Study On Activity Of Mutant Staphylococcal Enterotoxin B With Histindine Substitution

Staphylococcal enterotoxin B (SEB) is one of the members of Gram-positive pyrogenic exotoxins which possess superantigen activity. A minima of SEB can stimulate 5%～20% T lymphocyte not specifically and induce a mass of cytokines(IL-2,IL-4,IL-10,INF-γ and TNF-α) release, promote CTL cells to functional cells and activate NK cells, lead to tumor cells lysis. But SEB also has toxicity, it would cause vomiting, diarrhea, abdominal cramps in man and animal, even lead to death. In order to study the relationship between the histidine in SEB and it's toxicity, and to reduce the toxicity of SEB, the site-directed mutagenesis was used to alter some amino acid related to the toxicity domain of SEB based on the crystal structure. we constructed 6 mutant SEB, and then detected the activity change of the mutant SEB through a series of experiment. The purpose of our study is to obtain some mutated proteins which have lower toxicity and maintained the T-cell stimulating activity, and it can be a potential drug to tumor therapy or to the study on immunity disease. This study including two parties:1: Obtaining the mutant proteins In this party, the four histidine sites of SEB are substituted by "overlap" PCR and six mutants are constructed: SEB-H12D/H32D, SEB-H105D/H21D, SEB-H12D/H32D /H105D/ H121D, SEB-H32D, SEB-H32Q and SEB-H32L, then the DNA segments comprised of mutated sites were subcloned into the pET32a(+) prokaryotic expression vector and the mutant proteins were expressed in E.coli after induction with IPTG. Then the recombinant proteins were identified by Western-blot after purification according to the process we designed.2: Evaluation of the activity of the mutant proteins Examine the superantigen activity of the mutant proteins by tumor inhibition experiment,mouse spleen T-cells proliferation assay and Raji cell affinity experiment. In order to detect the toxicity of the mutated proteins, further studies were carried on with little cat vomit and mouse death experiment.The results showed that we successfully get the mutant proteins which expressed in soluble form. The purified proteins have high purity (>95%) Tumor inhibition experiment and the spleen T cells proliferation assay suggested that the mutant proteins also have the activity of stimulating T cells ; and the mutants kept restrain activity to tumor cells, but the activity is lower than that of the wild SEB. All the mutants have the ability of cross-linking to MHCⅡ, especially the ability(fluorescence intensity is 220.32) of AB mutant (H12D/H32D/H105D/H121D) is two times of that of the wild-type SEB. But the ability of C3(fluorescence intensity is 52.33) is lower than that of wild SEB. All mutant proteins can not cause the animal vomiting or diarrhea. Mouse death experiment showed that the mortality ratio of mutant proteins groups were reduced compared to the wild-type SEB group, especially the AB mutant group.These experiments revealed that there do have correlation between the histidine of SEB and its activity of causing animal vomit. But the mutant protein's activities of stimulating T lymphocyte are reduced irregularly, the reason may be exist in the alteration of the SEB's spatial structure. In those mutants, C3 has the lowest toxicity and highest activity of stimulating T lymphocyte proliferation compared with other mutants, The result suggest that mutant C3 can be a potential candidate drug for tumor therapy.