Cloning,Expression And Immunoprotective Analysis Of Glutathione Peroxidase EnGPX In Eimeria Necatrix

Coccidiosis is a worldwide poultry disease caused by one or more of at least seven species of Eimeria in the genus Apicomplexa,and with morbidity and mortality rates up to 80%.The main symptoms include depression,fluffy feathers,standing together,reduced drinking water,weight loss and hemorrhagic diarrhea.Among them,E.tenella has the strongest pathogenicity,followed by E.necatrix.After infection,it can lead to severe intestinal lesions,severe hemorrhagic diarrhea and high mortality,and causing huge economic losses to the poultry industry.At present,the prevention and treatment of chicken coccidiosis mainly preventively use anticoccidial drugs and vaccine immunization.However,the emergence of drug-resistant strains and drug residues make the prevention and treatment of anticoccidial drugs facing challenge.Therefore,it is urgent to develop new strategies to control coccidiosis.In the early stage,our research group conducted a comparative proteomic analysis of the wall forming body protein and oocyst wall protein that E.necatrix coccidia,and obtained the differentially expressed protein EnGPX,which has glutathione peroxidase activity and catalyzes a series of redox reactions,such as glutathione reduction of H2O2 and promotes the formation of disulfide bonds and dityrosine cross-linking between oocyst wall proteins,which play a role in the formation of oocyst wall.In this study,the EnGPX coding gene was cloned and sequenced,and a prokaryotic expression vector was constructed for in vitro expression,immunofluorescence localization analysis and its transcriptional dynamic analysis in worm body.Finally,the immunoprotective effect of rEnGPX immunized chickens was analyzed.This study will provide a new target antigen for the development of a new coccidial subunit vaccine.1.Cloning and sequence analysis of glutathione peroxidase En GPX from Eimeria necatrixThe gametophyte of E.necatrix was isolated and purified to extract total RNA.The EnGPX gene was amplified by RT-PCR,and was ligated to the pGEM-T-Easy vector to transform into DH5α.The positive clones were screened by blue-white screening to be sequenced.The EnGPX gene was 753 bp,encoding a total of 250 amino acids,with a molecular weight of about 27.7 ku and an isoelectric point of 9.46.It contains 12 antigenic determinants and has no signal peptide.It has 99.1%to E.necatrix Houghton strain EnGPX gene（XM₀13581216）.The amino acid function prediction of EnGPX showed that it had glutathione peroxidase activity,and the 96～111 amino acid was its GPX active site.It contained a conserved cysteine residue Cys108.which participated in the redox reaction to form a stable intermolecular disulfide bond,and also contained a thioredoxin domain（70～235 animo acid）.The active center of the domain uses FAD to catalyze the redox mechanism of two Cys-SH reversible oxidation to disulfide disulfide bonds by NADPH to interact with the protein and catalyze the formation and rearrangement of disulfide bonds during protein folding.2.Prokaryotic expression of glutathione peroxidase EnGPX of E.necatrix and its transcriptional dynamic analysisAccording to the EnGPX gene sequence,two restriction sites Not I and EcoR I were selected,and specific primers were designed.The target gene was amplified by PCR using the successfully sequenced pGEM-T-Easy-EnGPX positive plasmid as a template.The PCR product and pET-28a（+）vector were digested with Not I and EcoR I to be ligated to each other,and transformed into competent cells BL21（DE3）for in vitro expression.The recombinant protein rEnGPX mainly existed in the form of inclusion bodies,with a size of about 30 ku.The optimal expression condition was a final glucose concentration of 0.5%,37℃ for 2.5 h;final IPTG concentration of 0.20 mmol/L,37℃ for 5 h.The purified rEnGPX was used to immunize mice to prepare mouse polyclonal antibody,and the serum antibody titer was good verified by indirect ELISA.The results of Western-blot showed that rEnGPX could be recognized by 6×HIS tag monoclonal antibody,and could be recognized by rehabilitation serum from E.necatrix,E.maxima and E.tenella disease chicken,with good reactogenicity and cross-reactivity.The results of immunofluorescence localization showed that the EnGPX mainly distributed in the wall forming body II in the gametophyte and oocyst wall.The transcription level of EnGPX was low in the second-generation merozoites and the thirdgeneration merozoites,high in gametophytes,unsporulated oocysts,and the highest in unsporulated oocysts.3.Evaluation of immune protection effect of rEnGPXThe purified refolded rEnGPX protein was mixed with Freund’s complete adjuvant and Freund’s incomplete adjuvant to immunize chickens.The experiment was designed as five groups:rEnGPX high-dose group（200 μg）,rEnGPX medium-dose group（100 μg）,rEnGPX low-dose group（50 μg）,non-immunized challenge group and non-immunized non-challenge group.The animal immune protection effect of rEnGPX was evaluated by survival rate,relative weight gain rate,relative oocyst yield,oocyst reduction rate,lesion score,anticoccidial index,oocyst sporulation rate and humoral immunity level.The results showed that immunized chicken flocks treated with rEnGPX protein exhibited a measurable increase in antibody production without compromising growth and development.Additionally,rEnGPX treatment mitigated intestinal lesions and reduced both oocyst output and sporulation rate.Among them,the high dose group of rEnGPX was found to provide the most effective immune protection,with an anticoccidial index of 161.81,indicating a moderate anticoccidial effect and the lowest oocyst sporulation rate.