Preparation of Polyclonal Antibody Containing CPN60-β for Controlling Rice Stripe Virus Disease

  Objective  To elucidate the regulatory function and mechanism of the pathogenic processes mediated by the rice stripe virus (RSV)-encoding NSvc4 protein in rice, the high titer concentrated polyclonal antibody containing the chloroplast-related chaperonin-60-beta (CPN60-β) protein against the RSV disease was prepared.

  Method   The CPN60-β fragment was amplified by RT-PCR from rice genome. The recombinant plasmid pET-CPN60-β was constructed with prokaryotic expression plasmid pET-28a (+) and further expressed in Escherichia coli BL21 cells. The CPN60-β fusion protein was purified and immunized into New Zealand rabbits to obtain polyclonal antibody. Titer and concentration of the polyclonal antibody against CPN60-β protein were detected by ELISA and SDS-PAGE for confirmation.

  Result   The approximately 657bp chloroplast-related CPN60-β fragment was successfully amplified by RT-PCR. The reconstructed plasmid pET-CPN60-β positively expressed the CPN60-β fusion protein in E. coli BL21 cells under IPTG concentration of 0.5mmol·L⁻¹ at 37 ℃ with a rotate speed of 220 r·min⁻¹ and an induction time of 5 h. The purified polyclonal antibody had a high titer of 1:10⁶ and a concentration of 300 μg·mL⁻¹ as detected separately by ELISA and SDS-PAGE.

  Conclusion   The conditions of CPN60-β prokaryotic expression were confirmed and the polyclonal antibody against chloroplast-related CPN60-β protein with high titer and concentration successfully prepared for further studies.