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

S436.611.11; 木聚糖酶(xylanase)基因VmXyl1在苹果树腐烂病菌一黑腐皮壳菌(Valsa mali)致病过程中发挥重要作用.为了揭示VmXyl1在腐烂病菌与寄主互作过程中的作用机制,本研究构建了原核表达载体pET-32a-VmXyl1,利用异丙基-β-D-硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)进行诱导,在大肠杆菌(Escherichia coli)表达菌株Rosetta (DE3)中成功表达了VmXyll融合蛋白;利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacry...

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Published in农业生物技术学报 Vol. 27; no. 2; pp. 315 - 322
Main Authors 孟璐璐, 于春蕾, 练森, 李保华, 梁文星, 王彩霞
Format Journal Article
LanguageChinese
Published 青岛农业大学植物医学学院/山东省植物病虫害综合防控重点实验室/山东省应用真菌重点实验室,青岛,266109 2019
Subjects
木聚糖酶基因
原核表达
苹果树腐烂病菌
多克隆抗体
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Abstract S436.611.11; 木聚糖酶(xylanase)基因VmXyl1在苹果树腐烂病菌一黑腐皮壳菌(Valsa mali)致病过程中发挥重要作用.为了揭示VmXyl1在腐烂病菌与寄主互作过程中的作用机制,本研究构建了原核表达载体pET-32a-VmXyl1,利用异丙基-β-D-硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)进行诱导,在大肠杆菌(Escherichia coli)表达菌株Rosetta (DE3)中成功表达了VmXyll融合蛋白;利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)检测融合蛋白的可溶性和纯化效果,免疫新西兰白兔(Oryctolagus cuniculus)制备多克隆抗体,并对其效价和特异性进行分析.结果显示,该蛋白主要以包涵体形式存在,且0.5 mmol/L IPTG、30℃诱导8h融合蛋白高效表达;经Ni-NTA柱亲和层析纯化后,获得了预期大小的融合蛋白.酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)和Western blot分析表明,所制备抗体效价达1:102 400,且抗体能特异性识别VmXyl1融合蛋白及发病苹果(Malus pumila)树皮组织中的木聚糖酶蛋白,说明所制备的抗体具有高效性和特异性.研究结果为深入探究腐烂病菌木聚糖酶的致病机理及其与寄主的互作机制提供了基础资料.
AbstractList S436.611.11; 木聚糖酶(xylanase)基因VmXyl1在苹果树腐烂病菌一黑腐皮壳菌(Valsa mali)致病过程中发挥重要作用.为了揭示VmXyl1在腐烂病菌与寄主互作过程中的作用机制,本研究构建了原核表达载体pET-32a-VmXyl1,利用异丙基-β-D-硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)进行诱导,在大肠杆菌(Escherichia coli)表达菌株Rosetta (DE3)中成功表达了VmXyll融合蛋白;利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)检测融合蛋白的可溶性和纯化效果,免疫新西兰白兔(Oryctolagus cuniculus)制备多克隆抗体,并对其效价和特异性进行分析.结果显示,该蛋白主要以包涵体形式存在,且0.5 mmol/L IPTG、30℃诱导8h融合蛋白高效表达;经Ni-NTA柱亲和层析纯化后,获得了预期大小的融合蛋白.酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)和Western blot分析表明,所制备抗体效价达1:102 400,且抗体能特异性识别VmXyl1融合蛋白及发病苹果(Malus pumila)树皮组织中的木聚糖酶蛋白,说明所制备的抗体具有高效性和特异性.研究结果为深入探究腐烂病菌木聚糖酶的致病机理及其与寄主的互作机制提供了基础资料.
Author 李保华
于春蕾
练森
孟璐璐
梁文星
王彩霞
AuthorAffiliation 青岛农业大学植物医学学院/山东省植物病虫害综合防控重点实验室/山东省应用真菌重点实验室,青岛,266109
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Author_FL MENG Lu-Lu
LIAN Sen
YU Chun-Lei
LIANG Wen-Xing
WANG Cai-Xia
LI Bao-Hua
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DocumentTitle_FL Prokaryotic Expression of Xylanase VmXyl1 Gene in Valsa mali and Preparation of Polyclonal Antibody
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Keywords 木聚糖酶基因
原核表达
苹果树腐烂病菌
多克隆抗体
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Title 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
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