产气荚膜梭菌B型C58-1株β1毒素基因的克隆表达

利用PCR技术,从B型产气荚膜梭菌中国标准株C58-1株扩增出β1毒素基因,连接pMD18-T载体筛选阳性克隆,然后用限制性核酸内切酶BamHⅠ和SalⅠ对其进行酶切,回收927bp的β1毒素基因片段,将其定向克隆到载体pET-32a中,获得重组质粒pETβ927。将pETβ927转化至受体菌BL21(DE3)中,其表达产物经His-Trap FF预装柱纯化、SDS-PAGE检测目的蛋白大小和分布及Western blotting检测其反应原性。结果表明,完整的β1毒素基因大小为1 011bp,与GenBank发表的B型和C型产气荚膜梭菌β1毒素蛋白序列同源性达99.4%以上;SDS-PAGE结果显示重组目的蛋白在大肠杆菌中成功表达,融合蛋白大小为54ku,在超声波裂解上清和包涵体中均有分布,但以包涵体为主。Western blotting检测结果显示表达的重组蛋白可与特异性血清抗体发生免疫反应,表明β1毒素蛋白具有较好的反应原性。

Cloning and Expression of β1 Toxin Gene from Clostridum perfringens Type B C58-1 Strain

β1 toxin gene was amplified from Clostridum perfringens type B C58-1 strain by polymerase chain reaction (PCR),PCR products were connected to pMD18-T vector screening positive clones,and then cleaved with restriction endonucleases BamHⅠand SalⅠ,the 927 bp gene fragment was recovered and inserted into the same site of pET-32a vector.The recombinant plasmid pETβ927 was studied in detail by restriction endonuclease analysis and nucleotide sequencing.The recombinant plasmid could produce β1 toxin protein by SDS-PAGE.Expressed products were purified by pre-installed column of His-Trap FF,the size and distribution of the target protein were detected by SDS-PAGE,and its immunorectivity was confirmed by Western blotting.The results showed that the β1 toxin gene was 1 011 bp and the homologies with B and C type Clostridum perfringens protein sequences of GenBank were greater than 99.4%;In SDS-PAGE analysis,the fusion protein was 54 ku as expected and distributed in ultrasonic lysis supernatant as well as in inclusion bodies,but mainly existed in inclusion bodies.Western blotting analysis showed that the β1 toxin protein had a good immunorectivity with specific serum antibody.