腐败梭菌α毒素基因的克隆表达及其类毒素的免疫原性研究

根据GenBank公布的腐败梭菌α毒素基因序列,设计了一对引物,并以腐败梭菌基因组为模板,经PCR特异性扩增出腐败梭菌菌株HeB01的α毒素基因。序列分析表明,该基因产物大小为1323bp,与GenBank报道的4个腐败梭菌参考菌株α毒素基因序列同源性高于99·5%。将扩增的α毒素基因定向克隆到原核表达载体pQE30中,得到重组质粒pQE30-α,将重组质粒转化大肠杆菌M15中,得到重组菌株M15(pQE30-α),经IPTG诱导后,SDS-PAGE分析可见表达的48kD特异条带;Westernblot和ELISA检测实验表明:表达的α毒素与抗天然α毒素抗体发生特异性反应,说明α毒素蛋白具有较好的免疫原性。将表达的α毒素蛋白制成类毒素疫苗,免疫小鼠后,具有一定的保护能力,表明该重组菌株有望作为腐败梭菌基因工程类毒素疫苗的候选生产菌株。

The Study on the Cloning and Expression of Alpha Toxin Gene of Clostridium septicum and the Immunity of the Toxoid

In order to amplify alpha toxin gene of Clostridium septicum HeB01 strain, one pair of primers was designed according to the GenBank sequence, and a 1323bp alpha toxin gene fragment was obstained by PCR. Sequence analysis indicated that the homology of the nucleotid sequence of HeB01 strain to those other reference strains was more than 99.5% . The expression plasmid pQE30-alpha was constructed by inserting alpha toxin gene into the prokaryotic expression vector pQE30. The plasmid expressed when the recombinant strain M15(pQE30-alpha) was induced by IPTG. The specific 48 kD protein was detected SDS-PAGE and the immunogenicity of the expressed alpha toxin was confirmed by Western blot and ELISA. The expressed alpha toxin was transformed into alpha toxoid vaccine by adding 0.3% formaldehyde into alpha toxin. The protective immune response was proved after the mice was immunized with alpha toxoid vaccine. The results showed that the recombinanted strain M15 (pQE30-alpha) could be as a candidate of alpha toxoid vaccine to provide protective immune response against clostridium septicum infection.