The Study On Identification Of Protective Antigen In Fusobacterium Necrophorum And Gene Selection

Fusobacterium necrophorum is an important factor that leads to losing in feed of ruminant such as deer.cattle,sheep,etc.The best way to prevent and control it is vaccination. This study established mice model using FN(A)P2001, selected the immunogen and protective antigen, and proved that the hemolysin is the protective antigen and prime nosogenetic substance by sequencing of the amino acids of the protective antigen and gene selection.The result of this study administers to development of vaccine and study of immune mechanism.Below is the study:The mice were injected by suspensions that contained variant number of FN(A)p2001 from deer, observed state of infected mice every day.The result showed that FN(A)p2001 has strong infective ability.the MLD to mouse is 10⁶ cfu per mouse,and celiac injection is the best effective way. According to these,the mouse model is established.The antigens were respectively prepared using the supernate and the precipitation that were separated after FN(A)p2001 was broke up.The rabbits were injected by these antigens and the serum was collected. Immune component in the supernate and the precipitation were selected through SDS-PAGE and Western-blot. The positive protein bands were separated.The mice were respectively vaccinated by these proteins.The result gained through counter immunoelectrophoresis showed that FN(A) and FN(AB) respectively have three and one protein bands that have immunogenicity.The protein bands that have immunogenicity were cuted.The mice were respectively injected by these proteins.The immune effect was detected by counter immunoelectrophoresis.After four weeks,the mice were injected with corresponding FN(10⁷cfu per mouse).The result showed that 55KD antigen can induce effective immunity. Immunodiffusion between serum from survival of mice and antigens from broke up FN showed that there between antigens from FN (AB) and serum had not bands,but between antigens from five FN(A) strains and serum had bands.This result proved that five FN(A) strains have same antigenic determinant and can induce crossed immune reaction.The precipitation of two FN(A) strains were gained and diluted.After breaking FN(A),the supernate was salted out with 60%(NH₄)₂SO₄. The precipitation gained by dialysis was collected.The protective antigen was purified by column chromatography.The purpose bands were selected by 15%SDS-PAGE and Western-blot and transferred to PVDF membrane.The sequence of the amino acids in protein N-end showed that protective antigens from two FN(A) strains nearly have samesequence(sequencing ten amino acids.just first is variant).Corresponding sequence was not found in GenBank.This proved that this protein is a novel protein in FN.According to the sequence of the amino acids,primers were designed.The gene of the antigen was selected by random primer PCR,reverse PCR and enriching of purpose gene. Homologous analysis of the amino acid sequence, sequence contrast and the connection of ribonucleotide sequence were maked using BLAST and Clustal in NCBI and EBI.According to the conjecture of speciality and fuction of product of gene and the result of biological experiment,the gene that has 91% homologous sequence with hemolysin of Fusobacterium ATCC49256(gi27887417) is the purpose gene.