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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 botulinum neurotoxin A light chain (BoNT / ALC) fragment and lay the foundation for the preparation of antibody to BoNT / A and the establishment of detection methods. Methods: The BoNT / A light chain gene was amplified by PCR from Clostridium botulinum and inserted into pGEM T vector. After sequencing correctly, the recombinant plasmid pET21a was constructed with the expression vector pET21a and transformed into E. coli BL21 (pLys) The expression of the engineered cells was induced by the state cells, and the recombinant BoNT / A light chain was identified by Western blotting. Results: The coincidence of the BoNT / A light chain sequence obtained by PCR and the BoNT / A light chain gene sequence in GenBank was over 99.9%. Expression engineering bacteria BL21 / pET21a ALC at 37 ℃ induced by 0. 7mmol / LIPTG 6h, the target protein was highly expressed, accounting for 23% of the total bacterial protein. After purification by nickel ion affinity chromatography with Ni NTA, the purity of recombinant BoNT / A light chain can reach more than 97%. Western blot results showed that the recombinant BoNT / A light chain and anti-native BoNT / A horse serum specific antigen-antibody binding reaction. Conclusion: The BoNT / A light chain fragment was successfully cloned, expressed and purified, and its antigenicity is good.