| Staphylococcus aureus is a frequently isolated bacterial pathogen in both hospital-and community-acquired infections, and it is also an important causative agent for bovine mastitis. Toxic shock syndrome toxin (TSST-1) is one of the numerous exotoxins produced by Staphylococcus aureus. TSST-1 is a single-chain polypeptide of 22KD a molecular mass. It is secreted as a mature protein of 194 amino acids in length and belongs to a pyrogenic toxin superantigen, which is the key factor causing bovine mastitis. Notoxic and mutant superantigen Glutathione s-transferase and mutation Toxic shock syndrome Toxic 1 fusion protein (GST-mTSST-1) was obtained by M13 site-directed mutagenensis. The GST-mTSST-1 molecules were shown to be minimal toxicity fusion protein.To investigate the immunological reactivity, effects of physical and chemical factors on the immunological activity of the GST-mTSST-1 and whether immunization with GST-mTSST-1 fusion protein can protect against Staphylococcus aureues 834 strain infection, at the same time to confirm the best immune dosage of mice and set up the indirect Enzyme-linked Immunosorbent Assay (ELISA) method for detecting the specific auti-serum. After the E.coli DH5a cells harboring the pGXmTSST gene was induced by adding isopropyl-1-thio-β-D-galactopyranoside, the bacteria were collected by centrifugation. The GST-mTSST-1 fusion protein were isolated and purified by centrifugation and affinity Chromatography column. The rabbits were inoculated three times with purified GST-mTSST-1 fusion protein and the serum samples were collected and determined by gel double immunodiffusion assay. The immunological reactivity of GST-mTSST-1 with polyclonal rabbit anti-TSST-1 antibody and the effects of physical and chemical factors (temperature, different pH, repeated freeze-thaw etc.) on its immune activity were tested by using gel double immunodiffussion. The expression product and purity of GST-mTSST-1 were analyzed by Coomassie brilliant blue-stained sodium dodecylsulfate polyacrylanide gels (SDS-PAGE) and thin -layer chromatography(TLC). In the immuno-protective experiment, the purified GST-mTSST-1 was dissolved in sterile PBS and emulsified 1:1 in alum adjuvant. The immunized mice were injected with 200μl of the emulsion containing different GST-mTSST-1 fusion protein at two subcutaneous sites on their backs, the control mice were injected with adjuvant alone. Mice were inoculated 3 times with GST-mTSST-1 fusion protein and then were challenged at day 7 after with a lethal dose (5.0×10~7cfu) of S.aureus834 by intravenous injection. The death of mice were recorded over 15 days and the bacteria in the spleens,livers and kidneys were enumerated at day 3 after infection . Serum samples of the immunized mice were collected at day 14, 28, 42, 60, 90, 120 after the first inoculation. Production of the specific antibody in serum and the level of antibodies to GST-mTSST-1 were determined by indirect ELISA. We optimized the reaction conditions and established indirect ELISA detection method to detect the serum specific antibody of vaccine.The high purity and well immunogenic GST-mTSST-1 fusion protein were obtained. The fusion protein was the form of soluble protein and account for 25% in thallus protein. By analysis of SDS-PAGE and TLC, molecular weight of the expressed product was 47KD and the expression protein reached 95% in purity. The immunized rabbits produced high titer and specific antibodies proved by gel double-immunodiffusion assays. The immunological reactivity of GST-mTSST-1 and rTSST-1 with polyclonal rabbit antibodies were assayed by using gel immunodiffusion. The antibody reacted readily with purified GST-mTSST-1 and rTSST-1, and the precipitation lines between GST-mTSST-1 and rTSST-1 were united each other. These results indicate that GST-mTSST-1 retains the same antibody-binding epitopes as wild-type rTSST-1. By gel double-immunodiffusion assays, immune activity of GST-mTSST-1 fusion protein hadn't change at 4°C30d, 20°C72h, 7°C72h, and it could tolerate a number of times freeze-thaw and 5-11 domain of pH. These results show that GST-mTSST-1 was a better stable fusion protein. Mice were vaccinated subcutaneously 3 times with GST-mTSST-1 plus alum, or PBS plus alum and then intravenously challenged with S.aureus 834 at 5><107cfU /mouse. On day 8 after challenge, 80% of mice (20ug and lOOug) vaccinated with GST-mTSST-1 survived. Conversely, only 20% mice injected with alum alone survived. The experiment was terminated at day 15, 50% of the GST-mTSST-1 immunized groups III and IV still survived. Three days after inoculation, the numbers of bacterial cells in the spleens and livers of GST-mTSST-1-immunized mice were significantly fewer than in the organs of control mice (p<0.05). Taken together, these results confirm that the optimal immunization dosage of mice is 20 [xg with GST-mTSST-1. In addition, these experiments also clearly indicate that vaccination with GST-mTSST-1 provides efficient protection against lethal S.aureus challenge. We developed an indirect ELISA for the detection of antibodies against specific TSST-1 toxin by optimized reaction conditions. The high antibody level was produced in different stages serum by analysis of indirect ELISA .The result suggest that the specific antibodies might play an important role in host resistant againstS.aureus infection.We had obtained soluble and well immunologic GST-mTSST-1 fusion protein of S.aureus. The GST-mTSST-1 vaccine is highly effective, inducing high titers of TSST-1-specific antibodies. Both of the immunized groups produced the same level of immunoglobulin. The animal experiment indicates that protection might be mediated by neutralizing the S.aureus-pro&uctd TSST-1 pyrogenic toxin superantigens as well as by decreasing the mortality and bacterial growth rate, compared with control mice. This nontoxicity of GST-mTSST-1 and its specific antibodies must be useful in the control of S.aureus infection and development of Staphylococcus aureus genetic engineering subunit vaccine against mastitis in dairy cow. |
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