| Fusobacterium necrophorum(F. necrophorum) is an obligate anaerobe Gram-negative pleomorphic bacterium and has been associated with several diseases in humans and animals including footrot, liver abscesses, necrodermatitis and calf diphtheria in cattle, sheep and deer, Lemierre’s syndromeand in humans. The major approache to treat these diseases both at home and abroad is the use of antibiotics, because there is no commercial F. necrophorum vaccine. But antibiotics have brought about a lot of negative effect. For reseach of the biological characteristics of 120 ku hemagglutinin-related outer membrane protein, revealing its feasibility for prevent the necrobacillosis as genetic engineering subunit vaccine. The complete nucleotide sequence of 120 ku hemagglutinin-related outer membrane protein gene was amplified according to the partical known nucleotide sequence. Molecular characteristics of the protein were analyzed by bioinformatics tools. Based on the result of antigenic epitope analysis, specific primers were designed to amplified the three truncated overlapping gene fragments covering the whole open reading frame(ORF). Then expression the recombinant proteins P1, P2 and P3 in E.coli. Injecting the purified proteins into rabbits and testing the corresponding antibodies in serum. The main contents are as follows:1. The complete nucleotide sequence of 120 ku hemagglutinin-related outer membrane protein gene was amplified by genome walking according to the partical known nucleotide sequence(Gen Bank: AF529887.1). The sequence checked by high fidelity enzyme, considted of 4 247 bp, contained an ORF of 3 372 bp and encoding 1 123 amino acids, the molecular weight of protein was about 120 ku. Bioinformatics analysis showed that this protein might be a member of autotransporter family which had hemagglutination activity and antigenicity.2. Prokaryotic expression and purification of 120 ku hemagglutinin-related outer membrane protein from F. necrophorum. Three truncated overlapping gene fragments p1, p2 and p3 covering the whole open reading frame(ORF) were amplified and constructed recombinant plasmids p ET-28a-p1, p ET-32a-p2 and p ET-32a-p3, then induced expression in E.coli. The recombinant proteins P1, P2 and P3 were purified by AKTA purifier system, the molecular weight was about 48 ku, 59 ku and 62 ku, respectively. Western blot indicated that the recombinant proteins had good immunogenicity.3. Injecting the purified proteins P1, P2 and P3 into rabbits respectively and testing the corresponding antibodies in serum. The antibody titer in rabbits serum were 1:32, 1:16 and 1:16, respectively. This results showed the 120 ku hemagglutinin-related outer membrane protein from F. necrophorum had good immunogenicity and could stimulate the rabbits produce specific antibody.In conclusion, the cloning and antigenicity analysis of 120 ku hemagglutinin-related outer membrane protein from pathogenic Fusobacterium necrophorun will help us to further understand the biological function of this protein. In addition, the study provides a reference for further research on genetic engineering subunit vaccine. |
