Preparation of Polyclonal Antibody Containing <i>CPN</i>60-<i>β</i> for Controlling Rice Stripe Virus Disease

LAN Hanhong; CHEN Haihuang; HUANG Weixin; HE Xinyi

doi:10.19303/j.issn.1008-0384.2021.05.003

Volume 36 Issue 5

May 2021

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Fujian Journal of Agricultural Sciences > 2021 > 36(5): 512-517. > DOI: 10.19303/j.issn.1008-0384.2021.05.003

LAN H H, CHEN H H, HUANG W X, et al. Preparation of Polyclonal Antibody Containing CPN60-β for Controlling Rice Stripe Virus Disease [J]. Fujian Journal of Agricultural Sciences，2021，36（5）：512−517. DOI: 10.19303/j.issn.1008-0384.2021.05.003

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

School of Biological Sciences and Biotechnology, Minnan Normal University, Zhangzhou, Fujian　363000, China

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Received Date: September 16, 2020
Revised Date: January 21, 2021
Available Online: May 16, 2021

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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.
- rice,
- chloroplast,
- CPN60-β,
- polyclonal antibody,
- titer

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