Expression Of Recombinant Exfoliative Toxins From Staphylococcus Aureus And Studys On Antibody-Mediated Protection Against Exfoliative Toxins

Objective The exfoliative toxins （ETs） produced by Staphylococcus aureus （S. aureus） are the causative agents of staphylococcal scalded-skin syndrome（SSSS） . To carry on future studies, we need obtain the large quantities of purified recombinant ETA by genetic technology engineering.Methods The DNA fragment of the eta gene encoding the open reading frame corresponding to the mature form of ETA was obtained by utilizing PCR and DNA from ETA-producing S. aureus .A primer set was designed to amplify a 927 bp DNA fragment which inserted the original expression vector pQE30. The pQE-ETA was transformed into E.coli M15 and expressed in the presence of IPTG. The expression product was analyzed by SDS-PAGE and its antigenicity was confirmed by Western blot. After purified with Ni2+-NTA resin, the recombinant ETA protein was tested for activity in neonatal BALB/C mice.Results 1. Construction of recombinant expression plasmid pQE-ETA was successful. The gene fragment inserted was 927 bp, and corresponded with the eta gene sequence on GenBank database. 2. SDS-PAGE showed that expression of the relative molecular mass of 27kD, accounts for full expression of the total 12%. For maximal production of ETA, IPTG at a final concentration of 0.1 mM, and induction temperatureand time of 37°C and 4 h, respectively, was used. 3. Western blot showed that the recombinant protein could be recognized by sheep polyclonal anti-ETA IgG. 4. SDS-PAGE showed that the recombinant protein existed in the supernatant and precipitation of the lysed bacteria broken by ultrasonic wave, and its purity was over 90% after purified with Ni²⁺-NTA resin. 5. The exfoliative activity of recombinant ETA was confirmed by animal experiment.Conclusions The construction and expression of eta gene is successful, and obtain the high-grade recombinant protein which has favorable immunity and biological activity. The recombinant protein is the basis for diagnosing SSSS, researching the mechanisms of pathogenic and producing specific antibodies. Objective The role of the adaptive immune response was investigated using two experiments: the detection of antibodies to ETA and ETB in serum by ELISA and the protection of antibody for the mouse model of SSSS.Methods 1.ETA- and ETB-specific antibody titers in a commercial IVIg and in patient’s and general population’s serum were detected by Indirect ELISA. 2. The mouse model of SSSS was produced by injecting subcutaneously at the nape of the neck with rETA and the differences between the group with anti-ETA antibody and the group without anti-ETA antibody was compared.Results 1. Anti-ETA antibody titers were higher than Anti-ETB antibody titers in a commercial IVIg and the general population’s serum. 2. There was no significant difference of Anti-ETA antibody titers between with the SSSS group, the AD group and the normal children group. 3. There was significant difference of Anti-ETB antibody titers between with the SSSS group, the AD group and the normal children group, and Anti-ETB antibody titers of the SSSS group were much lower than the AD group and the normal children group. 4. According to the appearance of the skin, the histological examination and the mortality of the mouse model of SSSS, the group with anti-ETA antibody excelled the group without anti-ETA antibody. 5. The curative effect of employing anti-ETA antibody early was better than using antibody after 6 hour.Conclusions 1. ETB is associated with generalized SSSS, possibly owing to a lower titer of anti-ETB antibodies in the population. 2. Lacking enough protection of the specific antibodies in SSSS patients of which Anti-ETB antibody titers were much lower than the AD patients and the normal children may be one of the reasons for suffering SSSS. 3. Animal experiment indicated that the specific antibodies can play a important role in protecting against the effects of the ETs.