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Cloning of D.nodosus serotype C pili gene in Pseudomonas aeruginus and study on the immunogenicity of the expression pili subunit

Author: MiaoLiGuang
Tutor: WangKeJian
School: Chinese Academy of Agricultural Sciences
Course: Conservation and Utilization of Wild Fauna and Flora
Keywords: Deer paraganglioma Bacteroides the ciliary protein gene gene expression immunogenicity
CLC: S858.25
Type: Master's thesis
Year: 2001
Downloads: 82
Quote: 2
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Cilia proteins (Pili) is the major protective antigen of deer and other ruminants foot rot disease. C-type nodules Bacteroides is one of the cause of deer, cattle, sheep and other ruminants foot rot bacteria type. The study by Clonal C paraganglioma Bacteroides ciliary protein gene, to construct a the cilia protein gene expression vector the C section aneurysm Bacteroides, and in experimental animals on the preliminary evaluation of the immunogenicity of the expression product of the gene cilia proteins, so as to development and production of simple, low-cost deer foot rot C nodules Bacteroides the cilia protein genetically engineered vaccine laid the foundation, and the test results are as follows. Tumor Bacteroides strains extracted chromosomal DNA from the C-section. Was amplified by PCR with the immune protective antigen gene the 0.85 kb cilia protein gene (the Pili gene), the use of the gene construct of cilia protein gene expression vector. First C-section tumor Bacteroides under anaerobic conditions for 72 hours; bacteria reached 1 × 10 8 cells / ml or more, depending on the Gram-negative bacilli rapid DNA extraction method, mention DNA ; using the design of specific primers for PCR, pili gene was amplified from the DNA; the pili gene sequences cloned into a T Easy Vector System for sequencing amplified correctly; digested with EcoR1, low melting point of plastic recycling, the Klenow fill level, T 4 DNA ligase connection with the digested by Hpal dephosphorylation PPLλ intermediate vector pill gene into the vector at PPL; BamHI , BamHI + HindIll, Dra restriction enzyme digestion, cloning plasmid identified was inserted into the PPL into the vector; amplified into PPL-pi i recombinant plasmid, with the B. * Cut out. Ikb size of the fragment recovered fragment PME290 dephosphorylated BamHI digested expression plasmid, transforming a host cell had been PAK is carried out in a nutrient broth PI i gene expression. 18 DIAO 4 hours after culture, the culture supernatant was added 0. IM MGC; centrifugal extraction cilia proteins. C-type section with goat anti-rabbit tumor Bacteroides antiserum expression cilia protein extracted counter immuno electrophoresis test to prove that the cilia protein expression specificity. PAGE electrophoresis and W both t stare the nhi River to prove their cilia protein expression. In order to build the foot rot C-section tumor Bacteroides the Pil gene expression plasmid. C-section with gene expression in tumor Bacteroides cilia protein with an equal volume of Freund's complete adjuvant the mixed emulsion preparation C knots Bacteroides the cilia protein genetic engineering vaccine inoculated three rabbits; immunogenicity test; Bacteroides inactivated vaccine and bacterial the ciliary protein vaccine immunizing rabbits with nodules, the immune comparison test. C-section tumor Bacteroides cilia protein genetically engineered vaccine cultured in a nutrient broth containing 5 00Pg state control reed penicillin; MGC; precipitation method to extract cilia protein equal amount of Freund's complete adjuvant immune rabbits , 2 times, and asked every week, weekly blood test your immune rabbit serum antibody levels. Cilia protein genetically engineered vaccine serum agglutination within 7 days, 14 days up to -2 -. u2000 \C-section tumor Bacteroides cilia protein genetically engineered vaccine immune effect is better than the other two vaccines. while taking advantage of the C-shaped nodules Bacteroides cell lysate antigen and cilia protein genetically engineered vaccine antiserum in rabbits counter immuno electrophoresis test; Results from the 7th day until the 120th day has significant precipitation line C-type nodules proved Bacteroides the cilia protein genetic engineering vaccine immunogenicity.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Livestock > Deer
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