D型产气荚膜梭菌ε毒素基因表达及其免疫保护作用的初步研究

摘要

利用PCR技术,从D型产气荚膜梭菌标准株(C60-2)染色体DNA中扩增出ε毒素基因.该基因产物大小为906bp,序列分析结果表明,与GenBank报道的产气荚膜梭菌参考菌株ε毒素基因序列同源性大于99.将扩增的ε毒素基因定向克隆到原核表达载体pET32a中,得到重组质粒pET32a-ETX,将重组质粒转化大肠杆菌BL21 (DE3) plys中,经IPTG诱导后,SDS PAGE分析可见大小为54 ku的特异条带；经Western blotting和ELISA检测结果表明,表达的ε毒素能与抗天然ε毒素抗体发生特异性反应,说明ε毒素蛋白具有较好的反应原性.将表达的ε毒素蛋白用0.4％的甲醛溶液制成类毒素疫苗,免疫小鼠后,具有一定的保护力,这表明该重组菌株有望成为产气荚膜梭菌基因工程类毒素疫苗的候选菌株.%Epsilon-toxin (etoxin) gene was amplified from chromosomal DNA of Clostridium perfringens type D (C60-2) by polymerase chain reaction (PCR) .and a 906 bp epsilon toxin gene fragment was obtained. Sequence analysis indicated that the homology of the nucleotide sequence of the strain to those other reference strains was more than 99%. The expression plas-mid pET32a-ETX was constructed by inserting the epsilon toxin gene into the prokaryotic expression vector pET32a. The plas-mid pET32a-ETX was transformated into E. coli BL21 (DE3) plys and the recombinant strain BL21 (pET32a-ETX) was obtained. Then,the transformants were induced to express with IPTG. The specific 54 ku protein was detected by SDS-PAGE and the immunogenicity of the expressed epsilon toxin was confirmed by Western blotting and ELISA. The obtained recombinant protein was transformed into epsilon toxoid vaccine by adding 0. I% formaldehyde into epsilon toxin. The protective immune response was proved after the mice was immunized with epsilon toxoid vaccine. The results showed that the recombinan-ted strain BL21(pET32a-ETX) could be as a candidate of epsilon toxoid vaccine to provide protective immune response against C. Perfringens type D infection.