Prokaryotic Expression,Polyclonal Antibody Preparation and Activity Identification of Helicase RuvA from Mycoplasma hyopneumoniae
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
万方数据
维普
