论文部分内容阅读
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。
To test the expression of chitinase, a putative chitinase polyclonal antibody against Magnaporthe grisea was explored to explore its potential use. The chitinase gene of Magnaporthe oryzae was cloned into fusion expression vector pET-32a and induced in Escherichia coli BL21 (DE3). After being induced with 0.5 mmol / L IPTG for 4 ~ 6 h, the expressed protein was purified by Ni ~ (2 +) - NTA affinity chromatography to obtain soluble chitinase-His fusion protein. Polyclonal antibodies were prepared by immunizing New Zealand male rabbits with the fusion protein. ELISA analysis showed that the antibody titer of 1: 204.800, good specificity. The antibody was used to detect the four different strains of Magnaporthe grisea in different field strains, different strains of different expression levels. Western blot analysis of diseased leaves of rice blast infected with rice blast for three, five and seven days showed that the expression of chitinase was significantly different at different infection times. Chitinase expression differences with the strains of pathogenesis and other biological and physiological differences, is currently under further study.