Preparation Of Polyclonal Antibodies To α-Mannosidase And Chitinase Of Magnaporthe Grisea And Their Application

Magnaporthe grisea is not only the pathogenic fungus of rice but also a very important organism in the study of plant-microbe interaction. In the study of interaction between rice and rice blast fungus, elicitor is becoming a hot spot at present. Elicitors are some singal molecules that possess abilities to elecit plant defense responses. There are about 1000 secreted proteins in M. grisea through the whole genomic analysis. These secreted proteins are probably participating in the pathopoiesis process, and may have the function of elicitor. The test of extracellular protease in different physiology stages will improve understanding of the fungal biology, athogenetic mechanisms and molecular mechanisms of regulating the fungal pathogenicity as well as provide theories for cultivating durable resistance of rice and manage the disease integratedly.To prepare polyclonal antibody ofα-mannosidase, transformants of M.grisea that over-expressedα-mannosidase-His tagged fusion protein was cultured in liquid complete media. Solubleα-mannosidase was obtained from upper liquid and purified by Ni²⁺-NTA affinity chromatography column. The fusion protein was injected into New Zealand rabbits to get polyclonal antibody. ELISA analysis showed that the titer of this polyclonal antibody was 1: 51200 with good specificity. Culture filtrates of 30 M. grisea wild type strains were tested against this antibody with ELISA and Western Blotting. The result showed that expression levers ofα-mannosidase appeared to be significantly different among different strains as well as different stages after the strain 70-15 infected rice. Whether the expression lever ofα-Mannosidase is related to the differences of biological and physiological aspects such as pathogenicity among different strains requires further study.To prepare polyclonal antibody of chitinase of M.grisea the chitinase gene was cloned into the fusion expression vector pET-32a and expressed in Escherichia coli straia BL21 (DE3) host cells. After the expression, strain 118 was induced for 4-6 h by 0．5 mmol/LIPTG. Soluble chitinase was obtained when the extracelluar protein were collected from upper liquid and purified by Ni²⁺-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 was as high as 1: 204800 with good specificity for culture filtrates of 30 M.grisea wild type strains tested against this antibody with ELISA and Western Blotting. The result showed that the levers of expression level of the chitinase appeared to be significantly different among different strains as well as different stages after the strain 70-15 infected rice. Whether the expression levers of the Chitinase is related to the differences of biological and physiological aspects such as pathogenicity among different strains still remains unclear.