Isolation And Identification Of Pasteurella Multocida And Cloning And Expression Of OmpW And TbpA Proteins

Pasteurella multocida(Pm)is kind of septic gram-negative pathogen bacteria,which can infect a variety of animals and cause different degrees of damage.It mainly caused swine pneumonia and swine atrophic rhinitis,which brought huge economic losses to the pig industry.The prevention and control of the disease mainly relies on drug treatment and vaccine immunization,but in recent years,the abuse of antibiotics has led to a large number of drug resistance problems,which undoubtedly increases the difficulty of the prevention and control of porcine pasteurellosis.Therefore,in this experiment,the oral and nasal swabs and tissue samples of clinically ill pigs were collected to isolate,identify and biological characteristics analysis of Pasteurella multocida from large-scale farms in different regions,so as to evaluated the epidemic dominant serotype strains of Pasteurella multocida in pig farms in some regions of China.Through bioinformatics analysis,the highly conserved ompW and tbpA genes in different species of Pasteurella multocida and different serotype strains of Pasteurella multocida were found out.OmpW and TbpA proteins were cloned and expression by prokaryotic expression,so as to provide a basis for candidate immunogenic proteins of Pasteurella multocida from pigs.The specific contents are as follows:1.Isolation and identification of Pasteurella multocidaTo explore the prevalence of Pasteurella multocida in large-scale pig farms,firstly,the oral and nasal swabs and tissue samples of pigs with respiratory symptoms from largescale farms in Guangxi,Hubei and Inner Mongolia were isolated and cultured,stained and microscopic examination,biochemical tests and PCR detection and identification.The PCR results showed that six strains of Pasteurella multocida were isolated,including one strain of capsule serotype A(named GX-Pm A)and five strains of capsule serotype D(named GXPm D1,GX-Pm D2,GX-Pm D3,GX-Pm D4,NMG-Pm D5).Then twenty kinds of common drugs were selected for drug susceptibility test.The results showed that GX-Pm D1,GXPm D2,GX-Pm D3,GX-Pm D4 had no drug resistance,while NMG-Pm D5 showed obvious resistance to neomycin and gentamicin.The results mouse pathogenicity test showed that the virulence of the capsule serotype A strain was stronger than that of the capsule serotype D strain.Through the number,time and pathological changes of mice after challenge,the GX-Pm A and GX-Pm D4 strains with strong virulence were successfully screened.2.Cloning and expression of candidate immunogenic proteins OmpW and TbpA of Pasteurella multocidaThrough bioinformatics analysis of ompW and tbpA genes and their expression products,it is confirmed that the two genes are highly conservative.The analysis results of protein physical and chemical properties show that OmpW protein belongs to hydrophobic protein and TbpA protein belongs to hydrophilic protein,and the transmembrane region,signal peptide,antigen epitope,secondary structure and tertiary structure of the two proteins were successfully predicted.Based on this,the recombinant expression vectors p ET28 aompW,p ET28a-tbpA,p SUMO-ompW and p SUMO-tbpA of Pasteurella multocida from pigs were successfully constructed.The soluble expression of OmpW was successfully obtained through screening and condition optimization,which laid a foundation for the development of a new Pasteurella multocida vaccine.