Cloning And Expression The ToxA Gene Of Toxigenic Pasteurella Multocida And Biological Characterization Of The Expression Protein

In general, toxigenic Pasteurella multocida (T~+Pm) is a major pathogen of progressive atrophic rhinitis (PAR). T~+Pm secretes P. multocida toxin (PMT), a 146 kDa dermonecrotic toxin, which is encoded by toxA gene. Non-toxigenic P. multocida, which is toxA-negative, cannot cause the PAR. Injected little purified PMT in SPF pigs can cause the PAR. Obviously, PMT plays a key role in PAR, so the PMT detection became the key value of PAR. Extensive researches indicate that the N-terminal of PMT possesses cell-binding activity, the C-terminal has catalytic activity and the middle region is a transmembrane domain. The native PMT is pathogenic while the truncated toxin may lose its pathogenic activity but remain immunogenic activity. It was a good suggestion for PAR vaccine.The present study focused on the cloning, expression and application of the toxA gene of T~+Pm. The results were summarized as follows:1. The complete toxA gene and its 3'-terminal 2115bp fragment were amplified from the genome of the T~+Pm strain HN-13, an isolate from a swine farm with PAR in South China by our laboratory. The oligonucleotide primers were designed according to the sequence of the toxA gene, which published in GenBank. The amplified gene is 4019bp containing the open reading frame (ORF) of 3858bp, and it was submitted to the GenBank with an accession number of AY864768. The toxA gene was inserted into the prokaryote expression vector pGEX-KG, and the resulted recombinant plasmid pGEX-toxA was sequenced. This sequence processes a homology of more than 99.8% compared with the 6 published toxA sequences in GenBank. Similarly, the nucleotide homology of the 3'-terminal is more than 99.6%. The pGEX-toxA was transformed into E. coli BL21(DE3) competent cells. After induction by IPTG, a 173 kDa recombinant protein (named rPMT) was expressed. Western blot analysis revealed that the recombinant protein could react with antisera against T~+Pm. The recombinant protein could also induce high titer of antibody in Kunming mice, which could protect the mice from the challenge of the natural PMT.2. In order to study the biological activity of the toxin, we digested the toxA gene separately with 5 kinds of restrict endonucleases, 5 fragments, N1518, N3172, C2345, C2693 and C3388, were obtained. The 5 fragments and the 3'-terminal 2115bp fragment of the toxA gene were cloned into the prokaryote expression vectors system pET28a(b,c), and resulted plasmids were transformed into E. coli BL21(DE3) competent cells and then induced by IPTG Only 2 of them, pET28b-C2115 and pET28a-N1518, were highly expressed. The expressed proteins were 78kDa(nanled rPMT-C) and 57kDa (named rPMT-N) respectively, and the result of Western blot suggested that they had good immunological reactivity.3. Rabbits were respectively immunized with the rPMT-C and rPMT-N 5 times at interval of 10 days. Serum antibody titers reached 1/32 and 1/16, respectively, measured by gel diffusion test.4. The rPMT-C and rPMT-N recombinant proteins were mixed as coating antigens to establish an indirect ELISA to detect PMT antibodies. The optimum working concentration of antigen was 1.6/zg/mL, and the prefer dilution blood was 40, the limit of positive and negative was OD63o=0.291. 608 serum samples were tested by it, which were sented from farms, and the results showed the positive.raie was 69.6%.5. Four kinds of bacterin-toxiods were prepared as follows: formalin-inactivated TPm (3.3 X 108/mL) and Bb (5.0 X 108/mL), complemented respectively with (1) rPMT, (2) rPMT-C, (3) rPMT-N, (4) rPMT-C and rPMT-N, were adjuvanted with mineral oil according to the standard procedure. Pigs were vaccinated with the bacterin-toxiods, and the results showed that rPMT and rPMT-C were good candidates of PAR vaccine.6. A growth curve of 'TTm strain NH-13 was made. The results suggested the bacterium was in logarithmic phase, stationary phase and decline phase, after culturing 3hours, 7 hours and 15 hours. And the live bacterium value reached 3.4 X1010, when it was cultured 12 hours.In summary, the complete toxA gene and its 5'-termianal 1518bp and 3'-terminal 2115bp were cloned from the toxigenic P. multocida strain HN-13, and expressed in E. coli. The recombinant proteins were subjected to establish ?.n indirect ELISA, which was a good idea for detecting antibodies against PMT, and to be components of bacierin-toxiod vaccines of PAR.