Construction Of The Site-specific Mutants Of Staphylococcal Enterotoxin A Gene And Biological Activities Of Their Recombinant Expression Products

Background and Objective Staphylococcal enterotoxins, usually produced by Staphylococcus aureus, have been found so far to can be divided into five types SEA～SEE based on the differences of antigenicity. Of these five enterotoxins, SEA, SEB and SEC were well demonstrated to be the most effective stimulants for proliferation and activity of T lymphocytes of human and mammal origin. SEA～SEC at a very low dosages (ng or pg) can efficiently induce the mitosis of lymphoblast and promote T lymphocytes releasing multiple cytokines to kill various tumor cells by way of cytotoxicity. Therefore, SEA-SEC show a potential to be developed into a kind of peptide drug for increasing leukocyte and anti-tumor in human. However, SEA-SEC, even though at very low dosages, can cause diarrhea and vomiting in part of the tested populations due to their enterotoxicity and individual difference. It is well-known that the toxicity of peptide or protein toxins was generally dependent on the active sites in their molecules. It is a great possibility that SEA-SEC mutants with low or non-toxicity for practicalapplication can be obtained to change types of amino acid residuals in the active sites determining toxicity by using site mutation technique.SEA gene with whole length of 774bp encodes a peptide composed of 257 amino acid residuals and the reported sequences of SEA gene from different original S. aureus strains showed high conserved nucleotide and putative amino acid sequences. In this study, we would amplify entire SEA gene with no signal peptide sequence of S.aureus strain ATCC13565 genome DNA, according to Protein Data Bank and relative references , the site-specific mutants of SEA gene by PCR using mutant primers as well as prokaryotic expression systems of the SEA gene and its mutants were constructed. Those mutants with lower cytotoxicity, stronger promoting of splenocyte proliferation and inhibiting tumor cell growth,.we can choose them to lay a basis for further analysis of safty and effective SEA mutants medicine.Methods SEA gene with no signal peptide sequence of S.aureus strain ATCC13565 wasamplified and then cloned by using high fidelity PCR and T-A cloning method. The site-specific mutants of SEA gene by PCR using mutant primers as well as prokaryotic expression systems of the SEA gene and its mutants were were constructed. Ni-NTA affinity chromatography was performed to extract the expressed target recombinant proteins, then verified by SDS-PAGE. The purified recombinant proteins were recovered by mean of dialysis, then were degermed by filtration.The cytotoxicity to Vero cells of the recombinant proteins was measured using TCID50 titration method. MTT colorimetry was applied to examine the effects of the recombinant proteins at different dosages on promoting proliferation of mouse splenocytes and inhibiting growth of tumor cell lines KB and HL-60.Results The similarity of nucleotide sequence of the cloned SEA gene was 100%compared to the reported SEA sequences and the expected mutation of codons in each the seven mutants at the scheduled positions were present. All the recombiant protein expression outputs ofSEA gene and its mutants were approximate 52% of the total bacterial proteins. The TCIC50 value of rSEA's cytotoxicity to Vero cells was 4.1μg while the TCIC50 values of the recombinant of SEA gene mutants were 5.8～23.5μg, respectively. 1 and 5 μg/ml of rSEA and its five mutant proteins rSEA/H187A, rSEA/H225A, rSEA/D227A, rSEA/H187A/D227A and rSEA/H225A/ D227A showed remarkable effects to promote proliferation of mouse splenocytes (P＜0.05). The activities of promoting mouse splenocyte proliferation of 10 and 20μg/ml rSEA and the five mutant proteins were similar to that of 100 μg/ml PHA (P>0.05). The supernatants or the mixture of supernatant and splenocytes stimulated by 5～20 μg/ml rSEA or each the five mutant proteins could efficiently inhibit the growth of KB and HL-60 cells (P＜0.05).Conclusion The SEA gene mutants and their prokaryotic expression systems with high expression efficiency were successfully established in this study. Since rSEA/H225A, rSEA/D227A and rSEA/H225A/D227A display lower cytotoxicity, stronger promoting of splenocyte proliferation and inhibiting tumor cell growth, the recombinant proteins of three mutants have a potential used as candidates for developing leukogenic and anti-tumor SEA associated drugs.