Bioinformatics Analysis and Prokaryotic Expression Identification of Leukotoxin PL2 Protein of Fusobacterium necrophorum
To determine the composition,structure and bioinformatics properties of Fusobacterium necrophorum leukotoxin PL2 protein,according to gene sequence and the amino acid sequence of the leukotoxin PL2 of Fusobacterium necrophorum in the NCBI database,the physicochemical properties,B cell and T cell epitopes of leukotoxin PL2 protein were predicted by bioinformatics software,and recombinant plasmids pGEX-6p-1-PL2-1,pGEX-6p-1-PL2-2,pGEX-6p-1-PL2-3 were constructed by prokaryotic expression system,and the expression of leukotoxin PL2 protein was detected by SDS-PAGE.The obtained recombinant protein was validated by Western blot.PL2 protein was a stable hydrophilic protein rich in serine composed of 332 amino acids,and the secondary structure was mostly random curl and α helix.It was predicted that the protein contained 10 B cell dominant epitopes and 16 T cell dominant epitopes,and the pGEX-6p-1-PL2-1 obtained by PCR amplification was 228 bp,pGEX-6p-1-PL2-2 was 300 bp,and pGEX-6p-1-PL2-3 was 588 bp.The SDS-PAGE results demonstrated that 3 proteins were successfully expressed with a protein size of 34 kDa,and the western blot results showed that the recombinant protein PL2-1 had immunogenicity was proved.Leukotoxin PL2 protein of Fusobacterium necrophorum was an extramembrane protein,there was no transmembrane region,containing multiple phosphorylation sites and multiple B cell and T cell epitopes,and the recombinant PL2-1 protein obtained according to the prediction results of epitopes has immunogenicity was proved,which could be used as a potential antigen for the development of subunit vaccines for Fusobacterium necrophorum.
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维普
