稻瘟病菌—假定几丁质酶多克隆抗体的制备及其检测  

Preparation of polyclonal antibody of a chitinase from Magnaporthe oryzae and its usage for detection

作　　者：吴丽民[1,2] 连永乐[2] 王宗华[2] 汪世华[2] 鲁国东[2] 

机构地区：[1]福建生物工程职业技术学院,福州350002 [2]福建农林大学生物农药与化学生物学教育部重点实验室,福州350002

出　　处：《植物病理学报》2014年第3期247-253,共7页Acta Phytopathologica Sinica

基　　金：国家自然基金资助项目(31071654;30500325);国家科技支撑计划(2012BAD19B03);福建省教育厅科技项目(JA13412)

摘　　要：为检测几丁质酶的表达,制备稻瘟病菌的一假定几丁质酶多克隆抗体,探索其可能运用。将稻瘟病菌(Magnaporthe oryzae)几丁质酶基因克隆入融合表达载体pET-32a,并在大肠杆菌Escherichia coli BL21(DE3)中进行诱导表达。表达菌株经0.5 mmol/L IPTG诱导4~6 h后,用Ni^(2+)-NTA亲和柱纯化蛋白,得到可溶的几丁质酶-His融合蛋白。以该融合蛋白免疫新西兰雄兔,制备多克隆抗体。ELISA分析表明该抗体效价达1:204.800,特异性良好。用该抗体ELISA检测了稻瘟病菌4个不同田间菌株中,不同的菌株表达量不同。对稻瘟病菌侵染水稻3、5和7d后的病叶进行抗原Western验证,结果表明,稻瘟病菌不同的侵染时间其几丁质酶表达量有明显差异。几丁质酶表达的差异是否与菌株间致病型等生物学和生理学差异有关,目前正在进一步研究。The chitinase gene of Magnaporthe oryzae was cloned into the fusion expression vector pET-32a and expressed in Escherichia coli BL21（DE3） host cells.After the expression strain was induced for 4-6 hours by 0.5 mmoL IPTG, soluble chitinase were obtained from upper liquid and purifed by Ni^2＋-NTA affinity chromatography column. This fusion protein was injected into New Zealand rabbits to get polyclonal antibody. ELISA analysis showed that the titer of this polyclonal antibody with good specificity was 1： 204 800. Culture filtrates of 4 Magnaporthe oryzae wild type strains were tested with this antibody by ELISA and in vivo chitinase detection during the pathogen infection was carried by Western blot. The result showed that the expression level of the chitinase appeared to be significantly different among different strains as well as different infection stages of the strain 70-15. Whether the expression level of the chitinase is related to the biological and physiological characteristics such as pathogenicity among different strains still remains further study.

关 键 词：稻瘟病菌 几丁质酶 多克隆抗体 ELISA 

分 类 号：Q939.91[生物学—微生物学]