Study On Isolation, Identification And Its Leukotoxin Immunological Characteristics Of Bovine Origin Pathogenic Fusobacterium Necrophorum

Fusobacterium necrophorum(Fn), as a gram-negative, strict anaerobic polymorphic bacillus, is a common inhabitant of the enteron of humans and animals, which can be obtained by isolating from the gastrointestinal tract, oral cavity and genitourinary tract, especially from the infected abscesses focus and infected respiratory tract of humans and animals. As an opportunistic pathogen, it can cause a wide variety of necrobacillus among the wild animals and the livestock such as liver abscess in cattle, foot rot in ruminants, necrotic laryngitis and Lemierre's syndrome. There are about 3 million cases of the liver abscesses in cattle caused by Fn in America every year. The rate of confined cattle attacked by this disease reaches up to 25%～30%, which has resulted in the net loss of over 15 million dollars while the proportion of the productive forces has dropped to 7% and the percentage of the economic returns has fell to 4%～5%. Meanwhile, the rate of the foot rot disease is up to 10%～30% in China, which does harm to the performance of the milk cows seriously. Therefore, the foot rot, as one of the most harmful infectious diseases, causes the cattle industry to suffer a great loss.At present, the prevention of necrocacillus mainly depends on the usage of the killed vaccine throughout the world. It is very difficult to realize the production of this vaccine in a large scale to meet the needs of the products at low cost in market because of its strict anaerobic feature, the harsh nutrition request and the complex craft requirement. Additionally, the serious side effect of the whole bacterium vaccine limits the extension of the killed vaccine. Thus, to study Fn further and to develop new kinds of vaccines appear to be very insistent for preventing the foot rot. Fn biotype A, that is, necrophorum biotype (Fnn), is a major pathogenic bacteria type that can produce many virulence factors-leukotoxion (lkt), a secreted protein of high molecular weight against leukocytes. The lktA structural gene of Fn was cloned and sequenced, which shows that the leukotoxin does not have the sequence similar with any other bacterial leukotoxins. The gene consists of 3 structural subunits (lktB, lktA, lktC), of which the lktA gene with the length of 9,726 bp encodes the leukotoxin. Under the natural condition, the expression level of the entire lktA gene is low and its biologic activity is unstable. In China, there are few reports about virulence factor of Fn in cattle and the analysis of its epitopes. Therefore, it is necessary to develop the isolation and identification of Fn from domestic cattle and explore to develop new vaccine.In this study, it is the first time to isolate and identify the 4 domestic epidemic Fns and determine their biotypes. The major antigen coding genes (BSBSE gene and SH gene) of the lktA were cloned and expressed respectively. And the fusion expression plasmid pET32a-BSBSE-SH was constructed. The result of Western-blot indicated that the 3 recombinant target proteins had antigenicity.4 Fns marked by F1, F2, F3, F4 were isolated and identified through morphology, biochemistry experiment, rabbits challenged test and RNA polymerase beta subunit ( rpoB 276 bp ) specific PCR from the epidemic materials that were collected from the abscess tissue of the cattle's hoof infected by the foot rot. Adopting the above FnL ( deer Fn strain ) as the reference strain , the 16S rRNA gene(1500 bp) was amplified by PCR, proved to be Fn 16S rRNA by the analysis of blast with GenBank. The phylogenesis analysis was conducted through comparing F1, F2, F3, F4, FnL obtained with 16S rRNA reported at GeneBank (AF044948). The result of the homology analysis showed that the highest homology of comparing F1 with the reported 16S rRNA,FnL,F2,F3 and F4 reached to 94.8%, 97.8%, 96.6%, 98.5%, 97.6% respectively; among F4,F2,F3, FnL and the reported 16S rRNA, the highest homology was 99.6%, 99.4%, 99.4% and 99.6% respectively, which was all above 99.3%. The phylogenetic tree analysis also showed that F1 had a far phylogenetic relationship with others. However, F2, F3, F4 and FnL had a close genetic relationship with 16S rRNA. So they belonged to the same subspecies while F1 belonged to the other one. The lkt promoter region sequence was amplified further by PCR, whose outcome was clear that lkt promoter region (517 bp) of F2, F3, F4, FnL were positive except that of F1. The above results made it clear that F1 that could produce less lkt belonged to Fn subspecies funduliforme (biotype B) while F2, F3, F4 and FnL that could produce more lkt belonged to Fn subspecies necrophorum (biotype A).According to the lktA (AF312861) of the standard strain Fn reported at GenBank, Primer Premier 5.0 was utilized to design 2 pairs of primers of the BSBSE gene and the SH gene separately. The BSBSE and the SH of lkt were amplified by PCR when using the isolated F4 genome as the template, which were then cloned to the pMD18-T vector and sequenced respectively. The highest homologies of their nucleotide sequence compared with the BSBSE and the SH of lkt reported in GenBank reached to 99.1% and 98.1%. It is inferred that the amino acids homology of the BSBSE gene is 97.9% when amino acids distinction is 0.92%～2.1%;Amino acids homology of the SH is 98.3% when amino acids distinction is 1.4%～1.8%. The result stated that the BSBSE gene and the SH gene were conservative for there was a little difference in the aspect of the genes. ORF did not shift that ensured the protein accuracy.Then the BSBSE and SH fragments were subcloned into the multiple cloning sites of the pET32 to construct pET32a-BSBSE and pET32a-SH. After these positive plasmids were digested and testified by PCR, they were transformed into E. coli BL21 (DE3). With IPTG induction, the SDS-PAGE result showed that the expression product band of the lysic tropina BSBSE and SH fusion expression product band matched to the sample, the molecular weights of which were about 54.9 kD and 80.2kD respectively. The fusion proteins were purified by Ni-NTA affinity chromatography under the denature conditions, and above 90% and 95% of their respective purity was achieved. Bovine origin Fn positive serum that our laboratory preserved was used as antibody , and HRP Rabbit Anti-cattle IgG was used as antiantibody, BSBSE and SH presents the specificity hybrid signal with the positive serum, which confirmed BSBSE and SH expression products are the goal proteins, and have the good response activeness. In order to identify the biologic activity of the expression productions, the multiclone antiserum prepared by inoculated rabbits against BSBSE and SH . Used as an immunogen respectively, the fusion protein (500μg/animal) was homogenized with an equal volume of Freund's complete adjuvant and rabbits received the hypodermic injection. A booster dose was given in the 3nd week, the 5th week and the 7th week with Freund's incomplete adjuvant instead of Freund's complete adjuvant. Serum samples were collected from the heart after 10 days of the fourth immunity. The antiserum titers and specificity were determined by indirect ELISA. The purified proteins (BSBSE and SH) acted as the coat antigens.The serum was used as the antibody and HRP Goat Anti-Rabbit IgG was used as the antiantibody, and Set up the positive control and negative control .As a result, ELISA analysis indicated that BSBSE and SH all had the specific neutralizing activity with the rabbits antisera, So both BSBSE and SH had antigenicity. ELISA antibody titer of rabbit antisera against BSBSE and SH was 105. The truncated BSBSE and SH were very effective antigen shown by ELISA analysis. The antiserum prepared by BSBSE and SH have a specific reaction with the relevant purified antigens (BSBSE and SH) and the antisera titer was 105, which proved that it had a good specificity. The antiserum BSBSE and SH prepared by inoculated rabbits can be used as detection agent of Fn. To prepare a recombinant vaccine agaist necrobacillus, the recombinant expression plasmid pET32a-BSBSE-SH was constructed through the double digestion method by using BamHⅠsite as a linker. A blast research of the BSBSE-SH compared with the lktA at GenBank (AF312861) showed that the highest homology of the BSBSE-SH was above 97%. The result showed the linker did not increase the variation probability of the longer fusion fragment. In addition, the result of SDS-PAGE made it clear that the fusion protein of BSBSE-SH had been expressed. The molecular weight of the expression product was about 124.15 kD. Western-blot analysis indicated that BSBSE-SH had antigenicity.The study has laid the foundation of the theory to prepare for a new genetic engineering vaccine.