Characterization of vitellin and vitellogenin of giant tiger prawn Penaeusmonodon using monoclonal antibodies specific to vitellin subunits

Monoclonal antibodies specific to Penaeus monodon vitellin subunits were pr oduced from mouse immunized with sodium dodecyl sulfate (SDS) treated ovari an extract prepared fi om gravid ovaries. After fusion of mouse spleen cell s with P3X myeloma, hybridomas were selected by indirect immunoperoxidase E LISA against P. monodon ovarian extract. This was followed by dot-blotting against native and denatured proteins from ovarian extract, female haemolym ph, and male haemolymph, then by dot-blotting against each vitellin subunit . Hybridoma clones producing antibodies specific to each of vitellin subuni ts with a molecular mass of 83, 74, 104 and 58, 104 and 45 kD, antibodies s pecific to the 215 kD protein, an oocyte-specific protein, and one monoclon al antibody specific to haemocyanin were isolated. All monoclonal antibodie s could bind to both native and denatured proteins. Western blot analysis o f ovarian extract and female haemolymph from gravid ovary prawns separated by PAGE and SDS-PAGE revealed five vitellin subunits, molecular mass of 104 , 83, 74, 58 and 45 kD in ovarian extract, and four vitellogenin related po lypeptides, molecular mass of 200, 104, 83 and 74 kD in the female haemolym ph. From the immunoreactive relationships among these proteins, it could be assumed that vitellogenin may be released into the haemolymph in two forms , 200 and 74 kD, then the 200 kD polypeptide was either processed into the 104 and 83 kD polypeptides, or directly taken up into the oocyte. In the oo cyte, the 104 kD protein would be further cleaved into 58 and 45kD polypept ides while the 74 kD protein would undergo slight modification or remained unchanged. Western blot analysis of vitellin subunits at various stages of ovarian development revealed that the 200 kD protein appeared in the oocyte during early ovarian development the 45 and 58 kD proteins appeared during the late development.