INDUCTION,PURIFICATION AND CHARACTERIZATION OF VITELLOGENIN(VTG)FROM SILVER CRUCIAN CARP

Eight male silver crucian carps were treated by intraperitoneal injections every five day with 5mg estradiol-17β each fish Native PAGE(7.5%)of the serum indicated that three new bands which were absent in untreated male silver crucian carp appeared at four days after the first injection Two of them were identical to the specific serum protein in female The amount of the products of E2 treatment ascended with injection going on Twenty five days after the first hormone injection,serum was collected and prepared Aprotinin(1 TIU)and PMSF(0 1mol/L)were added to serum to inhibit protease activity High capacity anion exchanger Q connected to a fast protein liquid chromatography system(FPLC system)was developed to purify the products of E2 treatment from the serum The purification was conducted at low temperature(4℃)And PMSF was added to all solutions pumped onto the Q column The induced serum protein was eluted at 0 27mol/L NaCl Native PAGE(7.5%)suggested that the purity of the purified product was enough There were only two closely bands on SDS PAGE(7.5%),which suggested that the purified protein did not degrade during purification So the one step purification protocol was successful The purified protein was characterized as a phospholipoglycoprotein by native PAGE(7.5%),staining the gel for lipid with Sudan Black B,phosphoprotein staining method for phosphorus and DIG label method for carbohydrate Furthermore,it could be partly precipitated by Mg2+-EDTA And it was corresponding to the protein specific for female in silver crucian carp So it can be concluded that the induced product by E2 treatment is vitellogenin Silver crucian carp has two kinds of vitellogenin molecules