Construction, Expression, Purification And Function Of Single Chain Fragment Variable (ScFv) Against Ricin

In our experiments, the total RNAs were extracted from a hybridoma cell 4D12, producing murine monoclonal antibody against Ricin protein. The total RNAs were reverse transcription by the reverse transcriptase into cDNA sequence. Design primers, the variable region genes of light chain and heavy chain (VL, VH genes) of monoclonal antibody against Ricin protein were cloned by PCR from cDNA sequence. Add the Linker sequence to VL, VH genes, to gets the VL′, VH′genes. The VL′, VH′genes were spliced into ScFv by SOE-PCR. The ScFv gene was cloned into pMAL-p2X expression vector and sequenced. The nucleotide results showed that the VL gene shares 91% consistency with the Mus musculus Igκchain v-v region, and the VH gene shares 93% consistency with the Mus musculus 414.2 heavy chain variable region. It′s shows that the VL, VH genes were confirmed as functionally rearranged mouse immunoglobulin variable region genes. The ScFv gene was 750bp, VL gene was 324bp, encoding 108 amino acids; VH gene was 363bp, encoding 121 amino acids. The Linker sequence was between VL and VH which was 45bp. The results were conformity as the design.The pMAL-RT-ScFv recombinant plasmid was transformed into TB1 strain. The recombination protein was high expression by induced with IPTG at a low temperature, and the recombination protein was solubled. SDS-PAGE showed that the soluble protein had molecular weight approximately to 73kDa, was conformity as the theoretical value. Western-blot shows that, the fused protein purified by Amolose resin affinity column which can be tested specificity. The binding antigen affinity of the soluble protein was measured by ELISA and SPR. The results showed that the pMAL-RT-ScFv soluble protein had the activity of antigen binding.In our research, the variable region genes of light chain and heavy chain were extracted from a hybridoma cell 4D12, which producing murine monoclonal antibody against Ricin protein by RT-PCR. Spliced a ScFv by SOE-PCR and expressed in pMAL-p2X expression system. It is successfully to constitute the active ScFv gene which has combined with the ricin that provides perfect theory foundation for the researches related with both the application of the Ricin, the organism poisoned by rested toxin within the castor cakes or just the treatment after it, as well as the detoxication of the castor cakes.