Procaryotic Expression and Analysis of the EnGPX of Eimeria necatrix
The aim of this paper was to study the antigenicity of glutathione peroxidase EnGPX and its subcellular localization in Eimeria necatrix.Total RNA was extracted from the gameto-phyte of E.necatrix(Yangzhou strain),and the ORF coding sequence of EnGPX was amplified using RT-PCR.The prokaryotic expression plasmid pET-28a(+)-EnGPX was constructed and transformed into BL21(DE3)for in vitro expression,additionally,a mouse anti-rEnGPX poly-clonal antibody was prepared and used to analyze the recombinant protein through Western blot-ting,and laser confocal immunofluorescence localization analysis.The study found that the cod-ing region of the EnGPX gene was 753 base pairs in length and encoded a protein consisting of 250 amino acids.The resulting recombinant protein had a molecular weight of around 30 ku and was predominantly present in the form of inclusion bodies.The recombinant protein exhibited fa-vorable reactivity and cross-reactivity,as it was recognized by a 6XHIS-tagged monoclonal anti-body,a mouse anti-rEnGPX polyclonal antibody,and convalescent serum from E.necatrix,E.maxima,and E.tenella.EnGPX was detected in natural gametophyte proteins,with the enco-ded protein primarily localized to the type Ⅱ wall-forming body(WFBII)of the gametophyte and oocyst wall.This study successfully cloned and expressed the glutathione peroxidase(EnGPX)of E.necatrix.The recombinant protein exhibited good reactivity,and natural EnGPX protein was found to be localized on the gametophyte and oocyst wall.The results shed light on the molecular mechanism of EnGPX involvement in oocyst wall formation and identify potential targets for the development of novel subunit vaccines against coccidia.
Eimeria necatrixEnGPXcloning and expressionreactivityimmunofluorescence locali-zation
万方数据
维普
