Preparation Of EsxB Protein In Staphylococcus Aureus And Its Antiserum And Analysis Of Drug Resistance Of EsxB Antibody Positive Strains

Objective: To prepare of EsxB protein in Staphylococcus aureus (S. aureus) and itsantiserum, and to explore the distribution of EsxB protein in staphylococcus in patients andthe correlation between expression in patients with EsxB antibody-positive and drugresistance of the strains. Then to infer that EsxB protein can be as a candidate target of S.aureus vaccine, so for its use of the clinic further experimental on the basis.Methods:(1) The esxB gene DNA fragment was amplified by Polymerase ChainReaction(PCR) with designed specific primers esxB of S. aureus wild strain genomic DNAas a template.（2）The esxB gene was cloned into multiple cloning sites (BamH I and XhoI) in the pET-28a(+) prokaryotic expression vecto, to screen positive recombinant plasmids.(3) The plasmid was sequenced, to comparison of the result and esxB gene sequence.(4)The protein induced by pET-28a(+)/esxB recombinant plasmid was to identify by Westernblotting with anti-HisAb.(5) The prepared protein was used to immune in mice to obtainthe specific antiserum, and its titers were measured by ELISA and its specificity byWestern blotting.(6) The effective concentration of the antiserum were test by complementmediated antibody germicidal test.(7) The sera in50normal healthy controls,110patientswith other Staphylococcus infection and78cases of S. aureus infection were detected byindirected ELISA packed with EsxB protein.(8) The antimicrobial susceptibility tests ofthe related strains from the patients with EsxB positive-antibody were measured byautomatic microorganism system.Results:(1) The DNA fragment of the-315bp-length was amplified by PCR.(2)The positive-screened plasmid was recombinant cloned, and the expected size was in linewith the expectation. Then positive-plasmid was confirmed the sequencing, and it wascorrect.(3) The protein band was at about16KD and the purified protein was also in thesame position, confirmed as EsxB fusion protein.(4) The titer and specificity of theantiserum obtained by immune in mice were consonant with the expected results，and the effective bactericidal concentration was1:4000.(5) The number of patients with EsxBpositive-antibody in other Staphylococcus infection was7cases and its positive rate was6.4%; and the number of patients with positive-antibody in S. aureus infection was22andits positive rate was28.2%, then the difference was significant.(6) The multi-drug resistantof EsxB antibody-positive patients with S. aureus was significantly higher than that ofEsxB antibody-negative, and the MRSA was significantly increased, too.Conclusions:(1) The EsxB protein in S. aureus was prepared, purified andidentified,and it was consistent with the expected result.(2) Its titer and specificity ofEsxB protein antiserum were high, and its effective bactericidal titer reached therequirement.(3) The positive rate of EsxB protein antibody in patients with S. aureusinfection were significantly higher than the ones with the other staphylococcus.(4) Thetoxicity of S. aureus secreted EsxB protein is stronger, but also more prone to drugresistance.(5) EsxB protein can be a candidate target antigens of S. aureus vaccine, andwas the vital significance to the further research on the molecular pathogenesis, and alsolaid a good foundation for the research on vaccine.