C型产气荚膜梭菌β_1、β_2毒素基因的融合

Fusion of beta1-toxin gene and beta2-toxin gene from Clostridium perfringens type C

利用PCR技术 ,从C型产气荚膜梭菌染色体DNA中扩增出 β1 和 β2 毒素基因 ,构建了含 β1 - β2 融合基因表达质粒的重组菌株BL2 1(DE3) (pETXB1_2 )。经酶切鉴定和序列测定证实 ,构建的重组质粒pETXB1_2含有 β1 - β2 融合基因 ,且基因序列和阅读框架正确。经ELISA检测 ,重组菌株表达的 β1 - β2 融合蛋白能够被 β1 、β2 毒素抗体识别。免疫实验结果表明 ,用β1 - β2 融合蛋白免疫的小鼠可以抵抗 1MLD的C型产气荚膜梭菌C5 9_4 4毒素攻击 ,表明构建的重组菌株可以作为预防仔猪红痢基因工程亚单位苗的候选菌株。

Beta1-toxin and beta2-toxin genes from chromosomal DNA of Clostridium perfringens type C were amplified by PCR, PCR products were cleaved with restriction endonucleases and recovered. The recombinant plasmid pETXB1-2 containing β 1-β 2 fusion genes was constructed by recombinant technique and then transformed into Escherichia coli BL21(DE3). The β 1-β 2 fusion proteins were expressed in recombinant strain BL21(DE3) (pETXB1-2), and the expression level of the β 1-β 2 fusion proteins was about 15.36 of total cellular protein by SDS-PAGE and thin-layer gel scanning analysis. More importantly, immunization in a mouse model with crude preparation containing the fusion protein inclusion bodies or inactivated recombinant strain induced protection against at least 1MLD of the toxin from Clostridium perfringens type C. Hence the fusion proteins possess a good immunogenicity.The constructed recombinant strain BL21(DE3)(pETXB1-2) can be used as a candidate of vaccine strain.