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目的 :克隆、表达并纯化肉毒毒素E重链C端 (BoNT/EHC2 )保护性抗原片段 ,为BoNT/E的抗体制备和检测方法的建立奠定基础。方法 :采用PCR扩增BoNT/EHC2基因 ,插入表达载体 pET2 8a ,转化E .coliBL2 1(DE3) pLysS获得表达工程菌株 ,并对诱导表达条件和纯化条件进行优化。利用Western印迹和间接ELISA方法鉴定rBoNT/EHC2的抗原性。结果 :表达工程菌 pET2 8a EHC2 /BL2 1(DE3) pLysS于 37℃用 0 .2mmol/LIPTG诱导 6h ,表达的目的蛋白可以达到全菌蛋白的 37.9%。经过固定化金属螯合亲和层析一步纯化 ,rBoNT/EHC2的纯度可达 95 %以上。Western印迹和间接ELISA显示其具有良好的抗原性。结论 :首次克隆、表达并纯化了BoNT/EHC2片段 ,并可被抗天然BoNT/E马血清所识别 ,为制备相应的抗体及建立快速检测方法做好了准备。
OBJECTIVE: To clone, express and purify the fragment of protective antigen of botulinum toxin E heavy chain at the C terminus (BoNT / EHC2), which will lay the foundation for the preparation of antibody and detection method of BoNT / E. Methods: The BoNT / EHC2 gene was amplified by PCR, inserted into the expression vector pET2 8a and transformed into E. coli BL21 (DE3) pLysS to obtain the expression engineering strain. The induced expression conditions and purification conditions were optimized. The antigenicity of rBoNT / EHC2 was identified by Western blot and indirect ELISA. Results: The expressed recombinant protein pET2 8a EHC2 / BL2 1 (DE3) pLysS was induced by 0.2 mmol / L IPTG at 37 ℃ for 6 h, and the expressed protein reached 37.9% of the total bacterial protein. After purification by immobilized metal chelate affinity chromatography, the purity of rBoNT / EHC2 can reach more than 95%. Western blot and indirect ELISA showed good antigenicity. CONCLUSION: The BoNT / EHC2 fragment was cloned, expressed and purified for the first time and was identified by anti-BoNT / E horse serum in preparation for the preparation of the corresponding antibody and establishment of a rapid detection method.