摘要
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 pr ote ctive antigen of botulinum neurotoxin type E (BoNT/EH C2).Method s: A 437 bp gene fragment of BoNT/EH C2 was amplified from Clo stridium botulinum type E by PCR and inserted into the pET28a vector to constr uct an expression vector pET28a-EH C2. Then it was transformed into E.co li BL21 (DE3) pLysS. The expression and purification condition was optimized. The antigenicity of rBoNT/EH C2 was identified by Western blot and indirect ELISA. Results:The genetically engineering strain pET28a -EH C2/BL21(DE3)pLysS was induced for 6 h by 0.2 mmol/L IPTG at 37℃ and m ade a high-level expression which accounted for 37.9% of total protein. The rBo NT/EH C2 protein was one-step purified by immobilized metal-chelating aff inity chromatography (IMAC) and its purity was above 95%. Western blot and indir ect ELISA identified its good antigenicity. Conclusions:It is the first time to clone, express and purify BoNT/EH C2 successfully in domestic laboratory. It also can be detected by horse antiserum against native BoNT/E. It lays foundation for preparing antibody against BoNT/EH C2 and establishing a rapid detection method for BoNT/E.
出处
《军事医学科学院院刊》
CAS
CSCD
北大核心
2004年第5期417-419,423,共4页
Bulletin of the Academy of Military Medical Sciences
基金
国家自然科学基金资助项目 (3 0 3 70 0 81)
