| Pasteurella Multitocida(PM)is a zoonotic disease widely distributed worldwide,which is a kind of bacteria that can cause various animal upper respiratory tract infection diseases under specific conditions.PM can cause acute pneumonia in pigs,atrophic rhinitis in pigs and acute septic diseases in pigs.At the same time,PM has caused many troubles to the pig industry,whether it is economic losses or strengthening the management of animal epidemic prevention.At present,in the prevention and control of related diseases of Pasteurella infection,vaccine injection is one of the effective strategies to control the disease.At present,weak virus vaccine is commonly used in clinical practice in China.The stability of weak virus vaccine is not high and the cross-protection effect is limited.Therefore,it is more critical to develop a new vaccine that can prolong the protection period and has cross-immune protection.In this study,four PM outer membrane proteins with cross-immune protection,plp B,plp E,plp P and Omp87,were selected based on bioinformatics techniques,and the encoded four DNA fragments were cloned into p ET-28(+)vector plasmids using DNA recombination technology,and then the identified recombinant plasmids were transformed into E.coli BL21(DE3)competent to successfully construct PM recombinant proteins;the recombinant proteins were induced to express and purify,and the proteins were purified and prepared into biosynthetic subunit vaccines to construct mouse models to verify the immunogenicity of their proteins.In this study,the plp B,plp E,plp P and Omp87 gene sequences were amplified by polymerase chain reaction,which is commonly known as PCR technique,and the sequences were compared with Gen Ban K;then the gene fragments were cloned into p ET-28(+)vector plasmid and transferred into E.coli BL21(DE3)competent state,and finally IPTG inducer was added to induce expression.The results of SDS-PAGE protein electrophoresis showed that plp B was soluble,about 30 KDa;plp E,plp P and Omp87 were inclusion bodies,with sizes of 35 KDa,39 KDa and 85 KDa,respectively;soluble plp B was purified by Ni ion affinity chromatography,inclusion bodies plp E and plp P were refolded in 0.02 M PB buffer environment at p H 8.0,inclusion bodies Omp87were refolded in 1 m M EDTA buffer at p H 7.0,protein purified and prepared into subunit vaccine with ISA201 oil adjuvant,and pig-origin Pasteurella multocida A,B and D inactivated vaccine was prepared at the same time;a total of two immunizations were performed,14 days as a cycle,14 days after the second immunization,5 times the LD50 dose(about 1.25×10~3CFU/mouse)of Pasteurella multocida capsular B CVCC441 strain was used.The protective rate was 88.3%in mice,66.7%in group B and 50%in group P.In summary,this test showed that these four proteins could improve the host immune response to atypical serotypes,of which the antigen with the highest immune protection rate was plp E,and the combined immunization group also provided higher protection. |
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