稻瘟病菌一假定α-甘露糖苷酶多克隆抗体的制备及应用

摘要：【研究目的】为检测α-甘露糖苷酶的表达及其可能的功能，制备了稻瘟病菌一假定α-甘露糖苷酶多克隆抗体，并探索其可能应用。【方法】将与-His标签融合的过量表达一假定α-甘露糖苷酶蛋白的稻瘟病菌转化子，在液体完全培养基中培养，收集其上清液，经Ni2+-NTA亲和柱纯化后，得到可溶的融合蛋白α-甘露糖苷酶-His。【结果】纯化后的融合蛋白免疫新西兰兔，得到抗α-甘露糖苷酶蛋白的多克隆抗体，ELISA检测结果表明抗体效价达1：51200，特异性高。利用该多克隆抗体以ELISA方法检测并Western验证分析稻瘟病菌30个田间菌株培养液α-甘露糖苷酶的表达量。【结论】不同菌株α-甘露糖苷酶表达量有明显差异。进一步分析表明，菌株70-15接种水稻后，不同时期α-甘露糖苷酶的表达量差异明显。菌株间致病型等生物学和生理学差异可能与α-甘露糖苷酶表达的差异有关。

A polyclonal antibody of a putataive α-Mannosidase from Magnaporthe oryzae and its possible use

α Magnaporthe oryzae,a polyclonal antibody of a putative α-Mannosidase was prepared.A transformant of Magnaporthe oryzae,overexpressing a putative α-Mannosidase-His tag fusion protein was grown in liquid media.The α-Mannosidase fusion protein was purified by Ni 2 ＋-NTA affinity chromatography column from culture filtrate.The fusion protein was then injected into and used to immune New Zealand rabbits to produce polyclonal antibody.The titer of this polyclonal antibody was 1：51200 and with good specificity after ELISA analysis.Protein of 30 M.oryzae strains were tested against this antibody using ELISA and Western blotting show that the expression level of the α-Mannosidase appeared to be significantly different among strains as well as infection stages when inoculated with the strain 70-15.The difference expression level of the α-Mannosidase among different strains may be related to the physiological and biological characteristic such as pathogenicity of the fungus.