| 摘要: |
| 目的 构建含有跨膜肽(TAT)的抗B型肉毒毒素胞内抗体融合蛋白,在大肠埃希氏菌中诱导表达,进行重组蛋白纯化及初步活性测定。方法 从噬菌体抗体库Tomlinson I+J筛选出全人源单链抗体(ScFv)经PCR后酶切,克隆到原核表达载体(pET-28a-TAT)中,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。结果 成功构建TAT-ScFv表达载体,融合蛋白相对分子量为32.7KDa,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)×106L/mol,经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。结论 在大肠埃希氏菌高效表达了含有TAT的抗B型肉毒毒素重组抗体,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。 |
| 关键词: 跨膜肽(TAT) 重组蛋白 表达 蛋白纯化 亲和常数 乙酰胆碱 |
| DOI： |
| 投稿时间：2016-05-31修订日期：2016-06-17 |
| 基金项目:吉林省科技发展计划资助（20130206012yy） |
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| Preparation and study on the activity of anti B botulinum toxin in the treatment of cytoplasmic antibody |
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| (Jilin Agricultural University) |
| Abstract: |
| Objective Construction of anti B type botulinum toxin recombinant antibody protein containing Transmembrane peptide(TAT), induced expression in E.coli, Purification of recombinant protein and Preliminary activity assay. Methods The whole human single chain antibody (ScFv) was screened from phage antibody library Tomlinson I J. by PCR,Connection with carrier(pET-28a-PTD), 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. Results The TAT-ScFv expression vector was successfully constructed, and the relative molecular weight of the fusion protein was 32.7KDa, 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 0.273×106L/mol. The mouse neuronal acetylcholine quantitative determination experiments show that intracellular antibody has better anti toxin activity. Conclusion In Escherichia coli efficient expression of the recombinant botulinum toxin antibody against type B, which will lay a solid foundation for the type B botulinum toxin in the treatment of intracellular antibody development and treatment of botulism. |
| Key words: : Transmembrane peptide(TAT) Escherichia coli expression protein purification activity assay acetylcholine |
