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Molecular characterization and cloning of the Pasteurella
|摘要:||摘 要D型Pasteurella multocida產
P. multocida toxin (PMT)亦被証實為其主要毒力因子。本實驗選取P.
multocida D型、A型菌株及國外標準菌株進行實驗，以探討P. multocida
天竺鼠皮膚壞死試驗會有偽陽性或偽陰性發生，而以vero cell 細胞毒性
PCR (Random Amplified Polymorphic DNA; RAPD-PCR)研究其分子分型，
結果發現以10-mer random primer進行RAPD-PCR進行分析時，D型與A型菌
multocida。選取P. multocida type D產毒株Liu-48，利用PCR增幅完整
體pET system vector，以進行完整及部份PMT基因的選殖及表現。結果共
構築了Tox1 (2~486 a.a.)、Tox2 (486~987 a.a.)、Tox3 (987~1282 a.
a.)、Tox4 (2~987 a.a.)、Tox5 (486~1282 a.a.)、Tox6 (2~1282 a.a.)
及Tox7 (987~1282 a.a.)等七個選殖株，並且可成功地誘導其表現。所表
現產物除Tox1外，都可被swine anti-PMT polyclonal Ab所辯識，且各個
AbstractThe toxin-producing strain of Pasteurella multocida (type D) is characterized to be the major causative agent of atrophic rhinitis (AR) in swine. The P. multocida toxin (PMT) with a calculated molecular weight of 146 kDa has been identified to cause the atrophy of nasal turbinate bones. Both type D and type A of P. multocida including several wild isolates were selected to study the specificity for their toxin-producing activity. The results suggested the vero cell toxicity test and the PCR methods for screening PMT gene were more specificity if in comparison with both mouse lethality test and guinea pig skin test. The random amplification polymorphic DNA (RAPD) PCR method was also applied and successfully identified the type A and type D of toxin or non-toxin producing strains of P. multocida. The highly homogeneity of PMT genes between type D and type A were analyzed using PCR automated sequencing method and suggested the PMT gene was very conserved. Moreover, cloning full length and partial of PMT gene in pET- system expression vectors were applied for investigation the PMT biological!activity. Several clones contained partial or complete open reading frame of 3.8kb in size of PMT gene were successfully constructed into different vectors. Total four clones, Tox3, Tox5, Tox6 and Tox7 respectively, could be identified with both polyclonal and monoclonal antibody by Western blotting assay. Furthermore, the expression products of Tox4 and Tox5 can mount good protective immunity in experimental swine when they were challenged with high dose of PMT (>20 mg) and the neutralizing antibody titer was markedly increased up to 256X with vero cell toxicity test.
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