Recombinant fusobacterium necrophorum leukotoxin vaccine and preparation thereof

Abstract

The F. necrophorum gene expressing leukotoxin was sequenced and cloned. The leukotoxin open reading frame (lktA) is part of a multi-gene operon containing 9,726 bp, and encoding a protein containing 3,241 amino acids with an overall molecular weight of 335,956 daltons. The protein encoded by the gene was truncated into five polypeptides having overlapping regions by truncating the full length gene into five different sections and amplifying, expressing, and recovering the protein encoded by each of these sections. Additionally, a region upstream of the gene was sequenced and the polypeptide encoded by that nucleotide sequence was purified and isolated. These polypeptides along with the full length protein are then tested to determine their immunogenicity and protective immunity in comparison to the efficacy of immunization conferred by inactivated native leukotoxin in F. necrophorum culture supernatant.

1 . An isolated nucleotide sequence having a nucleotide sequence having at least about 50% sequence homology with a sequence that is a truncated form of SEQ ID No. 8. 
     
     
         2 . The sequence of  claim 1 , said sequence having at least about 60% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 8-14. 
     
     
         3 . The sequence of  claim 1 , said sequence having at least about 75% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 8-14. 
     
     
         4 . The sequence of  claim 1 , said sequence having at least about 87% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 8-14. 
     
     
         5 . The sequence of  claim 1 , said sequence having at least about 95% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 8-14. 
     
     
         6 . An expression vector containing a nucleotide sequence having at least about 50% sequence homology with a truncated sequence from SEQ ID No. 8. 
     
     
         7 . The vector of  claim 6 , said nucleotide sequence having at least about 60% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 8-14. 
     
     
         8 . The vector of  claim 6 , said nucleotide sequence having at least about 75% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 8-14. 
     
     
         9 . The vector of  claim 6 , said nucleotide sequence having at least about 87% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 8-14. 
     
     
         10 . The vector of  claim 6 , said nucleotide sequence having at least about 95% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 8-14. 
     
     
         11 . An isolated nucleotide sequence which differs from that of  claim 1  due to a mutation event selected from the group consisting of point mutations, deletions, insertions and rearrangements. 
     
     
         12 . A vaccine effective for conferring protective immunity against  F. necrophorum  comprising the protein expressed by a portion of SEQ ID No. 8 and a suitable pharmacologically compatible carrier. 
     
     
         13 . The vaccine of  claim 12 , said vaccine being prepared by a method comprising the steps of:
 a) providing the  F. necrophorum  gene which expresses leukotoxin;   b) truncating said  F. necrophorum  gene into a plurality of discrete nucleotide sequences, each of said discrete nucleotide sequences encoding for a respective polypeptide sequence;   c) expressing and recovering said encoded polypeptide sequence expressed by at least one of said discrete nucleotide sequences;   d) inactivating said recovered polypeptide sequence; and   e) combining said inactivated polypeptide sequence with said suitable pharmacologically compatible carrier to produce said vaccine.   
     
     
         14 . The vaccine of  claim 13 , said discrete nucleotide sequences having a sequence having at least about 50% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 9-14. 
     
     
         15 . The vaccine of  claim 13 , further comprising the step of expressing and recovering said respective polypeptides using said nucleotide. 
     
     
         16 . A recombinantly derived nucleotide sequence than encodes a polypeptide effective in conferring protective immunity against  F. necrophorum  infections in mice, said sequence comprising a truncated form of SEQ ID No. 8. 
     
     
         17 . The sequence of  claim 16 , said sequence having at least about 50% sequence homology with a sequence selected from SEQ ID Nos. 9-14.