The Study On Purification And Tissue Localization Of Prophenoloxidase From Ostrinia Furnacalis Guenée (Lepidoptera: Pyralidae) Larvae

Prophenoloxidase (PPO) was isolated from the hemolymph of Ostinia furnacalis larvae and purified to homogeneity. A 369.85-fold purification and 35.34% recovery of activity was achieved by employing ammonium sulfate precipitation, Blue Sepharose CL-6B chromatography and Phenyl Sepharose CL-4B chromatography. The purified enzyme exhibits a band with a molecular mass of 158 kDa on native PAGE and two spots with a molecular mass of 80 kDa and a pI of 5.70, and a molecular mass of 78 kDa and a pI of 6.50, respectively, on two-dimensional gel electrophoresis. The amino acid composition of purified PPO was similar to the PPO from Galleria mellonella. The phenoloxidase (PO) reaction was strongly inhibited by phenylthiourea, thiourea, dithiothreitol and ethylene diamine tetraacetic acid (EDTA), but poorly inhibited by diethyldithiocarbamate (DTC) and triethylenetetramine hexaacetic acid (THAA), and was not inhibited by o-phenanthroline and ethylene glycol-bis (?-aminoethylether) N,N,N`,N`-tetraacetic acid (EGTA). Both Mg2+ and Cu2+ stimulated PO activity when compared with controls. The ?-sheet content of PPO treated with Mg2+ and Cu2+ increased significantly (P<0.05). The purified PPO has magnisium level of 5.674±2.294μg/mg and copper level of 1.257±0.921μg/mg as determined with ICP-MS, suggesting that the purified PPO is a metalloprotein.Balb/C mouse was injected with purified PPO for 5 times to prepare polyclonal antibody. The antiserum was used as primary antibody to detect the antibody titer with indirect ELISA analysis.The cellular localization of PPO in the O. furnacalis larvae was detected with indirect immunofluorescent assay.The polyclonal antibody was used as a tool for the immunoelectron microscopy localization of PPO in the O. furnacalis larvae. The results showed that the antibody titer of two mouse is 10-5, one of them is 1:4.08×104. Western blotting analysis also showed that we have got the effective polyclonal antibody of PPO. The detection of indirect immunofluorescent assay indicated that the synthesis sites of PPO were oenocytoid and granulocyte. The colloidal gold particles were distributed in spotty group in the midgut and integument tissue of O. furnacalis, which indicated PPO existed in these tissue markedly.