| Pasteurella multocida is a resident flora in the upper respiratory tract and ocular mucosa of animals.Endogenous infections often occur when the body is stressed or infected with other respiratory pathogens.Bovine Pasteurellosis is common in cattle that are stressed or yak.The immunogen of the existing inactivated vaccine for bovine haemorrhagic septicaemia is mainly the Pasteurella multocida Capsular Serotype B.However,a large number of studies have shown that bovine respiratory tract infections caused by Pasteurella multocida Capsular Serotype A is more common,and the cross-protection effect between different capsular serotypes is not ideal.In this study,pathogenic bacteria were isolated from the diseased dead bovine lungs and sick cattle nasal swabs with severe respiratory infections in two large-scale dairy farms in Heilongjiang Province,and screening the epitope of the Pasteurella multocida transferrin binding protein Tbp A has important practical significance for the development of high-efficiency vaccines to prevent and treat the disease.Firstly,the lungs of sick cows and the nasal swabs of sick cows were collected aseptically,and the pathogen isolation culture,staining microscopy and biochemical tests,bacterial 16 S r DNA and Omp H gene PCR identification were performed.The capsular serotype and virulence factors of the isolates were determined by PCR.The results showed that two isolated strains,WC16551 and LY01,were Pasteurella multocida capsular Serotype A,and the drug sensitivity test showed that strains were highly sensitive to Enrofloxacin and ciprofloxacin.22 virulence factors were detected,and both strains carried 19 virulence factors(oma87,fim A,omp A,ton B,exb D,Fur,hgb A,sod C,Pm HAS,Tbp A,hsf-2,hsf-1,Pfh A,ptf A,nan H,plp B,exb B,and tad D),there are three types that are not detected(tox A,hgb B,and nan B).The LD50 of isolated strains were 2.3×108 CFU/m L and 2.0 8×105 CFU/m L?Secondly,the prokaryotic expression plasmid of p ET-32a-Tbp A protein was constructed for protein-induced expression and Western Blot detection;Purification of proteins with Ni-NTA,protein renaturation and concentration.The immunogen was prepared by using ISA206 adjuvant,and the mice were immunized to prepare high serum-free serum,followed by antibody purification.The results showed that the target protein was 104 KDa,and the expression of protein was higher when 37? induced 5 h at IPTG concentration of 0.1 m M.The target protein were expressed as inclusion bodies,and has good antigenicity.Thirdly,construct the fifteen peptide library plasmid and transfer the plasmid into the TG1 competent state with electric.The phage random fifteen-peptide library was prepared,then the diversity and reservoir capacity of peptide library were detected,the phage titer was calculated,and serum were affinity washed three times.The phage screened was sequenced,and homology analysis was performed with the Tbp A protein to finally obtain a Tbp A protein epitope.The results showed that fifteen-peptide plasmid and random fifteen-peptide library were successfully constructed,The capacity of peptide library was up to1.32×109 CFU/m L.After multi-anti-panning screening,two simulated binding sites of G103 and W232 were identified,and the epitopes were mainly distributed in the 95-265 and 573-765 protein sequences.In summary,this study determined that the pathogen of the onset cattle was Pasteurella multocida Capsular Serotype A,and successfully screened the Tbp A protein antigen epitope,which laid a foundation for the development of genetic engineering subunit vaccine for bovine Pasteurella multocida. |
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