Prokaryotic Expression of Xylanase VmXyl1 Gene in Valsa mali and Preparation of Polyclonal Antibody
MENG Lu-Lu*, YU Chun-Lei*, LIAN Sen, LI Bao-Hua, LIANG Wen-Xing, WANG Cai-Xia**
College of Plant Health and Medicine, Key Laboratory of Integrated Crop Pest Management of Shandong, Shandong Province Key Laboratory of Applied Mycology, Qingdao Agricultural University, Qingdao 266109, China
Abstract：Xylanase VmXyl1 plays an important role in the pathogenesis of Valsa mali. In order to reveal the action mechanism of VmXyl1 in the interaction of V. mali and host, a recombinant plasmid pET-32a-VmXyl1 was constructed in this study. The fusion protein VmXyl1 was expressed successfully in Escherichia coli Rosetta (DE3) strain when induced by isopropyl β-D-1-thiogalactopyranoside (IPTG). The solubility and purification effects of the fusion protein were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then, New Zealand white rabbits (Oryctolagus cuniculus) were immunized with purified VmXyl1 protein and polyclonal antibody was prepared. The titer and specificity of the antibody were analyzed by enzyme-linked immunosorbent assay (ELISA) and Western blot. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the fusion protein VmXyl1 mainly existed in the form of inclusion body, and the protein was expressed abundantly under induction by 0.5 mmol/L IPTG for 8 h at 30 ℃. The target protein with high purity was obtained by the Ni-NTA affinity chromatography. ELISA showed that the titer of prepared antibody was up to 1∶102 400. Western blot analysis demonstrated the polyclonal antibody could specifically recognize both the fusion protein VmXyl1 and xylanase protein in the apple (Malus pumila) bark tissue infected by V. mali, which indicated that the prepared antibody had high efficiency and specificity. The results in this study provide basic data for further exploring the pathogenesis of V. mali xylanase and its interaction mechanism with the host.
孟璐璐, 于春蕾, 练森, 李保华, 梁文星, 王彩霞. 苹果树腐烂病菌木聚糖酶VmXyl1基因的原核表达及多克隆抗体制备[J]. 农业生物技术学报, 2019, 27(2): 315-322.
MENG Lu-Lu, YU Chun-Lei, LIAN Sen, LI Bao-Hua, LIANG Wen-Xing, WANG Cai-Xia. Prokaryotic Expression of Xylanase VmXyl1 Gene in Valsa mali and Preparation of Polyclonal Antibody. 农业生物技术学报, 2019, 27(2): 315-322.
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