Staphylococcus Aureus Enterotoxin C2his118 Fixed-point Mutation Study

Superantigens are protein molecules with many kinds of immunological activities, which are extremely potent activators of T cells. In contrast to conventional antigens, superantigens needn't be processed by antigen presenting cell (APC), and can bind outside the classical antigen-binding groove of major histocompatibility complex (MHC) class II and Vβchain of T cell receptor (TCR) by intact protein form. Very low concentrations of SAgs are able to activate a large amount of resting T cells resulting in release of massive cytokines and pharmacody effects, thereby inducing the effect of superantigen dependent cell-mediated cytotoxicity (SDCC) and other immune effects, which can inhibit tumor cell growth in vivo. Staphylococcal enterotoxin C2 (SEC2) is one member of superantigens produced by Staphylococcus aureus, which has been applied to cure malignant tumors in clinic and significant curative effect such as lung cancer, esophageal cancer, ovarian cancer, liver cancer, cancer of colon, leukemia, carcinoma of urinary bladder and so on. As the potent gastrointestinal toxin, however, SEC2 could result in nausea, vomiting, diarrhea. This restricts the clincial application of SEC2 and cure of malignant tumors.Crystal structure of SE is related to pathopoiesis and pathogenicity. His118 is necessary for emetic activity and independent of T cells inducing. In this study, His118 is selected as substitutional site of site-directed mutagenesis, and the resultant mutants were expressed in E. coli. Mouse spleen lymphocytes proliferation activity and trypsin stability were used to analyze superantigen activity and character of trypsin stability. SEC2 was used as control, the anti-tumor effects were analyzed according to the results whether human colorectal cancer cell CX-1 was inhibited. Results showed as follows:Site-directed mutagenesis was performed using MutanBEST kit. The PCR primers were designed and used to amplify pET-28a-SEC2 by PCR. Two expression plasmids pET-28a-SEC2 (H118Y) was obtained and efficiently expressed in E.coli. The SEC2 mutants were purified with the Ni-NTA affinity chromatography and Sephadex G-75, and proteins shown as a single band respectively on SDS-PAGE were of high purity.To compare the general stability of SEC2 and its mutant derivatives, purified proteins were respectively incubated with trypsin at toxin: enzyme (w/w) ratio of 200:1. The extent of proteolysis was assessed by SDS-PAGE. Results showed that SEC2 and SEC (H118Y) exhibited the same stable characters. In addition, the mutated SEC2 still possessed superantigenic activity according to the results of mouse spleen lymphocytes proliferation assay. In vitro anti-tumor activity assay revealed that mutants could inhibit growth of human colorectal cancer cell CX-1 distinctly. The anti-tumor effect of SEC (H118Y) was similar to that of SEC2.