| Eimeria stiedae parasites in the liver,invades the bile duct epithelial cells,and causes severe liver coccidiosis.Rabbit coccidiosis is mainly controlled by anticoccidial drugs.With the emergence of drug-resistant strains and the advancement of reducing,replacing,and banning antibiotics in feed,the limitations of drug control gradually appear.Rabbit coccidiosis vaccine is still in the research and development stage,and no commercial vaccine has been registered and marketed.In this study,the prokaryotic expression of Eimeria stiedae ubiquitin-conjugating enzyme(UCE),elongation factor G(EFG),and dense granule protein(DG32)recombinant proteins and the evaluation of the immune protection effect were carried out to provide a reference for the research of Eimeria stiedae recombinant subunit vaccines.1.Transcriptional analysis and prokaryotic expression of UCE,EFG and DG32genes in Eimeria stiedaeIn this study,we successfully amplified Es-UCE,Es-EFG and Es-DG32 genes and performed multiple sequence alignment of the amino acid sequences of the three genes.The transcript levels of the three genes at different developmental stages(Unsporulated oocysts,Sporulated oocysts,Merozoites,Gametophytes)were analyzed using relative quantitative real-time PCR analysis(q PCR).Then prokaryotic expression,protein purification and Western blot analysis of the three genes were performed.The results showed that all three genes were highly expressed in the merozoites stage,and the amino acid sequences of UCE and EFG had high similarity,while the amino acid sequence of DG32 had low similarity.The recombinant UCE protein was expressed in the supernatant,while the EFG recombinant protein and the DG32 recombinant protein were expressed in the inclusion bodies;all three proteins were recognized by the positive serum of rabbits infected with Eimeria stiedae and had good immune responses.2.Evaluation of the immune protection effect of Eimeria stiedae(r Es-UCE、r Es-EFG、r Es-DG32)recombinant protein on rabbitsAfter a preliminary evaluation of the immune protection effect of three recombinant proteins of Eimeria stiedae(r Es-UCE,r Es-EFG and r Es-DG32)on rabbits,r Es-UCE was screened out for the immune protection test.Forty-two 45-day-old coccidia-free rabbits were divided into five groups(blank control group,challenge control group,vector protein control group,Quil-A adjuvant control group and r Es-UCE immunization group,8-10 rabbits in each group).The first immunization was performed by subcutaneous injection in the neck,and the second immunization was performed at the same dose 14 days later.14 days after the second immunization,all coccidia-free rabbits in each group except the blank control group were orally infected with Eimeria stiedae sporulated oocysts.After infection,the clinical symptoms,weighing,blood and fecal samples of the rabbits in each group were regularly observed,and necropsy was performed 21 days after the infection.The average weight gain of the r Es-UCE immunized group was 536±85.95 g,which was significantly greater than that of the challenge control group(P<0.05).The oocyst per gram was 0.26×10~6and average liver index was 3.95,which were significantly lower than those of the challenge control group(P<0.05).The relative weight gain rate of the r Es-UCE immunized group was90.69%,the oocyst reduction rate was 78.81%and the feed conversion ratio decreased.After immunization with r Es-UCE,the levels of IL-2,IL-10,IL-17,IFN-γand specific Ig G antibody in serum were significantly increased(P<0.05).Immunization with r Es-UCE induced cellular and humoral immune responses in rabbits. |
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