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以克隆表达的B型肉毒毒素轻链蛋白(Bo NT/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(Sc Fv),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-Sc Fv)。经PCR后酶切,克隆到原核表达载体(p ET-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-Sc Fv表达载体,融合蛋白相对分子量为32.7 k Da,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-Sc Fv的亲和常数为(1.133±0.273)×106L/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。
The full-length single-chain Fv antibody (Sc Fv) with high activity and high specificity was screened from the phage antibody library Tomlinson I + J by cloned and expressed Btotoxin B light chain protein (Bo NT / BL) Transmembrane single-chain antibodies (TAT-Sc Fv) were prepared in combination with a small fragment of transmembrane peptide (TAT) that efficiently carried the foreign protein through the biofilm. After being digested by PCR, cloned into the prokaryotic expression vector (p ET-28a-TAT), an anti-botulinum toxin B fusion protein containing transmembrane peptide (TAT) was constructed and expressed in E. coli. The purification process, the product concentration and purity, affinity constant determination and biological activity. The TAT-Sc Fv expression vector was successfully constructed. The relative molecular weight of the fusion protein was 32.7 kDa. The fusion protein was mainly expressed in soluble form with a purity of more than 95%. The intracellular antibody neutralized type B botulinum toxin pathogenic light chain to obtain TAT-Sc The affinity constant of Fv was (1.133 ± 0.273) × 106 L / mol. The quantitative determination of acetylcholine in mouse neurocytes proved that the intracellular antibody had good antitoxin activity. This result provides a basis for the development of botulinum toxin type B therapeutic intrabodies and the treatment of botulism.