Display Of AIV HA Protein And GPV VP3 Protein On The Surface Of Bacillus Subtilis And Preliminary Evaluation Of Their Immunogenicity

Since the implementation of"reducing and prohibiting the use of antibiotics"in China’s aquaculture industry,probiotics have gradually replaced antibiotics in feed additives.Bacillus subtilis（B.subtilis）,a non-pathogenic probiotic to humans and animals,has been recognized as a candidate strain for use as a probiotic feed additive due to its beneficial effects on promoting animal growth and development,enhancing immunity,and maintaining gut microbiota balance.Furthermore,B.subtilis is an excellent bacterial carrier with strong spore resistance,which can stably exist in the gastrointestinal tract to deliver antigens and enhance mucosal immunity.H9N2 Avian Influenza Virus（H9N2 AIV）and Goose Parvovirus（GPV）are two common pathogens in poultry farming,and mixed infections often occur in large-scale farms.HA and VP3 are the main immunogenic proteins of H9N2 AIV and GPV,respectively,and are the main targets for vaccine research.This study used wild-type B.subtilis with probiotic properties as a bacterial carrier,displaying HA and VP3 proteins on the spore and bacterial surface of B.subtilis,respectively.The resistance marker gene was knocked out using homologous recombination,and the immuneogenicity was evaluated by oral administration to goslings,laying a foundation for the development of functional probiotics.1.Isolation and identification of Bacillus subtilis and determination of its probiotic propertiesIsolation,identification,and probiotic characterization of Bacillus subtilis strains from straw Five Bacillus subtilis strains were isolated and screened from soil samples through morphological observation,physiological and biochemical tests,and 16sr DNA identification.These strains exhibited tolerance to high temperature,p H and bile salt conditions,with RC-B2strain demonstrating the highest tolerance.When administered orally to goslings for 14 days at a dose of 2.0×10¹⁰ CFU/m L,the isolated strains significantly improved the growth rate and immune organ indices of the goslings’thymus,spleen,and bursa of Fabricius（P<0.05）,with RC-B2 showing the most pronounced effect.The Kirby-Bauer test indicated that most common antibiotics were effective against all strains,and the drug sensitivity of RC-B2 and RC-B4strains met the standards for bacterial drug sensitivity in feed additives.In vitro antibacterial tests revealed that the isolated strains could inhibit Escherichia coli and Staphylococcus aureus to varying degrees,with RC-B2 showing the strongest inhibitory ability.2.Bacillus subtilis displaying AIV HA protein on their spore surfaceThe Cot C spore coat protein gene was used as the anchoring gene,and the HA gene was fused with it using overlapping extension PCR to construct the recombinant integration plasmid p DG1730-Cot C-HA.The plasmid was transformed into wild-type B.subtilis RC-B2 through natural transformation,and the fusion gene was successfully integrated into the genomic DNA of the bacterium to obtain the recombinant strain BCH,as confirmed by PCR and amylase activity verification after screening on spectinomycin-resistant plates.Western blot and indirect immunofluorescence assays were performed using mouse anti-H9N2 AIV serum as the primary antibody,and a single band was detected at 65k Da for the recombinant spore coat protein.The recombinant spores exhibited a specific fluorescence signal under a fluorescence microscope,indicating that the HA protein was successfully expressed on the surface of the RC-B2 spores.3.GPV VP3 protein is displayed on the surface of vegetative Bacillus subtilis cellsUsing the membrane proteinase gene spo IIIJ of Bacillus subtilis as an anchor gene,a recombinant plasmid p DG1730-spo IIIJ-VP3 was constructed by fusion of the VP3 gene through overlapping extension PCR.The recombinant plasmid was transformed into the RC-B2 strain by natural transformation.The recombinant strain BSV was verified by PCR and amylase activity,and was used for Western blot and indirect immunofluorescence assays using mouse anti-VP3 serum as the primary antibody.The recombinant bacterial protein showed a single band at 55k Da on Western blot and displayed specific fluorescence signals under a fluorescence microscope,indicating the successful expression of the VP3 protein on the surface of vegetative Bacillus subtilis RC-B2 cells.4.Bacillus subtilis co-displaying HA and VP3 proteinsThe fusion gene spo IIIJ-VP3 was constructed by seamless cloning into the p DG1730-Cot C-HA vector,generating the recombinant integration vector p DG-CHSV.The p DG-CHSV was transformed into the strain RC-B2 using natural transformation to generate the recombinant strain BCHSV.Western blot analysis of spore coat and cell lysate proteins from the recombinant strain revealed a single band at 65k Da and 55k Da,respectively,indicating successful expression of the fusion protein.Indirect immunofluorescence assay showed specific fluorescence signals in both the recombinant spores and cells,indicating that the HA and VP3 proteins were expressed on the surface of the spores and cells of the recombinant strain BCHSV.5.Traceless deletion of Spe antibiotic resistance gene in recombinant Bacillus subtilisTo delete the antibiotic resistance marker gene Spe from the recombinant strain,homologous recombination was employed.The Spe resistance marker gene and its flanking homologous arms were PCR amplified and cloned into the Gram-positive bacteria knockout vector p MAD,resulting in the recombinant knockout plasmid p MAD-ΔSpe.The plasmid was naturally transformed into the BCHSV strain,and the recombinant strain BCHSV::ΔSpe was obtained through homologous recombination and blue-white screening.The deletion of the Spe resistance marker gene was confirmed by PCR analysis and spectinomycin sensitivity verification.6.Immunogenic evaluation of recombinant Bacillus subtilisRecombinant strains BCHSV::ΔSpe were prepared as spores and vegetative cells.Seven-day-old goslings were orally immunized with a dose of 2×10¹⁰ CFU/m L.ELISA tests showed that effective specific Ig G antibody levels were detected in both the recombinant spore and vegetative cell groups 14 days after the initial oral immunization.By day 28,serum Ig G antibody levels reached their peak and were significantly different from the RC-B2 and PBS control groups（P<0.01）.42 days after the initial oral immunization,the specific SIg A antibody levels in the recombinant spore and vegetative cell groups were significantly higher than those in the RC-B2 and PBS control groups（P<0.01）.These results indicate that the recombinant Bacillus subtilis spores can induce the body to produce effective specific Ig G and SIg A antibodies.In conclusion,this study demonstrated that probiotic Bacillus subtilis strains isolated and identified as live carriers successfully demonstrated the HA and VP3 protein,and goslings could produce specific humoral and mucosal immune responses after oral administration.