Recombinant Staphylococcal Enterotoxin Type A Stimulate Antitumoral Cytokines (original) (raw)
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Applied and environmental microbiology, 1984
A monoclonal antibody capable of binding to determinants shared in common by staphylococcal enterotoxin serotypes A, B, C1, D, and E was developed. To accomplish this, BALB/c mice were immunized by alternating injections of serotypes A and D to enrich for spleen lymphocytes programmed to produce antibody to possible common determinants. These cells, fused with mutant myeloma cells (P3-X-63Ag8.653) produced hybrids that formed monoclonal antibodies to either serotype A only or to both serotypes A and D. A cloned hybridoma from the latter group produced an antibody (subclass immunoglobulin G1) which interacted with five serotypes. Highest affinity was to B and C1. An immunomatrix consisting of this antibody cross-linked to protein A-Sepharose CL-4B with dimethyl pimelimidate was capable of binding enterotoxin. Bound toxin was eluted with diethylamine. Because of its ability to interact with all five serotypes, the monoclonal antibody should prove useful in the development of a rapid m...
American Journal of Microbiological Research, 2017
Staphylococcus aureus (S.aureus) is the most common pathogen found in hospitals, community-acquired infections and colonizes up to 50% of humans, including mucous membranes and damaged skin. Some strains produce enterotoxins (Ent) as staphylococcal enterotoxin A (SEA) which is involved in 75% of food poisoning outbreaks. Few methods are sensitive and specific enough to confirm the diagnosis of staphylococcal food poisoning. In this study, a segment of Ent A-ORF with molecular weight 774 bp was amplified, cloned, sequenced and aligned with published Ent A-ORFs. Ent A-ORF was subcloned into bacterial expression vector (GST-PGEX4T1 vector) and expressed as a fusion protein with GST-tagged protein. A band size of 27 kDa of purified Ent A protein was cleaved from GST protein by thrombin. The expressed protein of Ent A was identified by strong reaction with commercialized polyclonal antibodies against Ent A. Antiserum against Egyptian recombinant Ent A protein was produced by immunization...
Heterologous expression of staphylococcal enterotoxin B (seb) gene for antibody production
Electronic Journal of Biotechnology, 2006
Staphylococcal food poisoning (SFP) is caused by the members of superantigen family called staphylococcal enterotoxins (SEs). About 20 different types of SEs are produced by Staphylococcus aureus out of which type A (SEA), B (SEB), C (SEC) and D (SED) are commonly implicated in SFP. Among these, SEB is the most potent toxin and has also gained the status of biological warfare (BW) agent. Therefore, detection of SEB is of utmost importance. Any immunological detection system for SEB requires specific and sensitive antibodies which inturn depends on the purity of the SEB. In the present investigation, seb gene of S. aureus was cloned and expressed in E. coli along with biotin as fusion partner to facilitate the purification process. The yield of purified recombinant SEB was 13.1 mg/L of culture broth. Biotin tag from the biotinylated toxin was removed by protease cleavage, and both biotinylated and non-biotinylated toxin types were used for raising hyperimmune antiserum. Antisera were also specific for SEB amongst different kinds of food poisoning agents tested by indirect plate ELISA and western blot analysis. The quality of the antisera raised in this study was found superior to the commercially available antiserum. The investigation suggests that construction of recombinant staphylococcal enterotoxin B is a good alternative for production of pure enterotoxin to be used in antibody generation.
Cancer Immunology Immunotherapy, 2009
Bacterial superantigen staphylococcal enterotoxins (SEs) tremendously stimulate polyclonal T cells bearing particular TCR Vβ domains when binding to MHC II molecules, suggesting that they could be a candidate of new antitumor agent. SEC2, an important member of superantigen family, has been used in clinical trial as an immuntherapy agent for cancer treatment in China, and obtained some encouraging effects. However, the presence of immunosuppression and endotoxic activity limits the therapeutic dosage of SEC2, and influences its antitumor effect in clinic. Therefore, the enhancement of superantigen activity and antitumor effect of SEC2 could effectively make compensation for the disadvantages mentioned above. In this study, a superantigen SEC2(T20L/G22E) mutant was generated by site-directed mutagenesis, and efficiently expressed in E. coli BL21(DE3). The results showed that SEC2(T20L/G22E) mutant exhibited a significantly enhanced superantigen activity and antitumor response, compared with native SEC2 in vitro. Further toxicity assay in vivo indicated that SEC2(T20L/G22E) mutant had no significant increase in emetic and pyrogenic activity compared with SEC2, which suggested that the mutant SEC2(T20L/G22E) could be used as a potentially powerful candidate for cancer immunotherapy, and could make compensation for the deficiency of native SEC2 in clinic.
Indian Journal of Microbiology, 2012
Staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 are the super antigens responsible for diseases such as staphylococcal food poisoning and toxic shock syndrome. At low serum concentrations, SEB can trigger toxic shock, profound hypotension and multi organ failure and hence is recognized as biowarfare molecule. In this study, a multidomain fusion protein (r-TE) was generated with specificity for SEB and toxic shock syndrome toxin (Tsst-1). The fusion gene comprising the conserved regions of seb and the tsst genes was codonoptimized for expression in Escherichia coli and encoded a 26 kDa recombinant multidomain chimeric protein (r-TE). Hyperimmune antiserum raised against r-TE specifically reacted with SEB (*28 kDa) and Tsst-1 (*22 kDa) components during Western blot analysis and by plate ELISA in confirmed toxin producing strains of S. aureus. The antigenicity of the SEB component of the r-TE protein was also confirmed using TECRA kit. The described procedure of creating a single protein molecule carrying components of two different toxins whilst still retaining the original antigenic determinants of individual toxins proved highly advantageous in the development of rapid, reliable and cost effective immunoassays and may also have the potential to serve as candidate molecule for vaccine studies.