PCR扩增葡萄球菌肠毒素A全长基因方法的建立及意义

为了分析和克隆葡萄球菌肠毒素（ＳＥ）Ａ全长基因,设计了一对针对ＳＥＡ全长基因的特异性引物,成功地用ＰＣＲ反应从标准产ＳＥＡ的葡萄球菌基因组中扩增出了一条约７７０ｂｐ的条带,为进一步用ＰＣＲ法克隆ＳＥＡ基因,并把它用于抗肿瘤研究奠定了实验基础     

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。     

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。     

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。     

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。     

葡萄球菌肠毒素的研究进展

葡萄球菌肠毒素是引起细菌性食物中毒的主要原因之一,葡萄球菌肠毒素按照血清学的方法可分为SEA、SEB、SEC1-3、SED和SEE等七个经典肠毒素。但仍有5％未知的新型肠毒素存在,其编码基因有seg、sei、sej、sek、sel等。葡萄球菌肠毒素也是一种超抗原,能刺激非特异性T细胞的大量增殖。本文对葡萄球菌肠毒素结构、编码基因和功能等方面的研究进展作了简要综述。     

PCR检测食品中金黄色葡萄球菌肠毒素基因

目的：检测食品中金黄色葡萄球菌肠毒素基因谱携带情况,探讨与食物中毒发病相关的金黄色葡萄球菌基因类型。方法：取食品中检出的金黄色葡萄球菌进行分离培养鉴定,通过PCR技术检测样本18种不同型的肠毒素基因的携带情况,并进行统计学分析。结果：食品中检出的金黄色葡萄球菌肠毒素基因有较高的阳性检出率,主要检出sea、seb、sei、seg、sel、sem、sen等基因,其中sea基因检出率最高,达到77.27%;sei、seg、sem、sen基因具有相关性,检出率为9.09%;seb、sel基因的检出率为4.55%。结论：在食品中主要检出金黄色葡萄球菌肠毒素sea、sei、seg、sem、sen、seb、sel等基因,其中,sei、seg、sem、sen基因的相关性还有待进一步深入研究。     

PCR技术检测食品中金黄色葡萄球菌肠毒素B基因

目的:金黄色葡萄球菌B型肠毒素是污染食品引起食物中毒的主要原因之一,针对进出口食品卫生监测的需要,研究一种简便、快速、准确的实验方法.方法:利用聚合酶链反应技术(PCR)采用特异的模板探针引物进行杂交,最后通过电泳技术与阳性对照进行比对,来判断阴阳性结果.结果:本方法检出率高,每克样品中有4个金黄色葡萄球菌即可检出,24小时即可报告结果.结论:PCR方法检测食品中金黄色葡萄球菌肠毒素B基因快速、准确,检测周期短,既可提高检出率,又可节省检测时间.     

Cloning and sequencing of the Clostridium perfringens enterotoxin gene

Several gene banks of Clostridium perfringens in E. coli were constructed. Using a mixture of synthetic 29-mer DNA probes clones were selected containing inserts from the C. perfringens gene coding for the enterotoxin. This has allowed sequencing of the complete gene and its flanking regions. The decuded amino acid sequence (320 a.a.) was found to differ at several sites from the sequence published previously by others. Two 40-mer DNA-probes were used to detect the toxin gene in C. perfringens strains isolated from the faeces of different non-symptomatic animals. Only 6% of the strains were found to possess the gene.     

金黄色葡萄球菌A型肠毒素双抗体夹心ELISA检测方法的建立及应用

【目的】建立一种快速、灵敏、特异的金黄色葡萄球菌A型肠毒素(Staphylococcal enterotoxin A,SEA)检测方法。【方法】以原核表达的可溶性重组SEA蛋白为免疫原,获得特异性强、亲和力高的单克隆抗体作为捕获抗体,同时制备抗SEA兔多抗血清作为检测抗体建立双抗体夹心ELISA(Double antibody sandwich ELISA,DAS-ELISA)检测方法。【结果】该方法对SEA的线性检测区间为2-128μg/L(y=1.102x-0.07,R2=0.994),检测下限为1.89μg/L,与SEB、SEC2和SED之间无交叉反应;鲜奶SEA人工污染试验测定回收率为94%-114%,变异系数小于10%。应用该方法对46株金黄色葡萄球菌水产品分离株和164株奶牛乳腺炎金黄色葡萄球菌分离株的体外培养上清进行检测,阳性率分别为4.4%和50.6%,表明SEA污染普遍存在。【结论】建立的DAS-ELISA方法特异性、灵敏度和稳定性好,为检测SEA的食源性污染提供了有效手段。     

Cloning and nucleotide sequence of the type E staphylococcal enterotoxin gene.

The gene for staphylococcal enterotoxin type E (entE) was cloned from Staphylococcus aureus into plasmid vector pBR322 and introduced into Escherichia coli. A staphylococcal enterotoxin type E-producing E. coli strain was isolated. The complete nucleotide sequence of the cloned structural entE gene and the N-terminal amino acid sequence of mature staphylococcal enterotoxin type E were determined. The entE gene contained 771 base pairs that encoded a protein with a molecular weight of 29,358 which was apparently processed to a mature extracellular form with a molecular weight of 26,425. DNA sequence comparisons indicated that staphylococcal enterotoxins type E and A are closely related. There was 84% nucleotide sequence homology between entE and the gene for staphylococcal enterotoxin type A; these genes encoded protein products that had 214 (83%) homologous amino acid residues (mature forms had 188 [82%] homologous amino acid residues).     

A型肉毒毒素轻链基因的克隆及其结构分析

以献报道的A型肉毒毒素基因全序列为标准,设计并合成一对引物,自肉毒梭菌基因组中扩增出肉毒毒素轻链基因片段,并将扩增产物与pGEM—T载体在体外连接,构建测序重组质粒,进行测序和基因结构分析。PCR扩增获得了产物为1 364bp的DNA片段,测序结果与DNA数据库对照检索分析证明,此基因片段与GenBank中的A型肉毒毒素LC基因的一致性达99．9％以上,可以认为克隆的基因为A型肉毒毒素LC基因。     

Nucleotide sequence of the type A staphylococcal enterotoxin gene.

We determined the nucleotide sequence of the gene encoding staphylococcal enterotoxin A (entA). The gene, composed of 771 base pairs, encodes an enterotoxin A precursor of 257 amino acid residues. A 24-residue N-terminal hydrophobic leader sequence is apparently processed, yielding the mature form of staphylococcal enterotoxin A (Mr, 27,100). Mature enterotoxin A has 82, 72, 74, and 34 amino acid residues in common with staphylococcal enterotoxins B and C1, type A streptococcal exotoxin, and toxic shock syndrome toxin 1, respectively. This level of homology was determined to be significant based on the results of computer analysis and biological considerations. DNA sequence homology between the entA gene and genes encoding other types of staphylococcal enterotoxins was examined by DNA-DNA hybridization analysis with probes derived from the entA gene. A 624-base-pair DNA probe that represented an internal fragment of the entA gene hybridized well to DNA isolated from EntE+ strains and some EntA+ strains. In contrast, a 17-base oligonucleotide probe that encoded a peptide conserved among staphylococcal enterotoxins A, B, and C1 hybridized well to DNA isolated from EntA+, EntB+, EntC1+, and EntD+ strains. These hybridization results indicate that considerable sequence divergence has occurred within this family of exotoxins.     

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.     

金黄色葡萄球菌肠毒素A(SEA)对小鼠体内肉瘤抑制效果

以实验室建立的$180小鼠肿瘤模型为研究对象,采用腹腔注射给药,观察葡萄球菌肠毒素A（SEA）在体内抑制肿瘤的效果。实验表明,SEA抑肿瘤率为40．18％,显示对肿瘤有一定的抑制作用;能显著刺激脾脏细胞增殖,使脾指数升高至11．3mg/g;使血清和脾组织中IL-2水平分别升至69．77pg/mL和682．43pg/mL;且能诱导肿瘤组织中产生大量的CD4^＋T细胞和CD8^＋T细胞。结果显示,SEA在机体内对免疫功能有正向调节作用,从而在一定程度上抑制了肿瘤细胞的生长。     

苹果蠹蛾线粒体DNA COⅠ基因克隆与序列分析

本研究采集新疆阿拉尔地区苹果蠹蛾Cydia pomonella(L.)幼虫,对其线粒体DNA细胞色素氧化酶Ⅰ亚基(COⅠ)基因进行了扩增、克隆和测序,并对COⅠ序列进行了分析。结果显示:苹果蠹蛾DNA扩增出的COⅠ基因序列片段长度为709bp,序列中A+T含量极高,占68.7%,而G+C的含量只有31.3%。经基因序列比对,与其它几种食心虫的同源性为85.4%～88.1%,遗传距离为0.130～0.162;采用NJ法构建了卷蛾科系统树,所得的聚类结果与传统的分类结果基本一致。本研究结果为苹果蠹蛾快速鉴定的DNA条形码技术研究提供重要基础。     

Degradation of staphylococcal enterotoxin A by a Pseudomonas aeruginosa isolate from raw milk

Recently, we found that staphylococcal enterotoxin A (SEA)-producing Staphylococcus aureus strains produced SEA in raw milk with microbial contaminants at high temperatures like 40 °C only. Moreover, the concentration of SEA produced in raw milk gradually decreased after the peak. The reason(s) for SEA degradation in raw milk was studied in this study. Degradation of SEA spiked in raw milk was observed at 40 °C, but not at 25 °C. A Pseudomonas aeruginosa isolate from raw milk degraded SEA spiked in broth at 40 °C. A sample partially purified with a chromatographic method from culture supernatant of the isolate degraded SEA. Two main proteolytic bands were observed in the sample by zymographic analysis with casein. These results suggested that the SEA in raw milk might be degraded by a protease(s) produced by the P. aeruginosa isolate. This finding might be the first report on SEA degradation by a proteolytic enzyme(s) derived from Pseudomonas bacteria to our knowledge.     

Tumor cells with B7.1 and transmembrane anchored staphylococcal enterotoxin A generate effective antitumor immunity

Staphylococcus enterotoxin A (SEA) stimulates T cells bearing certain TCR beta-chain variable regions, when bound to MHC-II molecules, and is a potent inducer of CTL activity and cytokines production. To decrease toxicity of SEA to the normal MHC-II(+) cells and to localize the immune response induced by SEA to the tumor site, my colleague previously genetically fused SEA with B7.1 transmembrane region (named as SEAtm) to make SEA express on the surface of tumor cells and tumor cells modified with SEAtm could induce efficient antitumor immunity in vitro. The tumor cell vaccines modified with multiple immune activators frequently elicited stronger antitumor immune responses than single-modified vaccines. In this study, we modified the tumor cell vaccine with B7.1 and SEAtm to improve efficiency in the application of SEA. First, SEAtm gene was subcloned from recombinant plasmid pLXSNSEP by PCR and murine B7.1 gene was cloned from splenocytes derived from C57BL/6 mice by RT-PCR. Then, the eukaryotic co-expression vector of SEA and murine B7.1 gene was constructed and named as pcDNA-BIS. B16 cell lines stably expressing SEA and/or B7.1 were established by screening with G418 after transfection and inactivated for the preparation of tumor cell vaccines to treat mice bearing established B16 tumors. The results indicated that the dual-modified tumor cell vaccine B16/B7.1+SEAtm (B16-BIS) elicited significantly stronger antitumor immune responses in vivo when compared with the single-modified tumor cell vaccines B16/B7.1 (B16-B7.1) and B16/SEAtm (B16-SEAtm), and supported the feasibility and effectiveness of the dual-modified tumor cell vaccine with superantigen and co-stimulatory molecule.