| Chicken coccidiosis is a parasitic protozoal disease caused by Apicompiexa and Eimeria in the intestinal epithelial cells of the gastrointestinal.There are seven main species infecting chicken,including E.necatrix,E.tenella,E.acervulina,E.brunetti,E.praecox,E.maxima and E.mitis.They are highly adaptable,widely distributed,and can reproduce asexually and sexually in chickens and infect different areas of the intestinal tract.Sick chickens mostly show loss of appetite,weight loss,depression,shrunken head and hemorrhagic diarrhea,which seriously endangers the healthy development of poultry industry.E.necatrix parasites in the middle 1/3 of the small intestine,and has more serious pathogenicity.At present,the clinical prevention and control of chicken coccidiosis mainly relies on traditional drug treatment.However,the former is not widely used due to the problems of drug-resistant strains,veterinary drug residues and antibiotic bans,which makes the control of chicken coccidiosis turned to vaccine immunization in some degree.Combined with the differential proteomic analysis of wall forming body and oocyst wall proteins of E.necatrix in the previous research of our group,the expression of EnOXIO1（Oxioreductase of E.necatrix）was highly expressed in the wall forming body and lowly expressed in the oocyst wall.According to the functional annotation of GO molecules,EnOXIO1 protein may participate in the redox reaction of CH-OH group releasing hydrogen ions,which promotes tyrosine cross-linking to form a solid oocyst wall.Therefore,this study cloned EnOXIO1 gene in vitro.Prokaryotic expression,immunolocalization analysis,and immune protective evaluation were employed to evaluate the function and characterization of the recombinant protein rEnOXIO1,aiming to lay a solid theoretical foundation for the development of a new subunit vaccine against chicken coccidiosis.1.Cloning and sequence analysis of EnOXIO1The the total RNA was extracted from E.necatrix gametocytes to amplify EnOXIO1 gene by RT-PCR.The "A" base was added to the 3’ end of the purified PCR product and cloned into pGEM-T easy vector for sequencing and sequence analysis.The full length of EnOXIO1 gene was 2535 bp,which has a complete open reading frame.It encoded a total of 844 amino acids with no signal peptide,and contained 34 antigenic decision clusters.Its molecular mass was about 93.51 ku and isoelectric point（PI）was 5.917.Functional prediction analysis of the amino acids revealed that there is a GMC（Glucose-Methanol-Choline）domain at each amino acid terminus,with an amino-terminal size of 47 amino acids（amino acids 158-204）and a carboxyl-terminal size of 70 amino acids（amino acids 765-834）,and is highly conserved.There is a FAD/NAD binding domain inside the amino acid,which covers two GMC structural domains.These structural domains are predicted to be closely related to the formation of disulfide bonds between amino acids in redox reactions.The homology of EnOXIO1 with other oxidoreductases genes of the genus Eimeria ranged from 73.6%to 99.6%.In conclusion,the obtained EnOXIO1 gene is an oxidoreductase encoding gene with high interspecific conservation.2.Prokaryotic expression of the oxidoreductase EnOXIO1 gene and Expression dynamics analysisThe positive recombinant plasmid pGEM-T-Easy-EnOXIO1 was cutted with EcoR Ⅰand Not Ⅰ to construct the prokaryotic expression vector pET-28a（+）-EnOXIO1,which was transformed into E.coli BL21（DE3）and expressed in vitro.The recombinant protein was purified.SDS-PAGE analysis showed that the recombinantly expressed protein was mainly in inclusion bodies with a size of about 100 ku,while a theoretical molecular mass size of about 93.51 ku.Western-blot analysis showed that the recombinant protein could be specifically recognized by 6×HIS labeled monoclonal antibody,the mouse anti-recombinant proteins polyclonal antibody,and the recovery serum from chickens infected with E.necatrix,E.tenella and E.acervulina sporulated oocysts,respectively.These results indicated that the recombinant protein had good antigenicity and cross-reactogenicity.Likewise,EnOXIO1 protein was detected in natural gametocyte proteins.Laser confocal immunofluorescence localization results showed that the EnOXIO1 protein mainly existed in the wall forming body in the gametocytes and participated in the formation of the oocyst wall.Real-time fluorescence quantitative PCR analysis showed that EnOXIO1 was transcribed at the highest level in the gametocyte stage.Western blot for protein expression showed that EnOXIO1 protein was highly expressed in the gametocyte stage than in other developmental stages.3.The immune protective effect of rEnOXIO1The recombinant proteins were mixed with adjuvant to prepare immunogen,and the chickens were immunized twice,respectively.The chickens were devided into three immune dose groups（200 μg,100 μg and 50 μg）.The two groups were used as positive（unimmunized and challenged with oocysts）and negative（unimmunized and unchallenged with oocysts）controls,respectively.The protective efficacy was evaluated based on the oocyst production,oocyst reduction rate,lesion score,body weight gain,disease mortality rate and humoral immunity level.The results showed that the rEnOXIO1 high dose group（200 μg/bird）had better immune protection compared with other immunization doses.It stimulated a certain level of humoral immunity in chickens,increased average weight gain,reduced oocyst production and lesion score,and had a moderate anticoccidial effect with an anticoccidial index of 172.36. |
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