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首页> 外文期刊>Nepal Journal of Biotechnology >Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains
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Cloning, Expression, Purification, and Characterization of Clostridium botulinum Neurotoxin Serotype F Domains

机译：肉毒梭菌神经毒素血清型F结构域的克隆，表达，纯化和表征

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摘要

The use of recombinant BoNT domains has been proposed as a means to develop strategies to treat and prevent botulism. Here, details on the molecular cloning, protein expression, purification, and immunoreactivity of BoNT/F domains from Clostridium botulinum are presented. Initially, full-length synthetic genes encoding recombinant BoNT/F domains (catalytic, translocation, and receptor binding) were designed and cloned into Escherichia coli for expression. Recombinant proteins were then purified through GST affinity chromatography preceding elution of GST-free recombinant domains by thrombin protease. Soluble recombinant proteins encoding catalytic light chain and translocation N-terminal heavy chain were subsequently used to perform in vivo immunization. Polyclonal mouse antibodies specific to these domains were raised, confirmed by Western blot analysis and elevated immunoreactivity was identified through indirect ELISA. In conclusion, availability of the recombinant protein provides an effective system to study the immunological aspects of BoNT/F and corresponding applications in pathogen detection and vaccine candidacy. Keywords: Clostridium botulunium; Botulinum Neurotoxin Type F (BoNT/F) domains; cloning; recombinant protein expression; immunoreactivity DOI: http://dx.doi.org/10.3126jb.v2i1.5634 Nepal Journal of Biotechnology Jan.2012, Vol.2(1): 1-15
机译：已经提出使用重组BoNT结构域作为开发策略来治疗和预防肉毒中毒的手段。在此，详细介绍了肉毒梭菌BoNT / F结构域的分子克隆，蛋白表达，纯化和免疫反应性。最初，设计了编码重组BoNT / F结构域的全长合成基因（催化，易位和受体结合），并将其克隆到大肠杆菌中进行表达。然后通过凝血酶蛋白酶洗脱不含GST的重组结构域，然后通过GST亲和色谱法纯化重组蛋白。随后将编码催化轻链和易位N末端重链的可溶性重组蛋白用于体内免疫。产生了针对这些域的多克隆小鼠抗体，通过蛋白质印迹分析确认，并通过间接ELISA鉴定了升高的免疫反应性。总之，重组蛋白的可用性为研究BoNT / F的免疫学方面以及在病原体检测和疫苗候选中的相应应用提供了有效的系统。关键字：肉毒杆菌F型肉毒杆菌神经毒素（BoNT / F）域;克隆;重组蛋白表达;免疫反应性DOI：http://dx.doi.org/10.3126jb.v2i1.5634尼泊尔生物技术杂志2012年1月，第2卷（1）：1-15

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