Construction Of A Plasmid Expressing A Fusion Construct Of D.nodosus Serotype A Pili Gene And Ovine IL-2 Gene

D.nodosus pili is the main protective immunogen against footrot in ruminants.IL-2 is known to be as an adjuvant in many areas. With the construction of fused Pili and ovine IL-2 genes.a recombinant fused protein vaccine was developedThe pili gene that dominates the main protective immunogen was amplified and cloned from D.nodosus serotype A by PCR.A recombinant plasmid .designated TE-Pilin,was constructed by inserting the Pili gene intoT-Easy which was digested with EcoRV.The ovine IL-2 gene is in pBluescript-IL-2 which is a plasmid constructed by Wang Ke Jian.To construct a fused plasmid,TE-Pili was digested with EcoRV ,which was inserted the IL-2 gene from pBluescript -IL-2 digested with EcoRV and BamHI.the recombinant plasmid was designated TE-Pilin-IL-2.A fused expression plasmid was constructed by inserting the fused Pilin-IL-2 gene into PME290.The plasmid harbouring Pilin-EL-2 sequence was designated PME290- Pilin-IL-2.The fused protein was expressed by transforming the PME290-Pilin-IL-2 into competent host cell PAK/2pfs.the fused protein was purified by MgCL₂ from the supernatant of the culture of transformed PAK/2pfs. Specific reaction between fused Pilin-IL-2 protein and the antiserum of D .nodosus serotype A Pilin was observed by cross electrophoresis.The molecular weight of the recombinant fused protein was about 33000 by SDS-PAGE.Healthy rabbits were inoculated each with vaccine of the recombinant fused Pili-EL-2 protein at 250ul per dose to potentiate the immunogenicity.Rabbits inoculated two times at one week interval. The blood of each rabbit was collected at days 0,7,14,2l,30.The antibody titers were evaluated by k-agglutination test.The results showed that lower agglutination titers was observed at day 7 and up to higher levels over 8000at day 21 induced by the vaccine of recombinant fused protein. These results suggested that the recombinant fused protein is of the good immunogenicity. It also means that the IL-2in the fused Pili-IL-2 construct may also have a potential role in stimulating vaccine antigen.