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肉毒毒素(BoNT)具有依赖锌的蛋白内切酶活性,可分解与神经传导有关的多肽,导致神经传导受阻,引起肌肉麻痹。本研究构建重组BoNT(rBoNT),采用点突变法去除维持内切蛋白酶活性的锌结合组氨酸基序,使rBoNT毒性消失而保留抗原性,并对其免疫保护作用进行了研究。含BoNT/C轻链基因的质粒pCL8用PCR扩增后用PstⅠ酶切,克隆到表达质粒pQE-30上,形成表达载体pQE-LC1。含完整rBoNT/C基因的表达载体pQE-TCl构建方法类似:先用PCR方法扩增3段带突变位点的rBoNT/c基因片段Ⅰ、Ⅱ和Ⅲ。其中IH~(229)
Botulinum toxin (BoNT) has a zinc-dependent endoproteinase activity that breaks down the neurotransmitter-related polypeptides, resulting in blocked nerve conduction and muscle paralysis. In this study, recombinant BoNT (rBoNT) was constructed and the point-mutation method was used to remove the zinc-binding histidine motif that retains endoprotease activity. The rBoNT toxicity was retained and the antigenicity was retained. The immunoprotection was also studied. The plasmid pCL8 containing the BoNT / C light chain gene was amplified by PCR, digested with PstI, and cloned into the expression plasmid pQE-30 to form the expression vector pQE-LC1. The construction method of the expression vector pQE-TCl containing the complete rBoNT / C gene was similar to that of the first rBoNT / c gene fragment Ⅰ, Ⅱ and Ⅲ with three mutation sites. IH ~ (229)