Homologous recombination mediated antigenic variation plays an important role in the frequent persistent infection of Mycoplasma hyopneumoniae of pigs.Allosteric regulation of Holliday junctions by helicase RuvA is a key step involved in homologous recombination.Given that,this study aims at expressing RuvAMhp recombinant protein,identifying its DNA binding ac-tivity and preparing polyclonal antibody against RuvAMhp.Prokaryotic expression plasmid pET21a-RuvA was constructed by molecular cloning.Following expression induction and purifi-cation,the RuvAMhp recombinant protein was obtained;The polyclonal antibody against RuvAMhp was prepared by immunizing rabbit,and the titer and specificity were determined by indirect ELISA and Western blot;The DNA binding activity of RuvAMhp was analyzed by electrophoretic mobility shift assay and surface plasmon resonance technique;The oligomerization of RuvAMhp was determined by electrophoretic mobility shift assay combined Western blot.RuvAMhp recombi-nant protein expressed by prokaryotic expression was about 26 ku;The prepared polyclonal anti-body against RuvAMhp had good specificity and high titer of 1∶256 000;RuvAMhp has strong DNA binding activity with an affinity of 624.4 pmol·L-1(KD)for Holliday junctions,and forms stable complexes with it mainly in octamers.In this study,the prokaryotic expression,polyclonal anti-body preparation and activity identification of RuvAMhp were achieved,which laid a foundation for further exploration of the molecular mechanism of helicase RuvA mediating antigenic variation of Mycoplasma hyopneumoniae by homologous recombination regulation.
国家科技期刊平台
NSTL
维普
万方数据
