Development Of The Recombinant Subunit Vaccine Against Cattle And Sheep Footrot Using Fusobacterium Necrophorum Leukotoxin

Footrot is a highly contagious disease caused by the associated infection of Fusobacteriumnecrophorum and Baeteroides nodosus. The disease is characterized by rot, diapyesis, necrosis,decompose, and forming cutin in hoof tissue of the infected animals. At present, footrot is a severedisease in Chinese goat and cattle industry and its incidences has been reached 50ï¼…in cattle andgoat nursery. Cow with footrot is characterized by lost appetite, decline of lactation production,deterioration of reproductive performance, and lead to a severe economic loss to the cattle industry.So it is important to prevent the footrot occurrence regarding cattle and goat, especially footrot ofcow. For the past years, treatment of footrot only depended on inactivated vaccine, and which wasconsidered to posses lower immunity, adverse reaction and difficulty for production. Thus, it isessential that a new genetically engineering vaccine was developed for prevention of footrotoccurrence about cattle and goat.It had beed reported that development of bacterium subunit vaccines were mainly based on itstoxin proteins and pilus proteins. Compared with Baeteroides nodosus, F necrophorum is a moresignificant etiological agent during hoof damage. Several studies implicate that leukotoxin isconsidered to be the important virulence factor. The importance of leukotoxin as a virulence factorin F. necrophorum infections is demonstrated by laboratory animals inoculated with the natureleukotoxin and protective effects of antileukotoxin antibody. So leukotoxin protein is a effectivecandidate antigen for development of the recombinant subunit vaccine against F. necrophorum. Inthis study, five fragments genes of leukotoxin protein, containing key antigenic epitope region,were expressed in E. coli BL21(DE3) or E. coli RosettaTM (plyss) with pGEX-6p-1 vector andpPRO-EX-HTa vector. These recombinant proteins were purified by using corresponding proteinpurification kit. To determine whether these purified recombinant proteins can be used as candicaterecombinant subunit vaccine against F. necrophorum, Kunming mices as experimental animal wereimmuned with these purified proteins and native leukotoxin protein. These immuned animals wereattacked with strong virulent Fusobacterium necrophorum strain A25 which derived from ATCC(USA). Immunogenicity and protective effects of truncated recombinant leukotoxin proteins ofFusobacterium necrophorum in mice were detected by mice mortality rate and liver pathologychange. To determine cytotoxicity of these recombinant proteins, apoptosis effects of these proerinsto goat neutrophilic leukocyte were analysed by flow cytometry. All experimental procedures as follows:1. Primers, which used to amplify five truncated overlapping fragments covering the entireleukotoxin gene of F. necrophorum strain H05 and F. necrophorum strain A25, were designed byPrimer Premier 5.0 software based on the nucleotides of lktA gene of goat F. necrophorum strainA25 (Accession, AF312861). PCR products of the five truncated leukotoxin gene were cloned intomultiple clone sites of pMD-18-T vector, and their positive plasmids were sequenced by dideoxymethod. The two entire leukotoxin genes spliced by the five overlapping fragments were comparedwith leukotoxin gene sequence of F. necrophorum strain A25 published in Genbank. The result ofsequence analysis revealed that concordance percentage of H05 strain with A25 strain published inGenbank in nucleotide was 99.75ï¼…and concordance percentage in amino acid was 99.63ï¼…, andconcordance percentage of cloning A25 strain with A25 strain published in Genbank in nucleotidewas 99.82ï¼…and concordance percentage in amino acid was 99.75ï¼…. this data showed thatleukotoxin gene of F. necrophorum was very conserved between Cattle and goat.So leukotoxin gene of F. necrophorum strain A25 was able to develop recombinant subunit vaccineagainst F. necrophorum for prevention of Cattle footrot.2. A series of recombinant plasmids, which were used to express five truncated leukotoxingenes, were construced with pET-28a, pET-30a, pET-32a, pQE30, pPRO-EX-HTa and pGEX-6p-1vectors. The E.coli BL21(DE3) or E. coli Rosetta^TM (plyss) containing recombinant plasmid wasinduced by IPTG. The result of SDS-PAGE showed that only lktA3 fragments was expressed inpPRO-EX-HTa vector, and lktA1, lktA2, lktA4 and lktA5 fragments were not able to be expressedin six kinds of prokaryotic expression vectors. Further to express truncated fragments regardinglktA1, lktA2, lktA4 and lktA5 fragments, main antigenic epitope regions about the five fragmentswere predicted by Protean procedure of DNAstar software. These epitope antigenic regions aboutlktA1,lktA2, lktA4 and lktA5 fragments were designated as PL1, PL2, PL4 and PL5. Using thesame process, a series of recombinant plasmids, which were used to express PL1, PL2, PL4 andPL5 fragments, were constructed by six kinds of vectors described above. These recombinantE.coli BL21 (DE3) strains or E. coli RosettaTM (plyss) strains were induced by IPTG. The result ofSDS-PAGE showed that PL1, PL2, PL4 and PL5 fragments were all expressed in pGEX-6p-1vectors. Western blot analysis demonstrated that PL1, PL2, lktA3, PL4 and PL5 recombinantproteins were able to react with anti-sera of F. necrophorum.3. Purified PL1, PL2, lktA3, PL4 and PL5 recombiant proteins as candidate recombinantsubunit vaccine against F. necrophorum were injected into Kunming mice. Native leukotoxin wascarried out as positive control, and PBS buffer was carried out as negative control. After eachbooster immunization, the serum antibody titers of immuned animals were tested by ELISA. Afterfourth booster immunization, immuned aminals were attacked with F. necrophorum strain A25.These results showed that the five recombinant proteins were all able to induce specific antibodiesagainst E necrophorum, and their antibodies titers were higher than native leukotoxin; Theantibodies induced, by five recombinant proteins were able to degrade the damage in liver, and all immuned animals were survived. In those recombinant proteins, mmunogenicity and protectiveeffects of PL1, lktA3 and PL4 recombiant proteins were better than recombinant proteins andnative leukotoxin.4. Apoptosis effects of the five recombinant proteins to goat neutrophilic leukocyte wereanalysed by flow cytometry. The result indicated that the five recombinant proteins were all able toinduce apoptosis of goat neutrophilic leukocyte, the apoptosis effects of the five recombinantproteins were markedly lower than native teukotoxin, and the apoptosis effects induced by PL1 andlktA3 recombinant proteins were conspicuously stronger than other recombinant proteins. Inaddition, apoptosis effects of the five recombinant proteins gradually weakened followingconcentration decrease of the recombinant proteins.