IDENTIFICATION AND CHARACTERIZATION OF MELOIDOGYNE SPECIES-SPECIFIC PROTEINS
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Date
2012
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Punjab Agricultural University, Ludhiana
Abstract
Root knot nematodes (RKNs) of genus Meloidogyne are among the most widely
spread parasitic nematodes which attack a wide range of crops and limit their production. The
egg masses of Meloidogyne nematodes were cultured on the tomato plants and were identified
on the basis of perineal pattern of females by Scanning Electron Microscopy. Only single
species of Meloidogyne i.e. M. incognita was identified, indicating that the maintained culture
was pure. The crude homogenate/antigen was prepared from the egg masses of established
pure culture by homogenization, sonication and centrifugation. Quantitative analysis of
prepared homogenate showed an average protein content (mg/gm, wet weight of nematodes)
of 128.43 ± 11.10. Protein profile of this crude protein extract by SDS-PAGE indicated a total
of seven bands (five major and two minor) observed within a range of molecular weight 27
kDa to 118 kDa. Major band was observed between 50-90 kDa followed by a band between
36-50 kDa. Two minor bands were also observed between 27-36 kDa and 50-90 kDa. Healthy
rabbits were immunized with crude protein/antigen of M. incognita egg masses and
hyperimmune serum showed significantly higher levels (% of total protein) of Ig from
2.37±0.32 (0 DPI) to 6.98±1.06 (60 DPI), and CIC from 3.48±0.80 (0 DPI) to 12.13±0.56 (60
DPI). Double immunodiffusion technique indicated the production of antibodies, as
precipitation line was observed due to the formation of lattices of antigen-antibody complexes.
Western blotting resulted in the transfer of single band (lying between 50-90 kDa) from the
protein profile of crude antigenic extract of M. incognita analysed on SDS-PAGE on the
nitrocellulose membrane probed with anti-Meloidogyne antibodies, thus indicating the
antigenic specificity of this protein
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