Construction of a Single Chain Variable Fragment(ScFv) Antibody against Ricin and Expression in E.coli

To construct and express a single chain variable(ScFv) fragment against ricin,VH and VL genes of anti-murine RT monoclonal antibody were cloned by RT-PCR from hybridoma cell secreting anti-RT McAb. ScFv gene was spliced by sequence overlap extending (SOE) PCR. ScFv gene was cloned into pMAL-p2X expression vector and transformed into TB1 E.coli. After positive clones were induced by IPTG for 18 hours,the identification of recombinant protein was detected by Western blotting. ScFv gene of VL-(Gly4Ser)3-VH was constructed successfully. The VH chain consisted of 363 bp and encoded 121 amino acids. The VL chain consisted of 324 bp and encoded 108 amino acids. SDS-PAGE and Western blotting analysis showed that the RT-ScFv gene was expressed in TB1 E.coli. The ScFv antibody expressed by pMAL-p2X fused with MBP tag protein and the relative molecular mass of fusion protein was about 75 ku. The expression vector of pMAL-RT-ScFv fusion protein was constructed successfully,and procured high performance expression.