HU Dong, GUI Jian fang. INDUCTION,PURIFICATION AND CHARACTERIZATION OF VITELLOGENIN(VTG)FROM SILVER CRUCIAN CARP[J]. ACTA HYDROBIOLOGICA SINICA, 2002, 26(2): 109-115.
Citation: HU Dong, GUI Jian fang. INDUCTION,PURIFICATION AND CHARACTERIZATION OF VITELLOGENIN(VTG)FROM SILVER CRUCIAN CARP[J]. ACTA HYDROBIOLOGICA SINICA, 2002, 26(2): 109-115.

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

  • Received Date: December 13, 2000
  • Rev Recd Date: November 13, 2001
  • Published Date: March 24, 2002
  • 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
  • [1]
    Joseph J K,Michael D K,Kathleen M J,et al.Fathead minnow vitellogenin:complementary DNA sequence and messenger RNA and protein expression after 17 β-estradiol treatment[J]. Environmental Toxicology and Chemistry,2000,19(4):972-981[2] Tyler C R,van Aerle R,Hutchinson T H,et al.An in vivo testing system for endocrine disruptors in fish early life stages using induction of vitellogenin[J].Environmental Toxicology and Chemistry,1999,18:337-347[3] Teo B Y,Tan N S,Lam T J,et al.Synergistic effects of nuclear factors-GATA,VBP and ER in potentiating vitellogenin gene transcription[J].FEBS Letters,1999,459:57-63[4] Teo B Y,Tan N S,Lam T J,et al.A novel piscine vitellogenin gene:structural and functional analyses of estrogen-inducible promoter[J].Molecular and Cellular Endocrinology,1998,146:103-120[5] Brooks S,Tyler C R,Sumpter J P.Egg quality in fish:what makes a good egg? [J].Reviews in Fish Biology and Fisheries,1997,7:387-416[6] Corthesy B,Claret F -X,WahlI W.Estrogen receptor level determines sex-specific in vitro transcription from the Xenopus vitellogenin promoter[J].Proc.Natl.Acad.Sci.USA,1990,87:7878-7882[7] Wahli W.Evolution and expression of vitellogenin genes[J].Trends in Genetics,1988,4:227-232[8] Mellanen P,Soimasuo M,Holmbom B,et al.Expression of the vitellogenin gene in the liver of juvenile whitefish(Coregonus lavaretus L.s.l.)exposed to effluents from pulp and paper mills[J].Ecotoxicology and Environmental Safety,1999,43,133-137[9] Harries J E,Sheahan D A,Jobling S,et al.Estrogenic activity in five United Kingdom rivers detected by measurement of vitellogenesis in caged male trout[J].Environmental Toxicology and Chemistry,1997,16:534-542[10] Parks L G,Cheek A O,Denslow N D,et al.Fathead minnow(Pimephales promelas)vitellogenin:purification,characterization,and quantitative immunoassay for the detection of estrogenic compounds[J] Comp.Biochem.Physilo.C,1999,123:113-125[11] Bon B,Barbe U,Rodriguez N,et al.Plasma vitellogenin levels during the annual reproductive cycle of the female rainbow trout(Oncorhynchus mykiss):establishment and validation of an ELISA[J].Comp.Biochem.Physiol.B,1997,117(1):75-84[12] Brion F,Rogerieux F,Noury P,et al.Two-step purification method of vitellogenin from three teleost fish species:rainbow trout(Oncorhynchus mykiss),gudgeon(Gobio gobio)and chub(Leuciscus cephalus)[J].Journal of Chromatography B,2000,737:3-12[13] 桂建芳.银鲫天然雌核发育机理研究的回顾与展望[J].中国科学基金.1997,11(1):11-15[14] Zhou L,Wang Y,Gui J F.Genetic evidence for gonochoristic reproduction in gynogenetic silver crucian carp(Carassius auratus gibelio)as revealed by RAPD assays[J].J.Mol.Evol.2000,50,in press[15] Wiley H S,Opresko L,Wallace R A.New methods for purification of vertebrate vitellogenin[J].Analytical Biochemistry,1979,97:145-152[16] Silversand C,Hyllner S J,Haux C.Isolation,immunochemical detection,and observations of the instability of vitellogenin from four teleosts[J].Journal of Experimental Zoology,1993,267:587-597[17] Debruyne I.Staining of alkali-labile phosphoproteins and alkaline phosphatases on polyacrylamide gels[J].Analytical Biochemistry,1983,133:110-115[18] Matsubara T,Ohkubo N,Andoh T,et al.Two forms of vitellogenin,yielding two distinct lipovitellins,play different roles during oocyte maturation and early development of barfin flounder,Verasper moseri,a marine teleost that spawns pelagic eggs[J].Developmental Biology,1999,213:18-32[19] Janeiro-Cinquini T R F,Ribolla P E M,Winter C E,et al.Vitellogenin and yolk protein processing in Bothrops jararaca(Wied),a tropical venomous snake[J].Comparative Biochemistry and Physiology.B,1999,122:189-198[20] Roubal W T,Lomax D P,Willis M L,et al.Purification and partial characterization of English sole(Pleuronectes vetulus)vitellogenin[J].Comp.Biochem.Physiol.B,1997,118:613-622[21] Yao Z,Crim L W.A biochemical characterization of vitellogenins isolated from the marine fish ocean pout(Macrozoarces americanus L.),lumpfish(Cyclopterus lumpus)and Atlantic cod(Gadus morhua)[J].Comp.Biochem.Physiol.B,1996,113(2):247-253[22] Carnevali O,Belvedere P.Comparative studies of fish,amphibian,and reptilian vitellogenins [J].Journal of Experimental Zoology,1991,259:18-25

    Joseph J K,Michael D K,Kathleen M J,et al.Fathead minnow vitellogenin:complementary DNA sequence and messenger RNA and protein expression after 17 β-estradiol treatment[J]. Environmental Toxicology and Chemistry,2000,19(4):972-981[2] Tyler C R,van Aerle R,Hutchinson T H,et al.An in vivo testing system for endocrine disruptors in fish early life stages using induction of vitellogenin[J].Environmental Toxicology and Chemistry,1999,18:337-347[3] Teo B Y,Tan N S,Lam T J,et al.Synergistic effects of nuclear factors-GATA,VBP and ER in potentiating vitellogenin gene transcription[J].FEBS Letters,1999,459:57-63[4] Teo B Y,Tan N S,Lam T J,et al.A novel piscine vitellogenin gene:structural and functional analyses of estrogen-inducible promoter[J].Molecular and Cellular Endocrinology,1998,146:103-120[5] Brooks S,Tyler C R,Sumpter J P.Egg quality in fish:what makes a good egg? [J].Reviews in Fish Biology and Fisheries,1997,7:387-416[6] Corthesy B,Claret F -X,WahlI W.Estrogen receptor level determines sex-specific in vitro transcription from the Xenopus vitellogenin promoter[J].Proc.Natl.Acad.Sci.USA,1990,87:7878-7882[7] Wahli W.Evolution and expression of vitellogenin genes[J].Trends in Genetics,1988,4:227-232[8] Mellanen P,Soimasuo M,Holmbom B,et al.Expression of the vitellogenin gene in the liver of juvenile whitefish(Coregonus lavaretus L.s.l.)exposed to effluents from pulp and paper mills[J].Ecotoxicology and Environmental Safety,1999,43,133-137[9] Harries J E,Sheahan D A,Jobling S,et al.Estrogenic activity in five United Kingdom rivers detected by measurement of vitellogenesis in caged male trout[J].Environmental Toxicology and Chemistry,1997,16:534-542[10] Parks L G,Cheek A O,Denslow N D,et al.Fathead minnow(Pimephales promelas)vitellogenin:purification,characterization,and quantitative immunoassay for the detection of estrogenic compounds[J] Comp.Biochem.Physilo.C,1999,123:113-125[11] Bon B,Barbe U,Rodriguez N,et al.Plasma vitellogenin levels during the annual reproductive cycle of the female rainbow trout(Oncorhynchus mykiss):establishment and validation of an ELISA[J].Comp.Biochem.Physiol.B,1997,117(1):75-84[12] Brion F,Rogerieux F,Noury P,et al.Two-step purification method of vitellogenin from three teleost fish species:rainbow trout(Oncorhynchus mykiss),gudgeon(Gobio gobio)and chub(Leuciscus cephalus)[J].Journal of Chromatography B,2000,737:3-12[13] 桂建芳.银鲫天然雌核发育机理研究的回顾与展望[J].中国科学基金.1997,11(1):11-15[14] Zhou L,Wang Y,Gui J F.Genetic evidence for gonochoristic reproduction in gynogenetic silver crucian carp(Carassius auratus gibelio)as revealed by RAPD assays[J].J.Mol.Evol.2000,50,in press[15] Wiley H S,Opresko L,Wallace R A.New methods for purification of vertebrate vitellogenin[J].Analytical Biochemistry,1979,97:145-152[16] Silversand C,Hyllner S J,Haux C.Isolation,immunochemical detection,and observations of the instability of vitellogenin from four teleosts[J].Journal of Experimental Zoology,1993,267:587-597[17] Debruyne I.Staining of alkali-labile phosphoproteins and alkaline phosphatases on polyacrylamide gels[J].Analytical Biochemistry,1983,133:110-115[18] Matsubara T,Ohkubo N,Andoh T,et al.Two forms of vitellogenin,yielding two distinct lipovitellins,play different roles during oocyte maturation and early development of barfin flounder,Verasper moseri,a marine teleost that spawns pelagic eggs[J].Developmental Biology,1999,213:18-32[19] Janeiro-Cinquini T R F,Ribolla P E M,Winter C E,et al.Vitellogenin and yolk protein processing in Bothrops jararaca(Wied),a tropical venomous snake[J].Comparative Biochemistry and Physiology.B,1999,122:189-198[20] Roubal W T,Lomax D P,Willis M L,et al.Purification and partial characterization of English sole(Pleuronectes vetulus)vitellogenin[J].Comp.Biochem.Physiol.B,1997,118:613-622[21] Yao Z,Crim L W.A biochemical characterization of vitellogenins isolated from the marine fish ocean pout(Macrozoarces americanus L.),lumpfish(Cyclopterus lumpus)and Atlantic cod(Gadus morhua)[J].Comp.Biochem.Physiol.B,1996,113(2):247-253[22] Carnevali O,Belvedere P.Comparative studies of fish,amphibian,and reptilian vitellogenins [J].Journal of Experimental Zoology,1991,259:18-25

Catalog

    Article views PDF downloads Cited by()
    Related

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return