产气荚膜梭菌ε毒素蛋白高效表达体系的构建及其免疫保护性的研究

ε毒素是由B和D型产气荚膜梭菌产生的,能够引起牛、羊肠毒血症,给畜牧业造成巨大的经济损失。本试验将ε毒素基因片段(972 bp)以正确的阅读框架定向连接到pET-28a(+)质粒上,然后将重组质粒导入受体菌株,成功构建重组菌株BL21(DE3)。SDS-PAGE和Western blotting检测结果表明,重组的菌株BL21(DE3)能以包涵体的形式高效表达ε毒素(占菌体总蛋白的32.83%)。在此基础上,用有效剂量0.2 mg包涵体粗提物免疫家兔,抗血清间接ELISA检测结果显示,在第4周抗体效价达到最高值为8.7log2。因此,在本研究中构建的重组菌株BL21(DE3)能高效表达ε毒素,而且该毒素具有较高的免疫保护性。

Construction of Efficient Expression System of Clostridium perfringens Epsilon Toxin Protein and Analysis of its Immune Protection

The epsilon toxin produced by type B and D Clostridium perfringens,could lead to fatal illness in a variety of livestock animals,causing financial loss.In this study,pET-28a(+) plasmid harboring epsilon toxin gene was transferred into E.coli strain BL21(DE3)to efficiently produce epsilon toxin.Results of SDS-PAGE and Western blotting showed that recombinant E.coli BL21(DE3) could highly expressed ε toxin(about 32.83% of total cellular protein).On this basis,rabbits were immunized with 0.2 mg of the insoluble recombinant protein to evaluate immune protection of the expressed epsilon protein.Results of indirect ELISA demonstrated that anti-ε antibody titer reached the maximum value,8.7log2 in the fourth week.In conclusion,the recombinant strain BL21(DE3) could highly express ε toxin,and the toxin had excellent immune protection.