocytes. The crystal structure of staphylococcal enterotoxin type A (SEA), a 27 kDa monomeric protein, was determined to 1.9 A resolution with an R-factor of 19.9% by multiple isomorphous replacement. SEA is a two domain protein composed of a beta-barrel and a beta-grasp motif demonstrating the same general structure as staphylococcal enterotoxins SEB and TSST-1. Unique for SEA, however, is a Zn2+ coordination site involved in MHC class II binding. Four amino acids including Ser1, His187, His225 and Asp227 were found to be involved in direct coordination of the metal ion. SEA is the first Zn2+ binding enterotoxin that has been structurally determined.     

表达超抗原SEA基因的溶瘤腺病毒载体的构建与鉴定

目的:构建表达超抗原SEA基因的溶瘤腺病毒载体并鉴定.方法:采用PCR技术,从产SEA的葡萄球菌标准菌株ATCC13565基因组DNA中获得SEA全长基因序列,酶切后克隆入pCA13质粒,构建重组病毒质粒pCA13-SEA.将鉴定正确的pCA13-SEA与含有腺病毒右臂的质粒pBHGE3通过Lipofectamine2000共转染HEK293细胞,经同源重组产生重组腺病毒Ad-SEA.Ad-SEA在293细胞中大量扩增并通过氯化铯密度梯度离心法纯化、测定其滴度.结果:经PCR扩增、酶切鉴定、序列测定证实,SEA基因成功克隆到溶瘤腺病毒载体中,可实现SEA基因的表达.结论:成功构建了表达超抗原SEA基因的溶瘤腺病毒载体,为进一步研究该病毒对膀胱肿瘤靶向治疗的作用奠定了基础.     

J亚群鸡白血病病毒AH09/2株的gp85基因的克隆与原核表达

利用PCR方法扩增出J亚群禽白血病病毒(ALV-J)AH09/2株的gp85基因全长930 bp DNA片段。经T载体克隆测序并连接到pGEX-6p-1载体上,构建了重组表达质粒pGEX-6P-1-gp85,在IPTG的诱导下进行表达。Western-blot结果分析表明,gp85融合蛋白表达产物分子量大小约61 kDa,并能与ALV-Jenv基因单抗发生特异性反应。这些结果为深入研究GP85蛋白的生物学功能及研制ALV-J检测ELISA试剂盒奠定了基础。     

Production of enterotoxin A by supposedly nonenterotoxigenic Staphylococcus aureus strains.

The production of staphylococcal enterotoxins A (SEA) and B (SEB) was studied by inoculating six well-defined staphylococcal collection strains into cow's, goat's, or sheep's milk (individually or as a 50% mixture of cow's + goat's or cow's + sheep's), into brain heart infusion, and into a medium generally used to enhance the synthesis of enterotoxins (3+3 medium). Four of the strains used are considered to be SEB producers, another is considered an SEA producer, and the remaining strain is nonenterotoxigenic but produces large quantities of staphylococcal protein A. Staphylococcal protein A masked the results in most cases. Only one strain secreted exclusively SEB, while the other three SEB producers synthesized SEA in different amounts. We conclude that enterotoxin production depends on the natural substrate and may differ from the results obtained when the strain is grown on cellophane over agar to determine its toxigenicity.     

Production of enterotoxin A by supposedly nonenterotoxigenic Staphylococcus aureus strains

The production of staphylococcal enterotoxins A (SEA) and B (SEB) was studied by inoculating six well-defined staphylococcal collection strains into cow's, goat's, or sheep's milk (individually or as a 50% mixture of cow's + goat's or cow's + sheep's), into brain heart infusion, and into a medium generally used to enhance the synthesis of enterotoxins (3+3 medium). Four of the strains used are considered to be SEB producers, another is considered an SEA producer, and the remaining strain is nonenterotoxigenic but produces large quantities of staphylococcal protein A. Staphylococcal protein A masked the results in most cases. Only one strain secreted exclusively SEB, while the other three SEB producers synthesized SEA in different amounts. We conclude that enterotoxin production depends on the natural substrate and may differ from the results obtained when the strain is grown on cellophane over agar to determine its toxigenicity.     

斜纹夜蛾核多角体病毒VP39-GST融合蛋白的原核表达

用PCR的方法扩增得到斜纹夜蛾核多角体病毒（Spodoptera litura multicapsid nucleopolyhedrovirus,SpltMNPV）vp39全长基因。将其克隆至原核表达载体pGEX-4T-1上,构建重组表达质粒pGEX-4T-vp39,转化大肠杆菌BL21（DE3）,在1 mmol/L异丙基硫代-β-D-半乳糖苷（IPTG）诱导下超量表达了与理论预测值相符的一个约60 kD的VP39-GST融合蛋白。VP39-GST融合蛋白的成功表达为进一步研究VP39在病毒侵染过程中与宿主细胞成分或病毒粒子蛋白间的相互作用奠定了基础。     

HIV-1整合酶蛋白的可溶性表达及功能研究

HIV 1整合酶是HIV病毒复制中一个重要的酶,也是治疗艾滋病药物的一个重要靶点。为了开展以整合酶蛋白为靶点的抑制剂筛选,构建HIV 1整合酶重组质粒,在原核细胞中进行可溶性表达和功能研究。通过重叠PCR技术引入F185K和C280S突变于HIV 1 B亚型标准株的整合酶cDNA片段中,PCR扩增片段克隆到pET 28a(+)表达载体中,构建重组质粒,在E. coli中进行整合酶基因表达,SDS PAGE鉴定表达产物,亲和层析纯化蛋白,酶联免疫吸附实验方法测定整合酶的生物学活性。结果构建的重组质粒获得高效稳定的可溶性表达,ELISA实验证实该蛋白具有整合酶的3′切割DNA和5′链转移的活性。HIV 1整合酶蛋白的可溶性表达和活性研究为建立以整合酶为靶点的抗HIV药物筛选平台打下了基础。     

Mutated SEA-D227A-conjugated antibodies greatly enhance antitumor activity against MUC1-expressing bile duct carcinoma

For the purpose of establishing a new adoptive immunotherapy for bile duct carcinoma (BDC), we have directed our attention to superantigens (SAgs), the most potent known activators of T lymphocytes. In our previous study, staphylococcal enterotoxin A (SEA) was conjugated chemically with MUSE11 mAb, which recognizes the MUC1 cancer-associated antigen, and shown to enhance the specific cytotoxic activity of T-LAK cells against MUC1-expressing BDC cells (TFK-1) in vitro and in vivo. However, it is probable that SEA might cause side-effects because of nonspecific binding to class II positive cells. In order to overcome these, we generated mutated SEA (mSEA) by changing Asp at position 227 of native SEA to Ala, which has reduced affinity to MHC class II molecules, but retains the potential for T cell activation. When mSEA-D227A was administered to rabbits to examine effects on blood pressure, 500 times more mSEA-D227A was tolerated than native SEA. This prompted us to construct a mSEA-D227A-conjugated mAb, reactive with MUC1. It augmented the antitumor activity of T-LAK cells significantly, and furthermore, mSEA-D227A could be conjugated to two bispecific antibodies, BsAb (anti-MUC1 x anti-CD3) and BsAb (anti-MUC1 x anti-CD28), which in combination had greater enhancing effects than mSEA-D227A-conjugated anti-MUC1 mAb, and combination of unconjugated BsAbs. These findings indicate a utility of mSEA-D227A-conjugated antibodies for targeted cancer immunotherapy.     

耐甲氧西林葡萄球菌PBP2a的质粒克隆与构建

目的克隆并构建耐甲氧西林金黄色葡萄球菌（MRSA）青霉素结合蛋白2a（PBP2a）全长及转肽酶区的原核表达质粒。方法登录基因文库查找获得mecA基因的编码序列,应用PCR技术扩增获得DNA片段,将此基因片段插入PET-32a载体,同时酶切鉴定阳性克隆,DNA序列测定验证序列正确性。结果 PCR扩增获得了mecA基因全长及转肽酶区DNA片段,成功插入到原核表达载体PET32a,双酶切鉴定及DNA序列测定证实插入片段正确。结论成功构建了PBP2a全长及转肽酶区片段表达质粒,为该蛋白的纯化表达和疫苗研究奠定了基础。