Isolation and Purification of Lipovitellin from Thymallus arcticus grubei and Preparation of Anti-serum

doi:10.7668/hbnxb.2011.S2.048

Abstract

Abstract: The yolk protein is the main nutrients during embryonic development.The quantity and quality of yolk is crucial for early larval growth.We have purified the egg curde extract of Amur grayling by Sephadex G-200.The results showed there were three protein peaks 1,2 and 3 by gel chromatographv at 4℃ 0.2 mmol/L PMSF and three protein peaks were relatively symmetric.Then protein peak 2 was purified by more than one time Sephadex G-200 eluting.The properties of yolk protein were analyzed by the methods of polyacrylamide gel electrophoresis and immuno diffusion.Based on the 7.5%Native-PAGE staining data,three brands were detected with molecular weights of 567.2,486.3,157.5 kDa.Based on the 12%SDS-PAGE staining data,nine brands were detected with molecular weights of 80.7,68.7,46.8,39,30,20.9,16.7,14.9,14.1 kDa.Protein peak 2 was lipovitellin and molecular weight was 486.3 kDa.Lipovitellin was composed of three subunits with molecular weights of 80.7,30,14.9,14.1 kDa,respectively.Polyclonal antibodies against the purified Lv from Amur grayling were produced in the New Zealand rabbit.Double immunodiffusion showed that the determination of the titre for anti-Lv serum was 1 ∶ 64.Double immunodiffusion showed that anti-Lv serum have sex-specificity.

Key words: Thymallus arcticus grubei, Dybowski, Yolk protein, Lipovitellin, Purification

CLC Number:

S965

SUO Li, QU Qiu-zhi, SUN Da-jiang, ZHANG Ying, WANG Xue, ZHAO Ji-wei. Isolation and Purification of Lipovitellin from Thymallus arcticus grubei and Preparation of Anti-serum[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2011, 26(S2): 229-234. doi: 10.7668/hbnxb.2011.S2.048.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.2011.S2.048

http://www.hbnxb.net/EN/Y2011/V26/IS2/229

References

[1] 董崇智.中国淡水冷水性鱼类[M].哈尔滨:黑龙江科学技术出版社,2001:63-65.
[2] 徐俐力.黑龙江省几种珍稀冷水鱼类生存现状[J].黑龙江科技信息,2007,13:137.
[3] 乐佩琦,陈宜瑜.中国濒危动物红皮书(鱼类)[M].北京:科学出版社,1998:44-46.
[4] 徐革峰,梁双,孙冬梅,等.黑龙江茴鱼的人工繁殖技术研究初报[J].水产学杂志,2006,19(2):51-53.
[5] 韩英,张澜澜,赵吉伟,等.黑龙江茴鱼胚胎的发育及仔、稚、幼鱼的生长[J].淡水渔业,2009,39(4):17-21.
[6] 马波,霍堂斌,姜作发.中国黑龙江水系茴鱼属一新纪录种(鲑形目,茴鱼科)[J].动物分类学报,2007,32(4):986-988.
[7] 古伟,王炳谦,赵吉伟,等.人工养殖黑龙江茴鱼肌肉营养成分的分析[J].东北农业大学学报,2009,40(11):86-90.
[8] 索力,赵吉伟,张颖,等.黑龙江茴鱼卵的营养成分分析[J].水产学杂志,2010,23(2):34-36.
[9] Hiramatsu N,Hiramatsu K,Hirano K,et al.Vitellogeninderived yolk proteins in a hybrid sturegeon,bester (Huso huso×Acipenser ruthenus):Identification,characterization and course of proteolysis during embryogenesis[J].Comp Biochem Physiol,2002,131A:429-441.
[10] Brown,Came A N,Chambers G.Purification partial characterization and peptide sequence of vitellogenin from a reptile the Tuatata (Sphenodon punctatus)[J].Comp Biochem Physiol,1997,117 B:159-168.
[11] Utarabhand P,Bunlipatanon P.Plasma vitellogenin of grouper(Epinephelus malabaricus):isolation and properties[J].Comp Biochem Physiol,1996,115C:101-110.
[12] Janeim-Cinquini T R F,Ribolla P E M,Capurro M L,et al.Vitellogenin and yolkprotein processing in Bothtops jararaca (wied) a tropical venomous snake[J].Comp Biochem Physiol,1999,122B:189-198.
[13] Holbech H,Andersen L,Petersen G I,et al.Development of an ELLSA for vitellogenin in whole body homogenate of zebrafish (Danio rerio)[J].Comp Biochem Physiol,2001,130C:119-131.
[14] Fenske M,Aerle R V,Brack S,et al.Devlopment and validation of ahomoologlous zebrafish (Danio rerio Hamilton-Buchanan) vitellogenin enryme-liked immunosorbent assay(ELISA) and its application for studies on estrogenic chemicals[J] Comp Biochem Physiol,2001,129C:217-232.
[15] Matsubara T,Sawano K.Proteolytic cleavage of vitellogenin and yolk proteins during vitellogenin uptake and oocyte maturation in barfin flounder(Verasper moseri)[J].Exp Zool,1995,272:34-45.
[16] 张年国,张颖,曲秋芝,等.施氏鲟卵黄蛋白的分离纯化及其性质[J].中国水产科学,2007,14(2):309-314.
[17] Matsubara T,OhkuboN,Andoh T,et al.Two forms of vitellogenin,yielding two distinct lipovitellins,play different roles during oocyte maturation and early development of barfin tlounder,Veraspermoseri,a marine teleost that spawns pelagic eggs[J].Devel Bio,1999,213:18-32.
[18] 李荷迪,周忠良,赵云龙.红螯光壳螯虾卵黄磷蛋白的分离纯化和鉴定[J].动物学杂志,2004,39(3):17-21.
[19] 穆淑梅,康现江,李彦芹,等.日本沼虾卵黄磷蛋白生化性质分析[J].河北农业大学学报,2006,29(5):72-75.
[20] Unuma T,Suzuki T.A protein identical to the yoll protein is stored in the testis in male red sea urchin Pseudocentrotus depressus[J].Biol Bull,1998,194:92-97.
[21] Hosono M,Ishikawa K,Mineki R,et al.Tandem reLeat structure of rhamnose-binding lectin from catfish(Silurus asotus) eggs[J].Bichem Biophys Acta,1999,1472:668-675.
[22] 郭尧君.蛋白质电泳实验技术[M].北京:科学出版社,2005:53-59.
[23] 汪家政,范明.蛋白质技术手册[M].北京:科学出版社,2000:77-122.
[24] Byrne B M,Gruber M.The evolution of egg yolk protein[J].Prog Biophys Mol Biol,1989,53:33-69.
[25] Chen J S,Sappington T W,Raikhel A S.Extensive sequence conservation among insect nematode and vertebrate vitellogenins reveals ancient common ancestry[J].Mol Evol,1997,44:440-451.
[26] Lazier C B,Mommsen T P.Hepatic estrogen receptors and plasma estrogen binding activity in the Atlantic salmon[J].Gen Comp Endoerinol,1985,57:234-245.
[27] 李颖,汝少国,邴欣.美国红鱼卵黄原蛋白的分离纯化及电泳性质鉴定[J].应用与环境生物学报,2007,13(1):61-65.
[28] 温茹淑,方展强,江世贵,等.剑尾鱼卵黄脂磷蛋白的纯化及免疫分析[J].水产学报,2007,31(5):648-654.
[29] 于坤,汝少国,邴欣,等.大菱鲆卵黄原蛋白的分离纯化及Native-PAGE、SDS-PAGE性质鉴定[J].海洋科学,2009,33(9):71-75.
[30] Hiramatsu N,Hara A.Relationship between vitellogenin and its related egg yolk proteins in Sakhalin Taimen (Hucho perryil)[J].Comp Biochem Physiol,1996,115A:243-251.
[31] 霍堂斌,张颖,孙大江,等.小体鲟卵黄雌性蛋白分离纯化及抗血清的研制[J].中国水产科学,2007,14(4):532-538.
[32] 师晓栋.玫瑰无须鲃卵黄蛋白原及卵黄脂磷蛋白的纯化和免疫学鉴定[D].青岛中国海洋大学,2003:53-69.
[33] Hara A,Yamauchi K,Hirai H.Studies on female-specific serum protein and egg yolk protein in Japanese eel (Anguilla japonica)[J].Comp Biochem Physiol,1980,65B:315-320.
[34] Hiramatsu N,Hara A.Relationship between vitellogenin and its related egg yolk proteins in Sakhalin Taimen (Hucho perryil)[J].Comp Biochem Physiol,1996,115A:243-251.
[35] 王凤,张颖,佟广香,等.哲罗鲑生殖周期中血清卵黄蛋白原浓度的ELISA检测[J].上海海洋大学学报,2009,18(6):673-679.
[36] Hara A,Hirai H.Comparative studies on immunochernical properties of female-specific serum protein and yolk proteins in rainbow trout (Salmo airdneri)[J].Comp Biochem Physiol,1978,48 B:389-399.
[37] Wildhaber M L,Papoulias D M,Delonay A J,el al.Development of methods to determine the reproductive status of pallid sturgeon in the Missouri river[D].Columbia:Columbia environmental research center,2006:1-88.
[38] 李康,周忠良,于静,等.鲫鱼(Carassius auratus)卵黄蛋白原的ELISA检测[J].中国环境科学,2003,23(3):276-280.
[39] Campbell C M,Idler D R.Characterization of an estradio-induced protein from rainbow rout as vitellogenin by the cross reactivity to ovarian yolk fractions[J].Biol Reprod,1980,22:605-617.
[40] Ruth C H,Jose J P,Joseph G K.Characterization of a heat-stable fraction of lipovitellin and development of an immunoassay for vitellogenin and yolk protein in winter flounder (Pleuronectes americanus)[J].Experimental Zoology,1997,278 (3):156-166.
[41] 温茹淑,方展强,江世贵,等.剑尾鱼卵黄蛋白原的ELISA检测[J].环境科学研究,2007,24 (4):144-149.
[42] 吴鹰,孟艳玲,周忠良.环境内分泌干扰物对河川沙塘鳢的雌激素效应检测[J].环境监测管理与技术,2006,18(6):13-18.

Please choose a citation manager

Content to export