Study On Eukaryotic Expression And Immunogenicity Of Eimeria Tenella AMA-1 And SO7 Gene

Immunization against coccidiostats and live vaccines is currently the main means of preventing and treating chicken coccidiosis.However,there are problems such as drug resistance of coccidia,drug residues,and poor safety of coccidia live vaccines,and the immune effect is not ideal.Therefore,the development of a new coccidiosis vaccine has become the main development direction for the prevention of chicken coccidiosis.In this study,the AMA-1 gene and the SO7 gene of E.tenella HB strain were ligated in tandem with IL-2 and pPIC9,respectively,and the immunoregulatory eukaryotic expression vectors GS115-pPIC9-IL-2-AMA-1 and GS115-pPIC9-IL-2-SO7 were constructed,which were used for protein expression,and the purified recombinant proteins IL-2-AMA-1 and IL-2-SO7 were used to conduct immunoprotective tests on chickens to evaluate their anticoccidial effect,immunogenicity and antioxidant capacity.To provide a theoretical basis for the future development of Eimeria tenella nucleic acid vaccine..The test results are as follows:1.The AMA-1 gene,SO7 gene cloning and identification and bioinformatics analysis of E.tenella HB strain showed that the AMA-1 gene and SO7 gene of E.tenella strain were successfully cloned.Among them,AMA-1 gene is 1 617 bp in length and encodes 535 amino acids.The predicted protein has signal peptide and transmembrane structure.The full length of SO7 gene is 645 bp,encoding 215 amino acids.The predicted protein has signal peptide but no transmembrane structure.2.E.tenella HB strain immunoregulatory eukaryotic expression vector GS115-pPIC9-IL-2-AMA-1,GS115-pPIC9-IL-2-SO7 construction results were showed that the E.tenella HB strain AMA-1 gene and SO7 gene were successfully recombined into the yeast cell genome to construct an immunoregulatory eukaryotic expression vector.3.The expression and purification of recombinant protein IL-2-AMA-1 and recombinant protein IL-2-SO7 showed that the protein has good immunogenicity.The recombinant protein IL-2-AMA-1 has a size of 153 kDa and the recombinant protein IL-2-SO7 has a size of 89 kDa.4.The anti-coccidial effect,immunogenicity and antioxidant capacity of the recombinant protein IL-2-AMA-1 and recombinant protein IL-2-SO7 of E.tenella HB strain showed that the ACI of the positive control group was only 130.68.The ACI of each immunized group was higher than that of the positive control group,and the immune recombinant protein IL-2-SO7 had the highest effect.When the immunization dose reached 50?g and 100?g,the ACI was 169.44 and 174.24,respectively,and the anticoccidial effect was good.The recombinant recombinant protein IL-2-AMA-1 group was immunized with the 150 ?g recombinant protein IL-2-SO7 group.Through the determination of humoral immunity level,cellular immunity level,T-SOD,GSHPx and T-AOC activity,it was shown that the two recombinant proteins have good immunogenicity and antioxidant capacity.The immunologically suitable dose of recombinant protein IL-2-AMA-1 was 50 ?g/head,and the recombinant protein IL-2-SO7 was 100 ?g/head.