Preparation of polyclonal antibody of a chitinase from <i>Magnaporthe oryzae </i>and its usage for detection

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Acta Phytopathologica Sinica ›› 2014, Vol. 44 ›› Issue (3) : 247-253.

ETIOLOGY

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

WU Li\|min^{1, 2}, LIAN yong\|le², WANG Zong\|hua², WANG Shi\|hua², LU Guo\|dong¹

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Abstract

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 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 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.

Key words

Magnaporthe oryzae / chitinase / antibody preparation / ELISA

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