银鲫卵黄原的诱导、分离纯化及其生化鉴定

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

以雄性银鲫为实验材料 ,通过雌二醇 (Estradiol- 1 7β,E2 )的多次诱导 ,使得 E2 诱导产物成为血清中的主要蛋白。而后 ,在快速蛋白液相色谱 (FPL C)系统上 ,利用高交换量的阴离子交换层析 Q柱 ,成功的从血清中提纯了与雌性特异蛋白相一致的 E2 诱导产物。糖、磷、脂蛋白分析表明 ,它是一类糖磷脂蛋白大分子。同时 ,它能被 Mg2 + - Ethylenediamine tetraaceticacid(Mg2 + - EDTA)部分沉淀 ,这进一步证明 ,与雌性特异蛋白相一致的 E2 诱导产物就是卵黄原 (Vitellogenin,VTG)。聚丙烯酰胺凝胶电泳表明 ,银鲫有两种 VTG分子。

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 E 2 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 E 2 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 Mg 2+ 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 E 2 treatment is vitellogenin Silver crucian carp has two kinds of vitellogenin molecules