Serological Investigation Of Five Important Epidemics Of Mutton Sheep And Prokaryotic Expression Of Pasteurella Multocida OmpA

Disease is one of the factors that affect the development of sheep and goats.The In each year,the economic losses caused by the occurrence of the epidemic are very serious.In this study,serological surveys of five epidemics were conducted on sheep serum samples from four provinces and autonomous regions in Gansu,Inner Mongolia,Shaanxi and Xinjiang using ELISA detection methods.The lung tissues of sheep lesions were collected,and the pathogenic bacteria were separated and identified by means of isolation culture and molecular biology methods,and Pasteurella multocida isolates were obtained.Biochemical experiments and drug sensitivity experiments were carried out on the isolates to explore the growth characteristics of the isolates.Due to the large number of types of Pasteurella multocida serum antigens and capsular antigens,different strains have different immunogenicity.Therefore,in this study,the OmpA gene with high homology was selected for prokaryotic expression,and the expression conditions of the protein were initially explored to lay the foundation for subsequent vaccine research.The main experimental results are as follows:1.Serological investigation of five kinds of diseases.450 sheep serum samples from Gansu,Inner Mongolia,Shaanxi and Xinjiang were collected,and five antibodies in the serum were detected by ELISA kit.The experiment found that the positive rates of infectious pleural pneumonia antibody and pasteurella antibody were higher,98% and 94.22%,respectively.The positive rates of antibodies against ORFV and Clostridium welchii were 6.44% and 7.78%,respectively.The positive rate of paratuberculosis antibody is the lowest,only 0.07%.After classifying the samples by region,it was found that the positive rates of antibodies in serum samples from Xinjiang,Gansu,Shaanxi and Inner Mongolia showed a similar trend..After counting the types of antibodies present in the serum,it was found that the amount of serum with two antibodies accounted for 83.78% of the total sample.Among them,the serum with Pasteurella antibodies and infectious pleural pneumonia antibodies accounted for 83.33%.Serum with antibodies to Clostridium welchii and antibodies to infectious pleuropneumonia accounted for 0.22%,Serum with antibodies to F.mutton virus and Clostridium welchii accounted for0.22%.It indicates that the flock is the most severely infected by the mixed infection of Pasteurella and Mycoplasma.2.Isolation of Pasteurella multocida and drug sensitivity analysis.The diseased sheep lung tissue disease material was collected and ground,and the filtrate was applied to serum TSA medium.After growing bacteria,repeat the streak culturemany times to obtain dominant colonies.The dominant colonies were identified by Gram staining and molecular biology,and a strain of Pasteurella multocida was finally obtained..After the drug sensitivity analysis of this strain,it was found that tetracycline,amoxicillin,kanamycin,ceftazidime,sulfamethoxazole and other drugs had an inhibitory effect on it.Based on this result,it can provide a reference for the treatment and control of Pasteurellosis.To analyze the pathogenicity of Pasteurella multocida,the mice were intraperitoneally injected with 100 ?L of Pasteurella multocida at different doses.It was found that when the injection volume reached 102 cfu/m L,the mice would begin to die within 6 hours,and all died within 24 hours,suggesting that this strain has strong pathogenicity3.Prokaryotic Expression of Pasteurella multocida OmpA Protein.The p ET-28 a prokaryotic expression system was used to express the OmpA gene of Pasteurella multocida.Firstly,the recombinant plasmid p ET-28a-OmpA was constructed,and the plasmid was transformed into E.coli BL21(DE3)to express the protein.It was found that about 40 ku of recombinant OmpA protein could be expressed.After optimizing the protein expression conditions,it was found that the optimal induction condition was 1m M IPTG and induction for 6 h..Finally,the antigenicity and solubility of the protein were analyzed,and it was found that the recombinant OmpA protein with His tag was an inclusion body protein.