重组肉毒神经毒素A轻链(BoNT/ALC)蛋白的表达、纯化与鉴定  

Expression of the recombinant light chain of botulinum neurotoxin serotype A (BoNT/A LC) in E.coli and its purification and identification

作　　者：贾宏丽[1] 王景林[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病原微生物与生物安全国家重点实验室,北京100071

出　　处：《军事医学科学院院刊》2005年第1期30-33,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(30370081)

摘　　要：　目的:克隆、表达并纯化肉毒神经毒素A轻链(BoNT/ALC)片段,为BoNT/A的抗体制备和检测方法的建立奠定基础。方法:以PCR方法自肉毒梭菌中扩增BoNT/A轻链基因,直接插入pGEM T载体,测序正确后再与表达载体pET21a构建重组表达载体pET21a 轻链,转化E.coliBL21(pLys)感受态细胞获得表达工程菌株并进行诱导表达,用Western印迹鉴定重组BoNT/A轻链。结果:经PCR获得的BoNT/A轻链序列与GenBank中的BoNT/A轻链基因序列的一致性达 99. 9%以上。表达工程菌BL21 /pET21a ALC于 37℃经 0. 7mmol/LIPTG诱导 6h,目的蛋白获得高表达,约占菌体总蛋白的 23%。经过镍离子螯合次氨基三乙酸 (Ni NTA)亲和层析一步纯化,重组BoNT/A轻链的纯度可达 97%以上。Western印迹结果表明,重组BoNT/A轻链与抗天然BoNT/A的马血清发生特异性的抗原抗体结合反应。结论:成功克隆、表达并纯化了BoNT/A轻链片段,其抗原性良好。Objective: To clone, express and purify the light chain(LC) of BoNT/A. Methods: A 1 364-bp gene fragment of BoNT/A LC was amplified from Clostridium botulinum type A by PCR and inserted into pET21a vector to construct a recombinant prokaryotic expression vector pET21a-ALC. Then it was transformed into E.coli BL21 (DE3) pLysS competent cells to express recombinant BoNT/A LC induced by IPTG. The antigenicity of recombinant BoNT/A LC was identified by Western blot. Results: BoNT/A LC gene was correctly amplified and inserted into the vectors as confirmed by sequencing and restriction analysis. The genetically engineered strain of E.coli BL21/pET21a-ALC was induced by 0.7 mmol/L IPTG for 6 h at 37℃ and a high level of expression was obtained. The target protein amounted to 23% of total cell protein and existed in inclusion body form. The recombinant BoNT/A LC protein was one-step purified by affinity chromatography with immobilized nickel-chelating NTA(Ni-NTA)and its purity was up to 97%. The good antigenicity was identified by Western blot. Conclusion: It is the first time to clone, express and purify BoNT/A LC successfully in domestic laboratories. The purified recombinant BoNT/A LC protein shows a good antigenicity. It lays foundation for preparing the antibody against BoNT/A LC and establishing a rapid detection method for BoNT/A.

关 键 词：肉毒神经毒素A(BoNT/A) 轻链 克隆表达 Ni—NTA亲和层析 

分 类 号：R996.1[医药卫生—毒理学]