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Objective To investigate the role of Zn 2+and Zn2+-coordinating glutamic acid residues ( 71Glu and 80Glu ) in the superantigenic activities of staphylococcal enterotoxin C2 ( SEC2 ) .Methods Over-lap PCR was used to amply genes encoding recombinant mutant proteins rSEC 2 ( E71A), rSEC2 (E80A) and rSEC2 (E71A/E80A).The mutant proteins were expressed in E.coli BL21 (DE3) and puri-fied by affinity chromatography .The differences of biological activities between rSEC 2 and its mutants were compared in vitro.The effects of Zn 2+on the superantigenic activities of rSEC 2 were evaluated by analyzing the proliferation of splenic lymphocytes in the presence or absence of ethylenediaminetetraacetic acid ( EDTA) .Results The substitution of glutamic acid residue at position 71 and 80 by alanine residue had no significant effects on the superantigenic activities and the conformational stability of rSEC 2 mutants.How-ever, traces of Zn2+(10μmol/L) could significantly enhance rSEC2-induced proliferation of splenic lympho-cytes, and only certain amount of Zn 2+could completely restore rSEC2-induced proliferation in the presence of EDTA.Conclusion This study indicated that mutations at Zn 2+-coordinating glutamic acid residues had no significant effects on the conformational stability and the superantigenic activities of SEC 2.Zn2+might play a role in regulating the superantigenic activities of SEC 2 through some indirect ways .
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Objective:To investigate the improved superantigen activity of SEC2(T20L/G22E) compared with recombinant staphylococcal enterotoxins C 2 ( rSEC2 ).Methods: The proliferation of spleen lymphocytes and T-cell subpopulations induced by rSEC2 and SEC2(T20L/G22E) were examined by WST-1 and flow cytometry separately,and the gene expression of cytokines and Vβspecificities were quantified by real-time PCR.Results: WST-1 and Flow cytometry assays showed that the superantigen activity of SEC2(T20L/G22E) was improved due to enhanced T-cell stimulating potency,resulting in massive activation of T-cells,particularly CD4+and CD8+T-cells.Quantitative real-time PCR assay showed that despite similar Vβspecificities induced by rSEC 2 and SEC2 (T20L/G22E),the quantities of activated T-cells bearing specific Vβwere different,and SEC2(T20L/G22E) could stimulate more gene expression of associated cytokines simultaneously.Conclusion: The results strongly suggested that the increased SEC 2 ( T20L/G22 E)-TCR-binding affinity contributed to more T-cells activation and cytokine release ,which elicit powerful immune activition.
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A tumor-targeting recombinant fusion immunotoxin B-L-SEC2 was constructed by fusing staphylococcal enterotoxin C2 (SEC2) and an anti-HER-2 single-chain Fv B1 through a peptide linker, and expressed in E. coli strain BL21 (DE3) with an improved expression vector pASK75-EX as inclusion body. The denatured inclusion body was purified with Ni-NTA chelate agarose, and then re-natured by dialysis. FACS and MTT assays indicated that the re-natured fusion immunotoxin B-L-SEC2 could target the HER-2 over-expressing breast tumor cell SK-Br-3 in vitro, and inhibit the growth of SK-Br-3.
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Objective:To obtain monoclonal antibodies(McAb) against Staphylococcal enterotoxin C2(SEC2) and establish method for detecting SEC2.Methods:The secreted SEC2 from staphylococcus aureus was used as antigen to immune BALB/c mice. Monoclonal antibodies against SEC2 were prepared by normal hybridoma technique. By identifying the characters of McAbs, the quantitative detection ELISA test method were established and were preliminarily applied.Results:Four hybridmas producing antibodies against SEC2 were obtained. IgG isotypes of four McAbs were IgG1. Their binding site was different except two McAbs that shared the same binding site.The McAbs were proved to be specific for SEC2.The sandwich ELISA method had good specificity, sensitivity and reproducibility, and it was founded to be able to detect SEC2 at concentration from 0.5 to 20 ng/ml. Its recovery ranged between 97.8% and 101%,and CV value ranged between 2% and 5%.Conclusion:The prepared McAb against SEC2 can be used for SEC2 immunoassay. This work provides a basis for controlling the quality of JINPUSU and researching staphylococcal enterotoxin.