论文部分内容阅读
目的:克隆、表达并纯化肉毒毒素B重链结合区C端(BoNT/B Hc)保护性抗原片段。方法:采用PCR扩增BoNT/B Hc基因,插入表达载体pET-H is,转化E.coliBL21(pLys)细胞获得表达工程菌株,并对诱导表达条件和纯化条件进行优化。利用W estern印迹和间接ELISA方法鉴定rBoNT/B Hc的抗原性。结果:表达工程菌BL21/pETH is-BHc于37℃经0.2 mmol/L IPTG诱导6 h后,目的蛋白获得高表达,约占菌体总蛋白的24%。经过N i-NTA亲和层析柱一步纯化,rBoNT/B Hc的纯度可达96%以上。W estern印迹和间接ELISA均显示其具有良好的抗原性。结论:成功克隆、表达并纯化了BoNT/B Hc片段,并可被抗天然BoNT/B马血清所识别,为建立快速检测方法和制备相应的抗体及亚单位疫苗奠定了基础。
OBJECTIVE: To clone, express and purify the C-terminal (BoNT / B Hc) protective antigen fragment of botulinum toxin B heavy chain binding domain. Methods: The BoNT / B Hc gene was amplified by PCR, inserted into the expression vector pET-His and transformed into E.coli BL21 (pLys) cells to obtain the expression engineering strain. The expression conditions and the purification conditions were optimized. The antigenicity of rBoNT / B Hc was identified using Western blot and indirect ELISA. Results: The recombinant protein BL21 / pETH is-BHc was highly expressed in E.coli BL21 / pETH is-BHc induced by 0.2 mmol / L IPTG at 37 ℃ for 6 h, accounting for about 24% of total bacterial proteins. After purification by N i-NTA affinity chromatography, the purity of rBoNT / B Hc can reach more than 96%. Both Western blot and indirect ELISA showed good antigenicity. CONCLUSION: The BoNT / B Hc fragment was successfully cloned, expressed and purified, and can be recognized by anti-BoNT / B horse serum. This study lays a foundation for establishing a rapid detection method and preparing the corresponding antibody and subunit vaccine.