Cloning, Expression Of Recombinant Botulinum Neurotoxin Serotype A And Its Preparation Of ELISA Kit

Botulinum neurotoxin (BoNT) produced by Clostridium botulinum, is a multimeric molecule composed of seven serotypes that have been assigned the letters A through G according to its antigenicity and BoNT/A, B, E are responsible for most of human botulism. BoNT molecule is divided into two parts structurally: light chain (LC) is the catalytic domain as zinc dependent metalloprotease; Heavy chain (HC) is the binding and translocation domain. The carboxy-terminal portion of HC plays a major role in interaction with the receptor located on target cells membrane. At present, BoNT is one of the important toxins in biological warfare agents; therefore, it is significant to perform an investigation into the recombinant BoNT antigenic fragments for developing ELISA Kit and preventive vaccine candidate.The purpose of this study was to develop cloning and expression of the gene of BoNT/A in E.coli by using PCR and DNA recombinant technology, and then to prepare monoclonal antibodies and polyclonal antibody. We purified and labeled antibodies for developing ELISA Kit, which were applied in the detection of BoNT toxin. The experimental results as follow:Recombinant botulinum neurotoxin serotype A binding domain [BoNT/A Hc-C], expressed in Escherichia coli cells. To improve the levels of expression in Escherichia coli, the gene encoding BoNT/A Hc-C was optimized by replacing rare codons with high-frequency ones and adjusting AT contents, then synthesized using overlapping PCR.The optimized BoNTAHc-C was subcloned into the pET-His vector. A 714-bp fragment of pET-His cloning vector was cut by BamH I and Hindâ…¢,and BoNTAHc was one-step purified and refolded by using Ni²⁺ affinity gel column chromatography .The purified recombinant BoNTAHc-C was be used to immunize the BALB/C mice. Three hybridoma cell lines, which secreted monoclonal antibodies (mAbs)against BoNTA consistently, were obtained through cell fusion, screening and cloning, Isotype of these three mAbs are all IgG1. The titres of the ascetic fluids of three mAbs measure by indirect ELISA were 1/10000. Their specific reaction is to BoNT/A only, not to BoNT/B, and SEB.Antiserum was prepared by immunizing rabbits,it was purified by caprylic method, their titers...