抗B型肉毒毒素治疗性胞内抗体的制备及活性研究

摘要

以克隆表达的 B 型肉毒毒素轻链蛋白（BoNT／BL）为抗原，从噬菌体抗体库 Tomlinson I ＋J筛选出得到活性高和特异性高的全人源单链抗体（ScFv），结合能够携带外源蛋白有效通过生物膜的小片段跨膜肽（TAT）制备跨膜单链抗体（TAT －ScFv）。经 PCR 后酶切，克隆到原核表达载体（pET －28a －TAT）中，构建含有跨膜肽（TAT）的抗 B 型肉毒毒素胞内抗体融合蛋白，并在大肠杆菌中诱导表达，进行纯化工艺，对产物进行浓度纯度、亲和常数测定及生物活性研究。成功构建 TAT－ScFv 表达载体，融合蛋白相对分子量为32．7 kDa，主要以可溶形式表达，纯度也达到95％以上，胞内抗体中和 B 型肉毒毒素致病轻链得到 TAT －ScFv 的亲和常数为（1．133±0．273）×106 L／mol，小鼠神经细胞乙酰胆碱定量测定实验证明胞内抗体具有较好的抗毒素活性。此结果为 B 型肉毒毒素治疗性胞内抗体的研制和肉毒中毒治疗奠定了基础。%Cloning and expression of botulinum toxin type B light chain protein (BoNT/BL)as antigen,the whole human single chain antibody (ScFv)with high activity and high specificity was obtained from phage antibody library I +J Tomlinson,Cross membrane peptide (TAT)preparation of transmembrane single chain antibodies (TAT -ScFv)by binding to a small fragment of a biological membrane capable of carrying a foreign protein by PCR, Connection with carrier(pET -28a -PTD),Construction of anti B type botulinum toxin recombinant antibody protein containing transmembrane peptide (TAT),And the expression was induced in E.coli.The purification process was carried out,and the concentration and purity of the product,the affinity constant and the search of biological activity.The TAT -ScFv expression vector was successfully constructed,and the relative molecular weight of the fusion protein was 32.7 kDa,which was mainly expressed in soluble form,and the purity reached more than 95%.Intracellular neutralizing antibodies against type B botulinum toxin light chain TAT -ScFv affinity constants for 1.133 +0.273 ×106 L/mol.The mouse neuronal acetylcholine quantitative determination experiments show that intracellular antibody has better anti toxin activity,which will lay a solid foundation for the type B botulinum toxin in the treatment of intracellular antibody development and treatment of botulism.