猪源D型产毒素多杀性巴氏杆菌toxA基因的克隆与表达

Cloning and Expression of the toxA Gene of Toxigenic Pasteurella multocida Serotype D from Swine

皮肤坏死毒素(Dermonecrotictoxin,DNT)是产毒素多杀性巴氏杆菌(ToxigenicPasteurellamultocida,T+Pm)的主要毒力因子和保护性抗原。以猪源D型T+Pm的基因组DNA为模板,用PCR方法扩增得到了编码DNT的toxA全基因编码序列,共4019bp。将其克隆到pMD18-T载体并测序,结果表明,toxA基因序列与GenBank已报道的5个toxA基因序列的同源性达99.8%以上。将该基因亚克隆到原核表达载体pGEX-KG的GST基因下游,转化BL21(DE3)大肠杆菌,经IPTG诱导表达,获得大小约173000的融合蛋白。Westernblot结果表明,该融合蛋白具有良好的反应原性。动物试验表明,该重组蛋白可以诱导小鼠产生高水平的抗体,并可抵抗致死剂量的天然DNT毒素攻击。

The dermonecrotic toxin （DNT） is one of the most important virulence factors and protective immunogens of the toxigenic Pasteurella multocida （T^＋Pm）, which encoded by the toxA gene. In this study, a 4019 bp fragment, containing the complete coding sequence of the toxA gene, was amplified from the genomic DNA of the T^＋Pm serotype D from swine by polymerase chain reaction （PCR）, and cloned into pMD18-T vector. Sequencing analysis showed that it shared more than 99.8% homology with the five corresponding sequences published in the GenBank. The toxA gene was then in-frame fused to 3＇-end of the GST gene of the prokaryotic expression vector pGEX-KG, and the resulted recombinant plasmid was transformed into E. coli BL21 （DE3） competent cells. A 173 000 fusion protein was expressed after IPTG induction. Western blot analysis showed that the fusion protein has specific immunological activity. Animal experiments revealed that a high level of antibody was detected in the mice immunized with the recombinant DNT, and the mice were protected from the challenge by a lethal dose of natural toxin. Our data suggest that the recombinant toxin may be a potential candidate for development of vaccines against the toxigenic Pasteurella multocida.