Study On The Influence Of ?-lactam Antibiotics Resistance On The NanP Expression In T.pyogenes

As a conditional pathogen,T.pyogenes is one of the major pathogenic bacteria causing cow endometritis,which leads to serious economic loss in the breeding industry.Beta-lactam antibiotics as the first choice for clinical treatment of infections caused by T.pyogenes,but due to overuse or even unreasonable use,antibiotic resistant strains become a real problem.Antibiotics resistance of bacteria not only reduces the efficacy of drugs,but also affects the virulence in bacteria.The virulence of T.pyogenes is determined by multiple virulence factors.Such as hemolysin(PLO),collagen binding protein(CbpA),FimA(FimA,FimC and FimG),and neuraminidase H(NanH)and P(NanP).Among them,NanP is one of the important virulence factors.In this study,the difference of the expression of NanP in ?-lactam antibiotics-resistant T.pyogenes isolates will provide a theoretical foundation for further study on the influence of antibiotics resistance on pathogenicity and clinical effective treatment.In this study,the structural domain,signal peptide and transmembrane region of NanP were analyzed and predicted by bioinformatics method.The result showed that that nanP gene encode 1737 amino acids,the relative molecular mass was 186.8 kDa,the theoretical isoelectric point was 5.56,and the grand average of hydropathicity(GRAVY)-0.351.The gene does not contain signal peptide,contains two transmembrane regions,42 phosphorylation sites and a total of 7 conserved domains.Analysis of sequence specificity found that its sequence specificity is good.By predicting the hydrophilicity,plasticity,accessibility and epitope of NanP,50-270 amino acids were selected for prokaryotic expression.These evidences provided theoretical basis for subsequent experiments.The prokaryotic expression plasmid pEASY-Blunt E1-nanP was successfully constructed,and the soluble protein was expressed in Escherichia coli.The expressed protein was purified by Ni-NTA kit.The purified His-NanP recombinant protein was immunized with New Zealand white rabbit to prepare rabbit polyclonal antibody against T.pyogenes NanP.The prepared polyclonal antibody has good reactivity with T.pyogenes NanP,and the titer of the antibody was 1: 64000.Western-blot technique was used to detect the expression of NanP in different anti-resistant phenotypes of T.pyogenes,and the expression of NanP was compared with that of standard strain.The results showed that the expression of NanP decreased as the the increase of resistance to ?-lactam antibiotics.This study will provide a theoretical foundation for further study on the influence of acquired resistance on the pathogenicity of T.pyogenes.