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[1]黄秦,曹瑜,吴鹏,等.盐生杜氏藻3-磷酸甘油脱氢酶不同结构域蛋白的表达纯化及其抗体的制备与分析[J].应用与环境生物学报,2009,15(02):202-206.[doi:10.3724/SP.J.1145.2009.00202]
 HUANG Qin,CAO Yu,WU Peng,et al.Purification and Antibody Preparation and Analysis of Different Domains of GPDH Proteins from Dunaliella salina[J].Chinese Journal of Applied & Environmental Biology,2009,15(02):202-206.[doi:10.3724/SP.J.1145.2009.00202]
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盐生杜氏藻3-磷酸甘油脱氢酶不同结构域蛋白的表达纯化及其抗体的制备与分析()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
15卷
期数:
2009年02期
页码:
202-206
栏目:
研究论文
出版日期:
2009-03-15

文章信息/Info

Title:
Purification and Antibody Preparation and Analysis of Different Domains of GPDH Proteins from Dunaliella salina
作者:
黄秦曹瑜吴鹏史岩张书乔代蓉曹毅
四川大学生命科学学院微生物与代谢工程四川省重点实验室 成都 610064
Author(s):
HUANG Qin CAO Yu WU Peng SHI Yan ZHANG Shu QIAO Dairong & CAO Yi**
(Microbiology and Metabolic Engineering Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu 610064, China)
关键词:
盐生杜氏藻3-磷酸甘油脱氢酶结构域纯化多克隆抗体Western Blot交叉反应
Keywords:
Dunaliella salina glycerol 3-phosphate dehydrogenase domain purification antibody Western Blot cross reaction
分类号:
Q949.206 : Q78
DOI:
10.3724/SP.J.1145.2009.00202
文献标志码:
A
摘要:
盐生杜氏藻(Dunaliella salina)是迄今为止世界上发现的最耐盐的真核光合生物,而甘油是盐藻用于调节细胞内外渗透压变化的关键调渗物质. 3-磷酸甘油脱氢酶(GPDH)是盐生杜氏藻甘油合成途径中的关键酶. 与已经报道的其它物种的GPDH基因不同,盐生杜氏藻3-磷酸甘油脱氢酶(Ds. GPDH)基因具有两个独立的功能结构域,即丝氨酸磷酸化酶结构域(SGPP domain)和3-磷酸甘油脱氢酶结构域(GPD domain). 本实验在已克隆的Ds. GPDH2基因的基础上,以含GPDH2全基因序列的T质粒载体(pMD18-T-GPDH2)为模板,PCR扩增分别得到两个单结构域片段(SGPP与GPD),以及双结构域片段(GPDH),构建了pET32a-SGPP、pET32a-GPD和pET32a-GPDH三种原核表达载体,并在大肠杆菌中成功表达. 纯化蛋白制备抗原,制备出3个多克隆抗体. Western Blot和交叉反应分析显示通过重组蛋白制备的多克隆抗体具有很高的特异性. 图7 参16
Abstract:
Dunaliella salina is one of the most salt-tolerant photosynthetic eukaryotic organisms. Glycerol is the key compatible solutes synthesized in saline cell for osmotic balance. Glycerol 3-phosphate dehydrogenase (GPDH) is a key enzyme in glycerol metabolism of D. salina. Unlike GPDH reported from other species, the glycerol 3-phosphate dehydrogenase from D. salina (Ds. GPDH) includes two independent domains: Ser-phosphatase domain (SGPP) and glycerol-3-phosphate dehydrogenase (GPD) domain. Based on the cloned Ds. GPDH2 gene, we used the pMD18-T plasmid containing the full length of GPDH2 (pMD18-T-GPDH2) as the template to amplify the segments of SGPP, GPD and GPDH which were used to construct the prokaryotic expression vectors of pET32a-SGPP, pET32a-GPD and pET32a-GPDH. The three recombinant proteins were expressed respectively in E. coli BL21 and purified successfully, and the antibodies of the three forms of proteins were prepared. The results of Western Blot and cross reaction indicated that the antibodies prepared by the recombinant proteins had high specificity. Fig 7, Ref 16

参考文献/References:

1 Avron M. The osmotic components of halotolerant algae. Trends Biochem Sci, 1986, 11: 5~6
2 Ben-Amotz A, Avron M. The role of glycerol in the osmotic regulation of the halophilic alga Dunaliella parva. Plant Physiol, 1973, 51: 875~878
3 Gmmler H, Möller E. Salinity-dependent regulation of starch and glycerol metabolism in Dunaliella parva. Plant Cell Environ, 1981, 4: 367~375
4 Albertyn J, Hohmann S, Thevelein JM, Prior BA. GPD1, which encodes glycerol-3-phosphate dehydrogenase, is enssential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway. Mol Cell Biol, 1994, 14: 4135~4144
5 Haus M, Wegmann K. Glycerol 3-phosphoate dehydrogenase (EC1.1.1.8) from Dunaliella tertiolecta. Plant Physiol, 1984, 60: 283~288
6 Giaever G, Chu AM, Ni L, Connelly C, Riles L, Véronneau S, Dow S, Lucau-Danila A, Anderson K, André B, Arkin AP, Astromoff A, El-Bakkoury M, Bangham R, Benito R, Brachat S, Campanaro S, Curtiss M, Davis K, Deutschbauer A, Entian KD, Flaherty P, Foury F, Garfinkel DJ, Gerstein M, Gotte D, Güldener U, Hegemann JH, Hempel S, Herman Z, Jaramillo DF, Kelly DE, Kelly SL, Kötter P, LaBonte D, Lamb DC, Lan N, Liang H, Liao H, Liu L, Luo C, Lussier M, Mao R, Menard P, Ooi SL, Revuelta JL, Roberts CJ, Rose M, Ross-Macdonald P, Scherens B, Schimmack G, Shafer B, Shoemaker DD, Sookhai-Mahadeo S, Storms RK, Strathern JN, Valle G, Voet M, Volckaert G, Wang CY, Ward TR, Wilhelmy J, Winzeler EA, Yang Y, Yen G, Youngman E, Yu K, Bussey H, Boeke JD, Snyder M, Philippsen P, Davis RW, Johnston M. Functional profiling of the Saccharomyces cerevisiae genome. Nature, 2002, 418: 387~391
7 Ansell R, Granath K, Hohmann S, Thevelein JM, Adler L. The two isoenzymes for yeast NAD+-dependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2 have distinct roles in osmoadaptation and redox regulation. EMBO J, 1997, 16: 2179~2187
8 Gee RW, Byerrum RU, Gerber DW, Tolbert NE. Differential inhibition and activation of two leaf dihydroxyacetone phosphate reductases: Role of fructose 2,6-bisphosphate. Plant Physiol, 1988, 87: 379~383
9 Kirsch T, Gerber DW, Byerrum RU, Tolbert NE. Plant dihydroxyacetone phosphate reductases: Purification, characterization, and localization. Plant Physiol, 1992, 100: 352~359
10 Klöck G, Kreuzberg K. Kinetic properties of a sn-glycerol-3-phosphate dehydrogenase purified from the unicellular alga Chlamydomonas reinhardtii. Biochim Biophys Acta, 1989, 991: 347~352
11 Gee R, Goyal A, Gerber D, Byerrum RU, Tolbert NE. Isolation of dihydroxyacetone phosphate reductase from Dunaliella chloroplasts and comparison with isozymes from spinach leaves. Plant Physiol, 1988, 88: 896~903
12 Gee R, Goyal A, Byerrum RU, Tolbert NE. Two isozymes of dihydroxyacetone phosphate reductase in Dunaliella. Plant Physiol, 1989, 91: 345~351
13 Gee R, Goyal A, Byerrum RU, Tolbert NE. Two Isoforms of dihydroxyacetone phosphate reductase from the chloroplasts of Dunaliella tertiolecta. Plant Physiol, 1993, 103: 243~249
14 Ghoshal D, Mach D, Agarwal M, Goyal A, Goyal A. Osmoregulatory isoform of dihydroxyacetone phosphate reductase from Dunaliella tertiolecta: Purification and characterization. Protein Expr Purif, 2002, 24: 404~411
15 He QH, Qiao DR, Bai LH, Zhang QL, Yang WG, Li Q, Cao Y. Cloning and characterization of a plastidic glycerol 3-phosphate dehydrogenase cDNA from Dunaliella salina. J Plant Physiol, 2007, 164: 214~220
16 Laemmli UK . Cleavage of structural proteins during the assemble of the head of bacteriophage T4. Nature, 1970, 227: 680~685

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备注/Memo

备注/Memo:
国家自然科学基金项目(Nos. 30740055, 30871321, 30771312)和教育部新世纪优秀人才支持计划(No. NCET-05-0785)资助 Supported by the National Natural Science Foundation of China (Nos. 30740055, 30871321, 30771312), and the Program for New Century Excellent Talents in Universities of China (No. NCET-05-0785)
更新日期/Last Update: 2009-05-05