Isolation And Identification Of Fusobacterium Necrophorum And Prokaryotic Expression And Immunogenicity Analysis Of Its Leukotoxin

Fusobacterium necrophorum is an important pathogen causing footrot disease and liver abscess.It has caused tremendous economic losses in sheep industry.The Leukotoxin is one of the strongest virulence factors in the secretion of toxins,which can inhibit the phagocytosis of neutrophils and provide help for bacteria invading the host,leading to the occurrence of the disease.As the epitope of leukotoxin is more dispersed,the nucleic acid sequence between 2407 ~ 2695 has not been reported.Therefore,in this study,isolate the Fusobacterium necrophorum from sheep footrot disease pathogen and carry out related research about it.All these research finding are benefit researching vaccine against Fusobacterium necrophorum.(1)Isolation and identification of Fusobacterium necrophorum from footrot disease of sheep with strict anaerobic environment.The morphological observation,PCR amplification and phylogenetic tree were used to identify and anaylis,The results showed that Fusobacterium necrophorum was isolated from the sheep footrot disease.The sequencing result of the isolated strain of Fusobacterium necrophorum was 99% homologous to that of Australian isolated strainA39A8(JX678850.1).(2)DNA star software was used to analyze the lktA gene.Based on its epitope and open reading frame to design a Leukotoxin gene of Fusobacterium necrophorum.By PCR,the target gene was successfully amplified and then cloned into pET-32a(+)expression vector.The recombinant lkt A35 was expressed by prokaryotic expression system.The results showed that only a major band of 35 ku was observed by SDS-PAGE.The western-blot result showed that the lktA35 could be combined with bacteria antiserum.It showed that lktA35 had reactionogenicity.(3)Used the recombinant to immunized the mice and got the polyclonal antibody.The indirect ELISA results showed that the antiboby titers of the second and third immunity were 1:64 000 and 1:128 000 for the polyclonal antibody,respectively.