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Bioscreening and expression of a camel anti-CTGF VHH nanobody and its renaturation by a novel dialysis–dilution method

机译:骆驼抗CTGF VHH纳米抗体的生物筛选表达及其复性的新方法

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摘要

The variable regions of the camel heavy chain antibody, also known as nanobody is the smallest antibody with antigen-binding efficiency. CTGF is considered important during extracellular matrix deposition which was involved in the pathogenesis of fibrosis related diseases. There are several anti-CTGF-C nanobody drugs under developing in pharmacy. In this study, we described the screening of a novel anti-CTGF-C nanobody from the peripheral blood of immunized camel by phage display. The screened nanobody was further expressed and purified from E. coli cells. A sophisticated dialysis–dilution method was designed for the in vitro refolding of the nanobody. The results showed that the expressed nanobody was consisted of 135 amino acid and mainly expressed as inclusion body in E. coli cells. The dialysis–dilution method was very effective and the recovery rate of the renaturation was more than 80 %. The ELISA result suggested the nanobody had been well refolded showing a superior CTGF binding activity to the commercial mouse anti-CTGF-C mAb. In conclusion, the anti-CTGF-C nonobody had been successfully screened by phage display. The dialysis–dilution refolding method was very effective and the recovery rate reached over 80 %.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0249-1) contains supplementary material, which is available to authorized users.
机译:骆驼重链抗体的可变区,也称为纳米抗体,是具有抗原结合效率的最小抗体。 CTGF被认为在细胞外基质沉积过程中很重要,该过程参与了纤维化相关疾病的发病机理。药学领域正在开发几种抗CTGF-C纳米抗体。在这项研究中,我们描述了通过噬菌体展示从免疫骆驼的外周血中筛选新型抗CTGF-C纳米抗体的方法。从大肠杆菌细胞中进一步表达和纯化所筛选的纳米抗体。设计了一种复杂的透析-稀释方法,用于体外重折叠纳米抗体。结果表明,表达的纳米抗体由135个氨基酸组成,主要表达为大肠杆菌细胞中的包涵体。透析稀释法非常有效,复性的回收率超过80%。 ELISA结果表明该纳米抗体已被很好地重折叠,显示出优于商用小鼠抗CTGF-C mAb的CTGF结合活性。总之,已经通过噬菌体展示成功地筛选了抗CTGF-C的非抗体。透析-稀释复折方法非常有效,回收率达到80%以上。电子补充材料本文的在线版本(doi:10.1186 / s13568-016-0249-1)包含补充材料,授权用户可以使用。

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