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。     

A staphylococcal enterotoxin B magnetoelastic immunosensor

A magnetoelastic immunosensor for detection of staphylococcal enterotoxin B (SEB) is described. The magnetoelastic sensor is a newly developed mass/elasticity-based transducer of high sensitivity having a material cost of approximately $0.001/sensor. Affinity-purified rabbit anti-SEB antibody was covalently immobilized on magnetoelastic sensors, of dimensions 6 mm x 2 mm x 28 microm. The affinity reaction of biotin-avidin and biocatalytic precipitation are used to amplify antigen-antibody binding events on the sensor surface. Horseradish peroxidase (HRP) and alkaline phosphatase were examined as the labeled enzymes to induce biocatalytic precipitation. The alkaline phosphatase substrate, 5-bromo-4-chloro-3-indolyl phosphate (BCIP) produces a dimer, which binds tightly to the sensor surface, inducing a change in sensor resonance frequency. The biosensor demonstrates a linear shift in resonance frequency with staphylococcal enterotoxin B concentration between 0.5 and 5 ng/ml, with a detection limit of 0.5 ng/ml.     

Transport and processing of staphylococcal enterotoxin A

A larger-molecular-weight precursor of enterotoxin A was found in membranes of Staphylococcus aureus and was shown to be the kinetic precursor to the extracellular form of the toxin. Subcellular fractionation revealed that mature enterotoxin A was transiently associated with the cell wall before being released to the extracellular environment.     

A DNA Spiegelmer to staphylococcal enterotoxin B

Bacterial staphylococcal enterotoxin B is involved in several severe disease patterns and it was therefore used as a target for the generation of biologically stable mirror-image oligonucleotide ligands, so called Spiegelmers. The toxin is a 28 kDa protein consisting of 239 amino acids. Since the full-length protein is not accessible to chemical peptide synthesis, a stable domain of 25 amino acids was identified as a suitable selection target. DNA in vitro selection experiments were carried out against the equivalent mirror-image D-peptide domain resulting in high affinity D-DNA aptamers. As expected, the corresponding enantiomeric L-DNA Spiegelmer showed comparable binding characteristics to the L-peptide domain. Moreover, the Spiegelmer bound the whole protein target with only slightly reduced affinity. Dissociation constants of both peptide-oligonucleotide complexes were measured in the range of 200 nM, whereas the Spiegelmer binding to the full-length protein was determined at approximately 420 nM. These data demonstrate the possibility to identify Spiegelmers against large protein targets by a domain approach.     

Inactivation of staphylococcal enterotoxin A by chemical modification

(3-¹⁴C)-serine was fed through cut ends of 10-day-old shoots of normal (Ganga-2) and opaque (opaque-2) maize ($\text{Zea}\text{mays}$ L.) and allowed to be metabolized in light for 24 hours. Subsequent methanolic extraction, radiochromatographic separation and radioautographic analysis showed that 10.4 and 10.3 percent of the absorbed ¹⁴C-serine activity was incorporated into L-tryptophan by the shoot tissues of the normal and opaque maize respectively.By coupling extraction of the enzyme with ammonium sulfate precipitation (75% saturation) it was possible to show a serine-dependent indole utilization by the enzyme preparations from coleoptiles (4-day-old), kernels (18 and 28 days after anthesis) and first nodal callus tissues (one-month-old) of the two corn varieties. Furthermore, the indole disappearance from reaction mixture occurred with a concomitant transfer of (3-¹⁴C)-serine appearance into tryptophan.     

Complete amino acid sequence of staphylococcal enterotoxin A

The amino acid sequence of staphylococcal enterotoxin A is presented. Staphylococcal enterotoxin A is a single-chain polypeptide which consists of 233 amino acid residues with a molecular weight of 27,078 and has the amino acid composition Cys2, Asp17, Asn19, Thr16, Ser13, Glu15, Gln12, Pro4, Gly15, Ala7, Val13, Met2, Ile10, Leu23, Tyr18, Phe8, His6, Lys24, Arg7, Trp2, with serine as both amino- and carboxyl-terminal amino acids. Automated sequence analysis of intact enterotoxin A, as well as characterization of the peptides obtained from cyanogen bromide treatment and trypsin and chymotrypsin digestion, led to the elucidation of the complete primary structure of this protein. Less structural homology is observed among staphylococcal enterotoxins A, B (Huang, I-Y., and Bergdoll, M. S. (1970) J. Biol. Chem. 245, 3518-3525), and C1 (Schmidt, J. J., and Spero, L. (1983) J. Biol. Chem. 258, 6300-6306) than that seen between enterotoxins B and C1.     

Transport and processing of staphylococcal enterotoxin A.

A larger-molecular-weight precursor of enterotoxin A was found in membranes of Staphylococcus aureus and was shown to be the kinetic precursor to the extracellular form of the toxin. Subcellular fractionation revealed that mature enterotoxin A was transiently associated with the cell wall before being released to the extracellular environment.     

Novel method for purification of staphylococcal enterotoxin A

A novel single-step procedure for the purification of staphylococcal enterotoxin A (SEA), namely, dye ligand affinity chromatography with the triazine dye Red A, was developed. SEA purified by this method produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield from 5 liters of culture supernatant was 0.113 g, corresponding to an overall yield of 55%. In some instances, purification of SEA from culture supernatants by dye ligand affinity chromatography produced two enterotoxin peaks that could be eluted from the column with 300 and 500 mM phosphate buffer (pH 6.8). Enterotoxin from these peaks produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but multiple bands were observed on isoelectric focusing gels. This method of purification represents a significant improvement in time, yields, and purity of enterotoxin over previously published purification methods.     

Double antibody solid-phase radioimmunoassay for staphylococcal enterotoxin A

Summary A double antibody solid-phase (DASP) radioimmunoassay for staphylococcal enterotoxin A is described. In the assay the antigen-antibody complex is precipitated by anti-rabbit serum which is adsorbed onto a solid carrier (cellulose). The method is sensitive to 200 pg of enterotoxin. It was possible to detect as little as 2–5 ng enterotoxin A/ml food extract from minced meat and sausage. Enterotoxins B and C were not found to inhibit the uptake of labeled enterotoxin A at a level which might distort the results of the enterotoxin A assay. The DASP technique is sensitive, rapid, and easy to perform and thus compares favorably with other radioimmunoassays for enterotoxin.     

Immunization of mice with melanoma cells transfected to secrete the superantigen, staphylococcal enterotoxin A

Immunization of mice with a melanoma vaccine coupled with staphylococcal enterotoxin A (SEA) inhibits the growth of primary melanoma tumors in mice. We have now successfully transfected B16 cells with the sea gene and have immunized C57BL/6 mice subcutaneously once per week for 4 weeks prior to tumor challenge with vaccines of irradiated B16 cells or, 4 weeks following tumor challenge of naïve mice with B16 cells, with irradiated B16 cells transfected with the sea gene. Primary tumor growth following both types of treatments was inhibited significantly. To characterize immune responses to these immunogens, we examined the production of antibodies to the B700 melanoma antigen, the stimulation of endogenous IL-2 production, the expression of CD4, CD8, Vβ and CD25 T cell markers, and the induction of NK activity. At 4 weeks following immunization of mice, there was a significant increase (P<0.05) in levels of interleukin-2 production by splenocytes from mice immunized with SEA-secreting B16 cells or with the parental B16 cells, compared to controls. Levels of antibodies to the B700 melanoma antigen were also significantly higher in mice immunized with the SEA-secreting B16 cells, as was expression of CD4, CD8, CD25 and Vβ T cell antigens, particularly CD4. Natural killer cell activity (at various E:T ratios) was tenfold higher in splenocytes of mice immunized with SEA-secreting B16 cells, and fivefold higher in mice immunized with the parental B16 cells, compared to controls.?These data confirm the possibility of using irradiated murine melanoma cells transfected to secrete SEA in vaccines targeted at preventing the development and growth of melanoma.     

Nature and reactivity of staphylococcal enterotoxin A monoclonal antibodies

Monoclonal antibodies from four clones (C5, C3, B2II, and B2I) directed against staphylococcal enterotoxin A were tested by the indirect enzyme-linked immunosorbent assay and double-gel immunodiffusion (micro-Ouchterlony) assay for the nature of heavy and light chain types. The reactivities of monoclonal antibodies were also tested by indirect enzyme-linked immunosorbent assay with various levels of purified staphylococcal enterotoxin A and various levels (dilutions) of monoclonal antibodies and saturation analysis-competitive indirect enzyme-linked immunosorbent assay. The heavy-chain isotype of monoclonal antibodies was found to be an unspecified subclass of immunoglobulin G1, and the light chain was the kappa type. Monoclonal antibodies from all of the clones exhibited high reactivity and nearly the same affinity to staphylococcal enterotoxin A in saturation analysis-competitive enzyme-linked immunosorbent assay. Purified immunoglobulin G from B2I yielded very high absorbance (1.2) at 405 nm with 1 ng of staphylococcal enterotoxin A as the coating antigen in the enzyme-linked immunosorbent assay. Monoclonal antibodies from B2I also neutralized the biological activity of staphylococcal enterotoxin A when tested by the kitten bioassay.