Purification of an inactive variant alpha-toxin HlaH35LH48Lexpressed in Escherichia coliand generation of antiserum in mice

Alpha-toxin (Hla) is a major cytotoxic agent produced by Staphylococcus aureus, playing a significant role in the pathogenesis of various infections and has been regarded as a promising antigen for vaccine development.This study aims to express HlaH35LH48L fused with His-tag in Escherichia coli BL21(DE3), purification of inactive form, and generation of antiserum in mice.The survey results indicated that the appropriate conditions for expressing the HlaH35LH48L protein were at 30C with an IPTG concentration of 0.05 mM, and harvesting 8 hours post-induction.Based on affinity chromatography between the 6xHis tag and Ni-NTA, the HlaH35LH48L protein was purified, obtaining a concentration of 1.17 mg/ml with a purity of 96%.Activity assays for the purified protein revealed that the HlaH35LH48L variant lost its ability to lyse rabbit red blood cells.After four injections in mice with a dose of 0.05 mg of HlaH35LH48L protein per mouse, the resulting antiserum was able to recognise the antigen with an antibody titer of 1:320,000.The results of this study serve as an important premise for further studies to neutralise Hla in vivo and develop a vaccine against S. aureus.