Bioactive Researches Of Staphylococcal Enterotoxin C2 Mutants Obtained Through Site-directed Mutagenesis

Staphylococcal enterotoxins (SEs) are proteins of highly biological activities produced by Staphylococcus aureus, which are also superantigens with the ability to activate T cells. Unlike conventional antigens, SAgs bind to major histocompatibility complex (MHC) class II molecules of antigen-presenting cells (APCs) and the variable region of theβchain (Vβ) of the T cell receptor (TCR) in their native form. SAgs are able to activate a large amount of resting T cells resulting in release of massive cytokines and pharmacody effect, thereby inducing the effect of superantigen dependent cell-mediated cytotoxicity (SDCC) and other immune effects. Utilizing the characteristic of superantigenic SEs, SEC2 was used as a new anti-tumor biological preparation, named"gaojusheng", by Shenyang Xiehe Group. It has been used to cure malignant tumor such as lung cancer, esophageal cancer, ovarian cancer, liver cancer, cancer of colon, leukemia, carcinoma of urinary bladder and so on, and received significant curative effect. As the potent gastrointestinal toxin, however, SEC2 could result in food poisoning, diarrhea and abdominal cramps, which restrict its clinical application to cure malignant tumors in a great extent.The structure of SEs is related with pathopoiesis and pathogenicity. The cystine loop (Cys93, Cys110) and His118 are necessary for emetic activity of SEs, but are independent of T cells inducing. In this study, Cys (93 and 110) and His118 were selected as substitutional sites through site-directed mutagenesis and the resultant mutants were expressed in E. coli. PBMC proliferation activity and anti-tumor activity in vitro were used to analyze superantigen activity and anti-tumor effect. Results as follows:1) The Cys110 was successfully replaced by Ala or Ser, His118 by Tyr, through site-directed mutagenesis. The plasmids with mutant gene were highly expressed by induction of IPTG in E. coli BL21 (DE3). After purification with the Ni-NTA affinity chromatography and Sephadex G-75, the fusion protein showed as a single band on SDS-PAGE.2) All mutants can stimulate the PBMC proliferation, and there is no significant difference between mutant SEC2 and SEC2. The most effective concentration is 200 ng/ml. 3) Results showed that these mutated proteins exhibited tumor-inhibition effect on human colorectal cancer cells (CX-1) at different extent, and the most efficient mutant is SEC2(C93S, C110S), which exhibited a better effect than SEC2 with the concentration between 2 ng/ml and 500 ng/ml.In conclusion, we successfully obtained SEC2 mutants, possessing similar superantigenic activity and anti-tumor activity with SEC2, through site-directed mutagenesis, in which the amino acid related with emesis were placed. It laid a foundation for empolder novel anti-tumor biological preparation of innocuity and no side-effect.