Prokaryotic Expression of FpgMBV1-P4 Gene and Preparation of Its Polyclonal Antibodies
LIU Dong-Wei1, SONG Jia-Qing1, PAN Xin1, YAN Shu-Wei1, LI Ke1, FAN Pei3, GAO Fei1, ZHANG Xiao-Ting1,*, DAI Jun-Li1,*, LI Hong-Lian1,2
1 College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, China; 2 State Key Laboratory of Wheat and Maize Crop Science, Zhengzhou 450046, China; 3 Colledge of Biology Engineering, Henan University of Technology, Zhengzhou 450001, China
Abstract：Fusarium pseudograminearum megabirnavirus 1 (FpgMBV1) is a mycovirus found in the hypovirulence Fusarium pseudograminearum strain FC136-2A. Four proteins were encoded in FpgMBV1. Among them, FpgMBV1-P4 composed of 268 amino acids with unknown function. Specific primers were designed and used in reverse transcription PCR (RT-PCR) to amplify the FpgMBV1-P4 gene from the hyphae of the strain FC136-2A in this study. Then a prokaryotic expression vector pGEX4T-FpgMBV1-P4 was constructed and transformed into Escherichia coli BL21(DE3) strain, and the recombinant protein was induced at 25 ℃ with 0.5 mmol/L isopropyl β-D-thiogalactoside (IPTG). Results showed that FpgMBV1-P4 in length of 807 bp was successfully amplified and the fusion protein with a molecular weight of about 60 kD was highly expressed . The purified fusion protein was used to immunize Zealand white rabbit (Lepus sinensis) to obtain antiserum. Indirect ELISA results showed the titer of this antiserum reached 1∶160 000. And the antiserum was used to specifically detect the FpgMBV1-P4 protein at about 36 kD from samples of the hyphae of FC136-2A. These results laid a foundation for the structural and functional analysis of FpgMBV1-P4, which would be meaningful in deciphering the hypovirulent mechanism of FpgMBV1.
刘东伟, 宋嘉庆, 潘鑫, 闫书味, 李轲, 范沛, 高飞, 张晓婷, 代君丽, 李洪连. FpgMBV1-P4基因的原核表达及多克隆抗体制备[J]. 农业生物技术学报, 2022, 30(11): 2246-2254.
LIU Dong-Wei, SONG Jia-Qing, PAN Xin, YAN Shu-Wei, LI Ke, FAN Pei, GAO Fei, ZHANG Xiao-Ting, DAI Jun-Li, LI Hong-Lian. Prokaryotic Expression of FpgMBV1-P4 Gene and Preparation of Its Polyclonal Antibodies. 农业生物技术学报, 2022, 30(11): 2246-2254.
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