288 39,, [11, 12] Vn, Vn, Vn (Enzyme-linked immunoabsorbent assay, ELISA) Vn [13, 14] Vn, Vn, Vn-ELISA, [13 16], Vn Vn, Vn, Vn ELISA, ( )

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1 39 2 Vol. 39, No ACTA HYDROBIOLOGICA SINICA Mar., doi: / ELISA 1 杨健 2 刘冬梅 1 何永吉 3 汉斯达姆斯 1 王兰 (1., ; 2., ; 3., 80708) : (Sinopotamon henanense), (Vitellin, Vn), SDS- Vn, Vn Vn Vn ELISA, Vn-ELISA Vn 5.8 mg/l, d, (1) SDS kd 3, Western-blotting, ; (2) Vn-ELISA, Vn , ng/ml, ng/ml; Vn 7.81%, ; (3) Vn, -,, : ; ; SDS- ; ; 中图分类号 : X174 文献标识码 : A 文章编号 : (2015) , (Sinopotamon henanense),, [1 4] [5],,, [6 8],,, (Vitellogenin, Vg) [9], Vg Vg (Vitellin, Vn), Vn, Vn, Vn [10] 收稿日期 : ; 修订日期 : 基金项目 : (No , No ); (No ); (No SXDX-SWX-003) 作者简介 : (1988 ),, ; ; yangjian333001@126.com 通信作者 : (1960 ),, ; ; lanwang@sxu.edu.cn

2 288 39,, [11, 12] Vn, Vn, Vn (Enzyme-linked immunoabsorbent assay, ELISA) Vn [13, 14] Vn, Vn, Vn-ELISA, [13 16], Vn Vn, Vn, Vn ELISA, ( ) (0.02 mol/l Tris-HCl, 0.1 mol/l NaCl, 0.01% EDTA, 0.1 mmol/l PMSF), g 30min, 1h g 30min,, 2 ml PBS (0.1 mol/l KH 2 PO 4, 0.1 mol/l Na 2 HPO 4 12H 2 O, 0.1 mol/l NaCl, 0.1 mol/l KCl, 0.01% EDTA, ph 7.7), - - 5, 1 ml PBS 260 ml ( =2.6 cm 100 cm, ), 220 ml ( Sephacryl TM S 100 HR, Pharmacia ), PBS, 16 ml/h, 1h 2 ml Vn, 16 ml/h, UVD ( ) 280 nm, 2 ml 1, 80 SpectraMax M5 ( Molecular Devices ) ( nm) 1.3 (SDS-PAGE, 10%, 5%), R-250, Tanon-2500 ( ), BandScan 5.0 Vn BALB/c, 1 Vn QuickAntibody 100 μl ( 25 μg), μl, 35, 1h, g 10min, 0.02% NaN 3, 80 (Western-Blotting) Vn SDS-PAGE, 72 V 1h , 1 ml, 1h; IgG-IRP(Catalog No. HSA0004, ), 1h 1 ml PBST (10 mmol/l PBS, 0.05% Tween-20, ph 7.2) 3, 10min; DAB ( Roche ) 3min Tanon-2500

2 : ELISA 289 1.5 Vn Vn-ELISA ELISA Vn Vn-ELISA (1) : Vn (0.05 mol/l Na 2 CO 3, 0.05 mol/l NaHCO 3, ph=9. 6) 1 μg/ml, 100 μl, 4 ; (2) : 200 μl PBST 3, 200 μl (1% ) 2h, PBST 3 ; (3) : Vn 1.. 1000 1.

3 2 : ELISA Vn Vn-ELISA ELISA Vn Vn-ELISA (1) : Vn (0.05 mol/l Na 2 CO 3, 0.05 mol/l NaHCO 3, ph=9. 6) 1 μg/ml, 100 μl, 4 ; (2) : 200 μl PBST 3, 200 μl (1% ) 2h, PBST 3 ; (3) : Vn % BSA, 100 μl, Vn 1% BSA, 37 2h, 200 μl PBST μl IgG-HRP, 37 1h PBST 3 ; (4) : 100 μl TMB ( ), 30min; (5) : 50 μl (2 mol/l H 2 SO 4 ); (6) : SpectraMax M5 450 nm, Vn ng/ml, 3, 3, A 450, Vn 1.6 5, 0. 1 g, 10, g 15min, 0.1 ml ELISA ELISA ; 3, ELISA mg/l, d 5, Vn- ELISA Vn SPSS 17.0 (SPSS Inc., Chicago, USA), ± Kolmogorov-Smirnov Levene, Dunnett s post-hoc Test (independent samples Test) Vn Vn, LSD, P< Vn 1, min 470 nm, Vn SDS- ( 1), 3, kd WesternBlotting, 3 Vn ( 1) 1 Vn SDS-PAGE Western-blotting Fig. 1 SDS-PAGE and Western-blotting of purified S. henanense vitellin 1. ; mg/ml; mg/ml; 4. Western-blotting 1. Molecular mass markers; 2. vitellin 0.6 mg/ml; 3. vitellin 0.8 mg/ml; 4. Vn Western-blotting 2.2 Vn 1 Vn ELISA ,

4 A , A , A %, Vn Vn, Vn A 450 表 1 河南华溪蟹 Vn 抗体不同稀释倍数的 ELISA 反应的 A 450 值 Tab. 1 The A 450 of ELISA by the different dilution rates of Vn antiserum Vn ng/ml, A 450, 4000 ng/ml 2000 ng/ml Vn, A 450, Vn ng/ml 2 Vn A 450 (y = x , R 2 = , P < , x y Vn A 450 ),,, Vn ng/ml Vn Vn anti-body dilution rate A 450 (Mean ± SD, n = 5) Coefficient of variation (%) ± 0.22 a ± 0.07 b ± 0.05 c ± 0.13 d 7.23 BSA ± 0.04 e 8.20 : (P < 0.05); n, Note: Values within a column having different superscript letter are significantly different (P < 0.05); n means sample number. The same applies bellow 2 Vn ng/ml, A 450 Vn 7.81 ng/ml, A 450 Vn ng/ml, A ng/ml, Vn ng/ml 表 2 不同 Vn 质量浓度的 A 450 值 Tab. 2 The A 450 of a serial of Vn concentration Vn Vn concentration (ng/ml) A 450 (Mean ± SD, n = 5) Coefficient of variation (%) Control 0.13±0.03 a ±0.01 a ±0.02 b ±0.04 c ±0.05 d ±0.05 e ±0.07 f ±0.03 g ±0.12 h ±0.08 i ±0.06 i Vn ELISA Fig. 2 The ELISA standard curve of purified Vn from the mature female S. henanense 2.3 Vn-ELISA, 5 3 3, 7. 81% Vn, ELISA 表 3 河南华溪蟹卵巢中 Vn 的含量 Tab. 3 The Vn contents in the ovary of female S. henanense mg/g wet weight, n = 5 Batch Vn Vn concentration (ng/ml) Coefficient of variation (%) ± a ± a ± a 9.43 Mean Vn 3,, Vn (P < 0.05),

5 2 : ELISA 291, Vn, 20d (31.42 ± 7.25) mg/g 3 Vn Fig. 3 Effect of cadmium on Vn in ovary of Sinopotamon henanense 3 [17, 18], Serrano-Pinto [19] Laino [20], Vn SDS 3, kd, kd, 2 6, (Eriocheir sinensis) 520 kd, kd [13] ; (Charybdis feriatus)vn kd [21] ; (Potamoum potamios) kd [22] ; (Macrobrachium nipponense ) kd [23] ;, kd [14] ; (Cambarus clarkii)vn 481 kd, ( kd) [24] Vn, Vn Western-blotting Vn Vn Vn-ELISA Vn Vn-ELISA, ng/ml Vn, ng/ml, Vn Vn-ELISA Volz [25] Leptocheirus plumulosus Vn-ELISA ng/ml, 1.9 ng/ml, Chen Vn-ELISA, ng/ml [13] (Crassostrea corteziensis)vn-elisa ng/ml [26] Vn-ELISA,,, Vn-ELISA,,,, Vn, Vn, [27] Vn, Revathi Rodriguez Vn,, [28, 29],,, Vg,,, Vg, Vg, (Penaeus semisulcatus) [30], Vg, (Oziothelphusa senex senex) [31], Vg

6 292 39,,, Vn : [1] Wang Q, Wang L, Xi Y Y, et al. The acute toxicity and accumulation of Cd 2+ in freshwater crab Sinopotamon yangtsekiense [J]. Journal Shanxi University (Natural Science Edition), 2003, 26(2): [,,,.. ( ), 2003, 26(2): ] [2] Wang L, Xu T, Lei W, et al. Cadmium-induced oxidative stress and apoptotic changes in the testis of freshwater crab, Sinopotamon henanense [J]. PLOS One, 2011, 6(11): e27853 [3] Liu D, Yan B, Yang J, et al. Mitochondrial pathway of apoptosis in the hepatopancreas of the freshwater crab Sinopotamon yangtsekiense exposed to cadmium [J]. Aquatic Toxicology, 2011, 105(3-4): [4] Jin F F, Xu T, Qin S J, et al. Effects of cadmium on hepatopancreas mitochondrial antioxidant enzyme activity and lipid peroxidation level in freshwater crab Sinopotamon yangtsekiense [J]. Acta Hydrobiologica Sinica, 2011, 35(6): [,,,.., 2011, 35(6): ] [5] Han T, Wang Q, Wang L. Ecological investigation of freshwater crab and river pollution in the basin of Qinhe River [J]. Sichuan Journal of Zoology, 2008, 27(5): [,,.., 2008, 27(5): ] [6] De Smet H, Blust R. Stress responses and changes in protein metabolism in carp Cyprinus carpio during cadmium exposure [J]. Ecotoxicology and Environmental Safety, 2001, 48(3): [7] Pierron F, Baudrimont M, Bossy A, et al. Impairment of lipid storage by cadmium in the European eel (Anguilla anguilla) [J]. Aquatic Toxicology, 2007, 81(3): [8] Lucia M, André J M, Gonzalez P, et al. Effect of dietary cadmium on lipid metabolism and storage of aquatic bird Cairina moschata [J]. Ecotoxicology, 2010, 19(1): [9] Wu X G, Yao G G, Yang X Z, et al. A study on the ovarian development of Portunus trituberculatus in East China Sea during the first reproductive cycle [J]. Acta Oceanologica Sinica, 2007, 29(4): [,,,.., 2007, 29(4): ] [10] Ghanawi J, Patrick Saoud I. Molting, reproductive biology, and hatchery management of redclaw crayfish Cherax quadricarinatus [J]. Aquaculture, 2012, : [11] Kim D H, Kim S K, Choi J H, et al. The effects of manipulating water temperature, photoperiod and eyestalk ablation on gonad maturation of the swimming crab, Portuuus trituberculatus [J]. Crustaceans, 2010, 83(2): [12] Tinikul Y, Poljaroen J, Tinikul R et al. Effects of gonadotropin-releasing hormones and dopamine on ovarian maturation in the Pacific white shrimp, Litopenaeus vannamei, and their presence in the ovary during ovarian development [J]. Aquaculture, 2014, : [13] Chen L, Jiang H, Zhou Z, et al. Purification of vitellin from the ovary of Chinese mitten-handed crab (Eriocheir sinensis) and development of an antivitellin ELISA [J]. Comparative Biochemistry and Physiology, Part B: Biochemistry & Molecular Biology, 2004, 138(3): [14] Zhang Y, Wu X G, Yang F, et al. Purification of vitellin and ELISA determination of vitellin of swinming crab (Portunus trituberculatus) [J]. Journal of Fisheries of China, 2011, 35(8): [,,,. ELISA., 2011, 35(8): ] [15] Allner B, von der Gönna S, Griebeler E M, et al. Reproductive functions of wild fish as bioindicators of reproductive toxicants in the aquatic environment [J]. Environmental Science and Pollution Research, 2010, 17(2): [16] Jones I, Kille P, Sweeney G. Cadmium delays growth hormone expression during rainbow trout development [J]. Journal of Fish Biology, 2001, 59(4): [17] Zheng H P, Zhang Q, Liu H L, et al. Cloning and expression of vitellogenin (Vg) gene and its correlations with total carotenoids content and total antioxidant capacity in noble scallop Chlamys nobilis (Bivalve: Pectinidae) [J]. Aquaculture, 2012, : [18] Jia X, Chen Y, Zou Z, et al. Characterization and expression profile of vitellogenin gene from Scylla paramamosain [J]. Gene, 2013, 520(2): [19] Serrano-Pinto V, Vazquez-Boucard C, Villarreal-Colmenares H. Yolk proteins during ovary and egg development of mature female freshwater crayfish (Cherax quadricarinatus) [J]. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2003, 134(1): [20] Laino A, Cunningham M L, Heras H, et al. Isolation and characterization of two vitellins from eggs of the spider Polybetes pythagoricus (Araneae: Sparassidae). Comparative Biochemistry and Physiology, Part B: Biochemistry & Molecular Biology, 2011, 158(2): [21] Mak A S C, Choi C L, Tiu S H K, et al. Vitellogenesis in the red crab Charybdis feriatus: Hepatopancreas speciflic expression and farnesoic acid stimulation of vitellogenin gene expression [J]. Molecular Reproduction and Development, 2005, 70(3):

7 2 : ELISA 293 [22] Pateraki L E, Stratakis E. Synthesis and organization of vitellogenin and vitellin molecules form the land crab Potamon potamios [J]. Comparative Biochemistry and Physiology, Part B: Biochemistry & Molecular Biologym, 2000, 125(1): [23] Mu S M. Yolk proteins of macrobrachium nipponense and effects on their accumulation by unilateral eyestalk ablation [D]. Thesis for Master of Science. Hebei University, 2004 [. (Macrobrachium nippoueuse).,, 2004] [24] Xie S, An J H, Liang X H, et al. Partial characterization of vitellin of crayfish, Procambius clarkii [J]. Journal of Hebei University (Natural Science Edition), 2006, 26(2): [,,,.. ( ), 2006, 26(2): ] [25] Volz D C, Chandler G T. An enzyme-linked immunosorbent assay for lipovitellin quantification in copepods: a screening tool for endocrine toxicity [J]. Environmental Toxicology and Chemistry, 2004, 23(2): [26] Arcos F G, Ibarra A M, Rodríguez-Jaramillo M C, et al. Quantification of vitellin/vitellogenin-like proteins in the oyster Crassostrea corteziensis (Hertlein 1951) as a tool to predict the degree of gonad maturity [J]. Aquaculture Research, 2009, 40(6): [27] Yang J, Liu D, Jing W, et al. Effects of Cadmium on lipid storage and metabolism in the freshwater crab Sinopotamon henanense [J]. PLoS One, 2013, 8(10): e7756 [28] Revathi P, Vasanthi LA, Munuswamy N. Effect of cadmium on the ovarian development in the freshwater prawn Macrobrachium rosenbergii (De Man) [J]. Ecotoxicology and Environmental Safety, 2011, 74(4): [29] Rodríguez E M, Lopez Grego L S, Fingerman M. Inhibition of ovarian growth by cadmium, in the fiddler crab Uca pugilator (Decapoda, Ocypodidae) [J]. Ecotoxicology and Environmental Safety, 2000, 46(2): [30] Avarre J C, Michelis R, Tietz A, et al. Relationship between vitellogenin and vitellin in a marine shrimp (Penaeus semisulcatus) and molecular characterization of vitellogenin complementary DNAs [J]. Biology of Reproduction, 2003, 69(1): [31] Girish B P, Swetha C H, Reddy P S. Hepatopancreas but not ovary is the site of vitellogenin synthesis in female fresh water crab, Oziothelphusa senex senex [J]. Biochemical and Biophysical Research Communications, 2014, 447(2): ESTABLISHED OF ELISA METHOD OF VITELLIN FROM FRESHWATER CRAB (SINOPOTAMON HENANENSE) AND EFFECT OF CADMIUM ON VITELLIN ACCUMULATION IN OVARY YANG Jian 1, LIU Dong-Mei 2, HE Yong-Ji 1, Hans-Uwe Dahms 3 and WANG Lan 1 (1. School of Life Science, Shanxi University, Taiyuan , China; 2. School of Basic Medical, Shanxi Medical University, Taiyuan , China; 3. School of Life Science, Kaohsiung Medical University, Kaohsiung 80708, Taiwan, China) Abstract: To explore the effect of cadmium (Cd) on oogenesis of freshwater crab (Sinopotamon henanense), vitellin (Vn) and the method of Vn-ELISA was studied and established. Vn was purified from mature ovaries of S. henanense by gel filtration chromatography. Using SDS- polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight and the quantity of Vn subunit were determined. Based on the purified Vn and Vn anti-serum, the reaction parameters for an enzyme-linked immunosorbent assay (ELISA) was developed. To explore the effect of cadmium on the Vn in the ovaries, freshwater crab S. henanense were treated with 5.8 mg/l Cd for 10, 15, and 20d. The results showed that Vn was composed of three subunits (116, 66 and 45 kd). Western-blotting confirmed the polypeptides had a specific reactivity with mouse polyclonal Vn anti-serum. The optimal dilution rates of Vn anti-serum were shown to be According to these optimal parameters, the standard linear equation was established for the determination of Vn concentration with a valid range of ng/ml, and qualitative detection can be ng/ml. In verification experiments, Vn content in the ovaries showed no significant differences, and the mean coefficients of variation of inter-assay were 7.81%, suggesting that the developed ELISA of this study was precise, stable and repeatable. Moreover, Cd caused a time-dependend down-regulation of Vn level, and showed significant effects of Cd on vitellogenesis, which suggest that Cd slows down oocyte maturation and vitellogenesis in S. henanense. Key words: Sinopotamon henanense; Vitellin; SDS-polyacrylamide gel electrophoresis; Enzyme-linked immunosorbent assay; Cadmium

<4D6963726F736F667420576F7264202D2031303130382DC9BDCEF7B4F3D1A72DC9FACEEFD1A7CAB5D1E9BDCCD1A7D6D0D0C4C9EAB1A8B9FABCD2BCB62DB8BDBCFE322E646F63>

<4D6963726F736F667420576F7264202D2031303130382DC9BDCEF7B4F3D1A72DC9FACEEFD1A7CAB5D1E9BDCCD1A7D6D0D0C4C9EAB1A8B9FABCD2BCB62DB8BDBCFE322E646F63> 十二五国家级实验教学示范中心申请书学校名称 : 学校主管部门 : 中心名称 : 中心负责人 : 山西大学山西省教育厅生物学实验教学中心王兰学校管理部门电话 : 0351-7018284 申报日期 : 2013.3.28 中华人民共和国教育部高教司制