Abstract:
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.
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