Objective: To express ricin toxin B(RTB)chain protein and prepare the antibody against RTB.Methods: The optimum design of
786 bp gene fragment was carried out by replacing rare×codons with high-frequency ones,and the 786 bp fragment was synthesized using
overlapping PCR by two steps.After confirmed by sequencing,6×His-tagged gene sequences were added to the C-termini of RTB gene fragments,which were subcloned to construct
recombinant expression vector pBV220-RTB.Then it was transformed into E.coli DH5α.The expression and purification condition was optimized. The antigenicity of rRTB was identified by Western blot and indirect ELISA.Results: Recombinant strain E.coli DH5α/pBV220-RTB was induced at 42℃ for 3 hours.A specific expression band with a relative molecular mass 29×10~(3) was detected by SDS-PAGE.Torget protein existed in form of inclusion and amounted to 65.48% of total protein.The purity of expressed protein purified by one-step Ni-NTA affinity chromatography could reach 96.21%,and 25 mg/L of recombinant protein was obtained.Both Western blot and indirect ELISA showed that the antiserum against native ricin had a specific affinity for the recombinant RTB protein.Conclusions: It is the first time to synthesize RTB gene using overlapping PCR,and recombinant protein is expressed successfully in E.coli DH5α cells.The purified recombinant RTB protein can be detected by multiclonal antibody against native ricin.This lays foundation for preparing specific antibodies against RTB and detecting ricin toxin.