Study On The Immune Activity Of Extraction And Prokaryotic Expression Of Outer Membrane Proteins From Fusobacterium Necrophorum

Fusobacterium necrophorum (F necrophorum, FN), a gram-negative polymorphous anaerobic bacillus, mainly existed in the oral and necrotic lesions of ruminants, causing acute or chronic zoonotic diseases. It is one of important infectious diseases affecting ruminants’health.In this study, Outer membrane proteins (OMP) of FN were obtained by using a modified extraction process. Its concentration was4.524mg/mL with the spectrophotometer. The results showed that only a major band of44.5kDa was observed by SDS-PAGE. The analysis of western blot showed that the OMP44.5can be combined with rabbit antiserum. It showed that OMP44.5had immunogenie activity. It was found that lesions of the mice organs and cell viability decreased were the causes of OMP by challenge experiments in mice and MTS reduction assay of AnA-1. It showed that OMP had a toxic effect for cells of mice.On this basis, OMP gene fragment was amplified from DNA genome of FN by polymerase chain reaction (PCR). It was inserted into pMD-18T Simple and it became recombinant plasmid pMD(?)18-T Simple-OMP. Then the ecombinant plasmid pMD(?)18-T Simple-OMP was sequenced. Comparison of the results of sequence and the gene sequence of GenBank, the homologous rate was100%. Recombinant plasmid of pET-28a-OMP was transformed into host bacteria E.coli BL21(DE3), induced expression with IPTG, the recombinant proteins purified by His Trap affinity columms. The proteins were identified by SDS-PAGE and western blot. The results showed that OMP fusion proteins were effectively expressed and had immune activity. It was the theoretical foundation of the study of OMP from/FN subunit vaccine.