Molecular Characterization And Protective Immunity Of Four Protein Of Eimeria Maxima

Coccidiosis is recognized as a major parasitic disease in chickens.Coccidiosis seriously impairs feed conversion efficiency,leading to loss of productivity.Eimeria maxima(E.maxima)is considered to be one of the highly immunogenic specie with respect to chicken coccidiosis.In the present study,four E.maxima sporozoites soluble proteins specific for invasion process,were cloned and their protective immune efficiency was evaluated.The findings of this study could help to screen new antigen candidate for developing vaccines against E.maxima.1.Molecular characterization and the protective immunity evaluation of E.maxima surface antigen proteinE.maxima SAG(EmSAG)open reading frame(ORF)which consisted of 645 bp encoding a protein of 214 amino acids was successfully amplified and sequenced.Subsequently,the EmSAG ORF was subcloned into pET-32a(+)and pVAXl,respectively.RT-PCR and immunoblot analyses confirmed that the target gene was successfully transcribed and expressed in vivo.Immunofluorescence analysis showed that EmSAG was expressed in both the sporozoites and merozoites.The animal experiments demonstrated that both rEmSAG and pVAX1-SAG could clearly alleviate jejunum lesions and body weight loss.The EmSAG vaccines could increase oocyst decrease ratio,as well as produce an anticoccidial index of more than 170.The percentages of CD4+and CD8+ in both the EmSAG immunized groups were much higher,when compared with those of PBS,pET32a(+),and pVAX1 controls(P<0.05).Similarly,the anti-EmSAG antibody titers of both rEmSAG and pVAX1-SAG immunized groups showed higher levels compared with those of PBS,pET32a(+),and pVAX1 controls(P<0.05).The IFN-γ,TGF-β,IL-17 and IL-4,levels showed significant increments in the rEmSAG and pVAX1-SAG immunized groups,when compared with those in the negative controls(P<0.05).These results demonstrated that EmSAG could be used as a promising antigen candidate for developing vaccines against E.maxima,2.Cloning,expression and the protctive immunity evaluation of E.maxima elongation factor2 proteinE.maxima elongation factor2 gene(EmEF2)open reading frame(ORF)which consisted of 2499 bp encoding a protein of 832 amino acids was successfully amplified and sequenced.Subsequently,the EmEF2 ORF was subcloned into pET-32a(+)and pVAX1,respectively.RT-PCR and immunoblot analyses confirmed that the target gene was successfully transcribed and expressed in vivo.The animal experiments demonstrated that both rEmEF2 and pVAX1-EF2 could clearly alleviate jejunum lesions and body weight loss.The EmEF2 vaccines could increase oocyst decrease ratio,as well as produce an anticoccidial index of more than 160.The percentages of CD4+ in both the Em EF2 immunized groups were much higher,when compared with those of PBS,pET32a(+),and pVAX1 controls(P<0.05).Similarly,the anti-EmEF2 antibody titers of both rEmEF2 and pVAX1-EF2 immunized groups showed higher levels compared with those of PBS,pET32a(+),and pVAX1 controls(P<0.05).The IFN-y levels showed significant increments in the rEmEF2 and pVAX1-EF2 immunized groups,when compared with those in the negative controls(P<0.05).These results demonstrated that EmEF2 could be used as a promising antigen candidate for developing vaccines against E.maxima.3.Molecular characterization and the protective immunity evaluation of E.maxima 14-3-3 proteinEimeria maxima 14-3-3(Eml4-3-3)open reading frame(ORF)which consisted of 861 bp encoding a protein of 286 amino acids was successfully amplified and sequenced.Subsequently,the Em 14-3-3 ORF was subcloned into pET-32a(+)and pVAX1,respectively.RT-PCR and immunoblot analyses confirmed that the target gene was successfully transcribed and expressed in vivo.Immunofluorescence analysis showed that Em 14-3-3 was expressed in both the sporozoites and merozoites.The animal experiments demonstrated that both rEml4-3-3 and pVAX1-14-3-3 could clearly alleviate jejunum lesions and body weight loss.The Em14-3-3 vaccines could increase oocyst decrease ratio,as well as produce an anticoccidial index of more than 165.The percentages of CD4+ in both the Em 14-3-3 immunized groups were much higher,when compared with those of PBS,pET32a(+),and pVAX1 controls(P<0.05).Similarly,the anti-Eml4-3-3 antibody titers of both rEml4-3-3 and pVAX1-14-3-3 immunized groups showed higher levels compared with those of PBS,pET32a(+),and pVAXl controls(P<0.05).The IFN-y and tumor growth factor-β(TGF-β)levels showed significant increments in the rEm14-3-3 and pVAX1-14-3-3 immunized groups,when compared with those in the negative controls(P<0.05).These results demonstrated that Em14-3-3 could be used as a promisingantigen candidate for developing vaccines against E.maxima.4.Molecular characterization and the protetive immunity evaluation of E.maxima rhoptry protein proteinE.maxima rhoptry protein(EmRON)open reading frame(ORF)which consisted of 2475 bp encoding a protein of 824 amino acids was successfully amplified and sequenced.Subsequently,the EmRON ORF was subcloned into pET-32a(+)and pVAXl,respectively.RT-PCR and immunoblot analyses confirmed that the target gene was successfully transcribed and expressed in vivo.The animal experiments demonstrated that both rEmRON and pVAX1-RON could clearly alleviate jejunum lesions and body weight loss.The EmRON vaccines could increase oocyst decrease ratio,as well as produce an anticoccidial index of more than 160.The percentages of CD4+in both the EmRON immunized groups were much higher,when compared with those of PBS,pET32a(+),and pVAX1 controls(P<0.05).Similarly,the anti-EmRON antibody titers of both rEmRON and pVAX1-RON immunized groups showed higher levels compared with those of PBS,pET32a(+),and pVAX1 controls(P<0.05).The IFN-γ,TGF-β and IL-4 levels showedsignificant increments in the rEmRON and pVAX1-RON immunized groups,when compared with those in the negative controls(P<0.05).These results demonstrated that EmRON could be used as a promising antigen candidate for developing vaccines against E.maxima.