抗球虫病枯草芽孢杆菌活载体口服疫苗ET seven的构建与鉴定OA

[Objective]This study aimed to construct a ET seven recombinant Bacillus subtilis plasmid encoding seven Eimeria tenella antigens(EtAMA1,EtAMA2,EtMIC3,EtMIC4,EtMIC13,EtROP5,and EtSAG1),and verify the expression and surface display of the fusion protein on bacterial spores,providing research ideas for the development of new oral coccidiosis vaccines.[Method]Using the ET seven eukaryotic expression plasmid and Bacillus subtilis genomic DNA,ET seven gene and the spore capsid protein encoding gene CotB were amplified,respectively.They were fused with the vector pDG364 using homologous recombination technology to construct the integrated recombinant plasmid pDG364-CoTB-ET seven,which was then transformed into Bacillus subtilis WB800N competent cells.The expression of ET seven fusion protein was detected by Western blotting and indirect immunofluorescence methods.Partridge chickens were immunized with recombinant Bacillus subtilis spores,and serum levels of specific IgG antibodies were detected by indirect ELISA.The immunoprotective effect was evaluated by a challenge test.[Result]PCR amplification obtained ET seven and CotB genes with sizes of 1 761 and 1 124 bp,respectively.The integrated recombinant plasmid pDG364-CotB-ET seven was successfully constructed and transferred into Bacillus subtilis WB800N competent cells,obtaining a stable and heritable recombinant Bacillus subtilis WB800N-CotB-ET seven.Western blotting results showed that the recombinant Bacillus subtilis WB800N-CotB-ET seven had a specific band at 90.6 ku,and the high immune serum of ET seven protein could specifically bind to the fusion expression protein,confirming the correct expression of the fusion protein.Indirect immunofluorescence detection results showed that specific fluorescent signals were detected on the spores.ELISA test results showed that specific IgG antibodies in the serum of immunized chickens were highly positive after secondary immunization.The results of the insect attack test showed that compared with mock-infected control group,the oocyst per gram of chickens immunized with recombinant Bacillus subtilis spore was decreased significantly(P<0.05).[Conclusion]This study successfully constructed a recombinant Bacillus subtilis strain displaying ET seven fusion protein on its surface,confirming that the recombinant spore had good immunogenicity and could induce specific immune protection,laying a foundation for the development of new oral vaccines against coccidiosis.