目的 构建艰难梭菌高毒力株2型毒素B(TcdB2)的枯草芽胞杆菌工程株,获得高效表达的生物活性TcdB2蛋白.方法 以ST1/RT027型菌株基因组DNA为模板,通过PCR扩增获得TcdB2全长基因片段,构建到PHT01载体中,转化枯草芽胞杆菌WB800N菌株,进行原核表达获得TcdB2全长蛋白,进一步通过细胞毒性试验验证重组TcdB2的生物活性.结果 成功构建了高效表达TcdB2的枯草芽胞杆菌工程株,并获得具有细胞毒性的重组蛋白TcdB2.结论 具有生物活性的重组TcdB2蛋白,为进一步研究艰难梭菌高毒力株的致病机制和作为疫苗候选靶标等奠定了基础.
This study was aimed at creating an engineered strain of Bacillus subtilis for efficient expression of biologically active type 2 toxin B(TcdB2)derived from a highly virulent strain of Clostridium difficile.The TcdB2 gene was cloned from ST1/RT027 strain genome DNA,incorporated into the PHT01 vector,and then transformed into B.subtilis strain WB800N for prokaryotic expression.Cell toxicity assays revealed that the recombinant TcdB2 exhibited cytotoxic effects in various cells.The engineered B.subtilis strain effectively expressed biologically active TcdB2,thus providing a basis for further exploration of the pathogenic mechanisms of highly virulent strains of C.difficile and establishing a foundation for potential vaccine can-didate targets.
林兴豪;张凯;王梦婕;杨鸣;顾汉阳;薛笑兰;罗永能;金大智;胡珲
杭州医学院检验医学院、生物工程学院,杭州 310053杭州医学院检验医学院、生物工程学院,杭州 310053||浙江省生物标志物与体外诊断转化重点实验室,杭州 310063
基础医学
艰难梭菌;毒素B;枯草芽胞杆菌;表达;细胞毒性
Clostridium difficile;toxin B;Bacillus subtilis;expression;cytotoxicity
《中国人兽共患病学报》 2024 (006)
498-503 / 6
Supported by the National Natural Science Foundation of China(No.8210030375)and the Foundation from Zhejiang Medical and Health Science and Technology(No.2021RC050). 国家自然科学基金(No.8210030375)与浙江省医药卫生科技项目(No.2021RC050)联合资助
10.3969/j.issn.1002-2694.2024.00.076
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