Cloning Of D.nodosus Serotype C Pili Gene In Pseudomonas Aeruginus And Study On The Immunogenicity Of The Expression Pili Subunit

Master Degree Candidate: Miao LiguangSupervisor:Wang KejianD.nodosus pili is the main protective immumogen against footrot in ruminants.A plasmid expressing pili gene of D.nodosus serotype C was constructed by which arecombinant phi vaccine was developed. The immumogenicity of the recombinantpili vaccine was evaluated through experimental animals.The pili gene that dominates the main protective immunogen was amplifiedand cloned from D.nodosus serorype C by PCR. An expression plasmid wasconstructed by cloning the pili gene into PME29O.The plasmas harbouring the pilisequence was designated PME29O-Pili.The PME29O-pili was transformed into thehost competent cell PAKI2pfs and the recombinant pill was expressed in thesupernatant of the cultures of the transformant cell PAK]2pfs. The recombinant pillwas purified by Mgcl2 from the supernatant of the culture of the transformedPAKI2pfs. The specific reaction of the recombinant pili and antiserum of D.nodosusserotype C pili was demonstrated by cross electrophoresis. The recombinant pili wasexpressed at high level in PAKI2pfs.Healthy rabbits were inoculated each with vaccine of the recombinant pillprotein at 250ug per dose to potentiate the immunogenicity. Rabbit inoculated twotimes at one week interval. The blood of each rabbit was collected at days 0,7, 14,21,30, 60, 90, 120. The antibody titers were evaluated by k-agglutination test. Theresults showed that the lower agglutination titers were observed at day 7 and up tohigher levels over 8000xat day 21 induced by the vaccine of recombinant phi.Above results suggested that the good immunogenicity of the recombinant pillvaccine could match with that previously measured of either the D.nodosus pill or the killed D.nodosus vaccine.