团头鲂卵壳蛋白的分离与纯化
EXTRACTION AND PURIFICATION OF ZONA RADIATA PROTEINS (ZRPS) FROM BLUNTNOSE BLACK BREAM (MEGALOBRAMA AMBLYCEPHALA YIH)
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摘要: 报道了一种从团头鲂卵壳中提取卵壳蛋白的有效方法.该提取液经SDS-PAGE电泳鉴定后,证明有三条主要的蛋白带,其分子量分别为6KDa,56KDa和52KDa,借助于制备型SDS-PAGE电泳和电洗脱分离纯化技术,获得了三种高纯度的卵壳蛋白.Abstract: This study deals with an effective procedure for extraction and purification of zona radiata proteins (Zrps) from eggshells of Bluntnose black bream (Megalobrama amblycephala Yih). To avoid harding resulting from parthenogenic activation, unfertilized eggs were immediately immersed in a large volume of ice cold buffer A. After homogenization and washing, the eggshells were frozen dry and solubilized in buffer B. The SDS-PAGE electrophoretic migration pattern shows the solubilized Zrps consiste mainly of three protein bands with the apparent molecular weight of 64KDa, 56KDa and 52KDa. To purify these three Zrps, the solubilized eggshell proteins were subjected to preparative SDS-PAGE. The protein bands were visualized using an ice-cold KCl solution. The opaque bands were excised from the gel and extracted using extraction buffer with electro-elution. SDS-PAGE pattern of purified Zrps shows highly purified Zrps were acquired and sufficient for further studies with immunoassay.
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Keywords:
- Bluntnose black bream /
- Zona radiata proteins /
- Extraction /
- Electro-elution /
- Purification
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[1] McLachlan J A. Estrogens in the environment II [M]. New York: Elsevier. 1985[2] Lang Y, Xu Y, Yang F X. et al. Induction, purification and electrophoretic comparison of vitellogenin from two cyprinid fish species: carp (cyprinus carpio) and wuchang fish (megalobrama amblycephalayih)[J]. Acta Hydrobiologica Sinica, 2002,26(4):317-321[梁勇,徐盈,杨方星等. 鲤和团头鲂幼鱼卵黄蛋白原的诱导、纯化及电泳比较. 水生生物学报,2002,26(4):317-321][3] Palmer B D, Palmer S K. Vitellogenin induction by xenobiotic estrogens in the red-eared turtle and African clawed Frog [J]. Environ. Health perspect, 1995,103(Supp1.4):19-25[4] Sumpter J P, Jobling S. Vitellogenesis as a biomarker for estrogenic contaminants of the aqqutic environment [J]. Environ. Health Perspect, 1995,103(Supp1.7):173-178[5] Arukwe A, Knudsen F R, Goksoyr A. Fish zona radiata (Eggshell) protein: a sensitive biomarker for environmental estrogens [J]. Enviro. Health Perspect, 1997,105:418-422[6] Arukwe A, Celius T, Walther B T, et al. Effects of xenoestrogen treatment on zona radiata protein and vitellogenin expression in Atlantic salmon (Salmo salar)[J]. Aquatic Toxicology,2000,49:159-170[7] Arukwe A, Celius T, Walther B T, et al. Plasma levels of vitellogenin and eggshell zona radiata proteins in 4-nonyphenol and o,p'-DDT treated juvenile Altanatic salmon (Salmo salar). Marine Environ. Res,1998,46(1-5):133-136[8] Oppen-Berntsen D O, Olsen S O, Rong C J et al. Plasma levels of eggshell zr-proteins, estradiol-17β, and gonadotropins during an annual reproductive cyclt of Atlantic salmon (Salmo salar) [J]. J. Experimental Zoology,1994,268:59-70[9] Oppen-Berntsen D O, Gran-Jensen Z, Walther B T. Zona radiate proteins are synthesized by rainbow trout (Oncorhynchns mykiss) hepatocytes in response to oestradiol-17β[J]. J. Endorcrinology, 1992,135:293-302[10] Hyllner S T, Oppen-Berntsen D O, Helvik J V, et al. Oestradiol-17β induces the major vitelline envelope proteins in both sexes in teleosts [J]. J. Endorcrinology,1991,131:229-236[11] Hierten S, Wu B L. Studies of the fish zona pellucida by high performance inoexchange chromatograph on agarose columns and free zone electrophoresis [J]. J. Chromatogr,1985,341:295-304［12］Yang S T, Gui J F. Comparative analysis on the protein composition of egg chorions between gynogenetic silver crucian carp and gonochoristic color crucian carp and the changes during fertilization [J]. Hereditas, 2000,22(4):221-224[杨书婷,桂建芳. 雌核发育银鲫与两性生殖彩鲫卵壳蛋白组分的比较分析及其受精前后的变化. 遗传,2000,22(4):221-224][13] Cotelli F, Andronico F, Brivio M, et al. Structure and Composition of the fish egg chorion (Carassiua auratus) [J]. J. Ultrstructure and Molecular Structure Research,1988,99:70-78[14] Oppen-Berntsen D O, Melvik J V, Walther B T. The major structural proteins of cod (Gadus morhua) eggshells and protein crosslinking during teleost egg harding [J]. Developmental Biology,1990,137:258-265 McLachlan J A. Estrogens in the environment II [M]. New York: Elsevier. 1985[2] Lang Y, Xu Y, Yang F X. et al. Induction, purification and electrophoretic comparison of vitellogenin from two cyprinid fish species: carp (cyprinus carpio) and wuchang fish (megalobrama amblycephalayih)[J]. Acta Hydrobiologica Sinica, 2002,26(4):317-321[梁勇,徐盈,杨方星等. 鲤和团头鲂幼鱼卵黄蛋白原的诱导、纯化及电泳比较. 水生生物学报,2002,26(4):317-321][3] Palmer B D, Palmer S K. Vitellogenin induction by xenobiotic estrogens in the red-eared turtle and African clawed Frog [J]. Environ. Health perspect, 1995,103(Supp1.4):19-25[4] Sumpter J P, Jobling S. Vitellogenesis as a biomarker for estrogenic contaminants of the aqqutic environment [J]. Environ. Health Perspect, 1995,103(Supp1.7):173-178[5] Arukwe A, Knudsen F R, Goksoyr A. Fish zona radiata (Eggshell) protein: a sensitive biomarker for environmental estrogens [J]. Enviro. Health Perspect, 1997,105:418-422[6] Arukwe A, Celius T, Walther B T, et al. Effects of xenoestrogen treatment on zona radiata protein and vitellogenin expression in Atlantic salmon (Salmo salar)[J]. Aquatic Toxicology,2000,49:159-170[7] Arukwe A, Celius T, Walther B T, et al. Plasma levels of vitellogenin and eggshell zona radiata proteins in 4-nonyphenol and o,p'-DDT treated juvenile Altanatic salmon (Salmo salar). Marine Environ. Res,1998,46(1-5):133-136[8] Oppen-Berntsen D O, Olsen S O, Rong C J et al. Plasma levels of eggshell zr-proteins, estradiol-17β, and gonadotropins during an annual reproductive cyclt of Atlantic salmon (Salmo salar) [J]. J. Experimental Zoology,1994,268:59-70[9] Oppen-Berntsen D O, Gran-Jensen Z, Walther B T. Zona radiate proteins are synthesized by rainbow trout (Oncorhynchns mykiss) hepatocytes in response to oestradiol-17β[J]. J. Endorcrinology, 1992,135:293-302[10] Hyllner S T, Oppen-Berntsen D O, Helvik J V, et al. Oestradiol-17β induces the major vitelline envelope proteins in both sexes in teleosts [J]. J. Endorcrinology,1991,131:229-236[11] Hierten S, Wu B L. Studies of the fish zona pellucida by high performance inoexchange chromatograph on agarose columns and free zone electrophoresis [J]. J. Chromatogr,1985,341:295-304［12］Yang S T, Gui J F. Comparative analysis on the protein composition of egg chorions between gynogenetic silver crucian carp and gonochoristic color crucian carp and the changes during fertilization [J]. Hereditas, 2000,22(4):221-224[杨书婷,桂建芳. 雌核发育银鲫与两性生殖彩鲫卵壳蛋白组分的比较分析及其受精前后的变化. 遗传,2000,22(4):221-224][13] Cotelli F, Andronico F, Brivio M, et al. Structure and Composition of the fish egg chorion (Carassiua auratus) [J]. J. Ultrstructure and Molecular Structure Research,1988,99:70-78[14] Oppen-Berntsen D O, Melvik J V, Walther B T. The major structural proteins of cod (Gadus morhua) eggshells and protein crosslinking during teleost egg harding [J]. Developmental Biology,1990,137:258-265
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