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[1]赵锐,金楠,陈卫民,等.盐藻LHCB3蛋白的表达纯化及抗体制备[J].应用与环境生物学报,2011,17(06):847-850.[doi:10.3724/SP.J.1145.2011.00847]
　ZHAO Rui,JIN Nan,CHEN Weimin,et al.Expression, Purification and Antibody Preparation of LHCB3 Protein in Dunaliella salina[J].Chinese Journal of Applied & Environmental Biology,2011,17(06):847-850.[doi:10.3724/SP.J.1145.2011.00847]
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盐藻LHCB3蛋白的表达纯化及抗体制备()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
17卷
期数:
2011年06期
页码:
847-850
栏目:
研究论文
出版日期:
2011-12-25

文章信息/Info

Title:
Expression, Purification and Antibody Preparation of LHCB3 Protein in Dunaliella salina
作者:
赵锐; 金楠; 陈卫民; 马根; 徐青锐; 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):
ZHAO Rui; JIN Nan; CHEN Weimin; MA Gen; XU Qingrui; 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; 光系统Ⅱ; 光捕获蛋白; 表达纯化; 抗体
Keywords:
Dunaliella salina;  photosystem Ⅱ;  light harvesting chlorophyll a/b-binding protein;  expression and purification;  antibody
分类号:
Q949.210.6 : Q78
DOI:
10.3724/SP.J.1145.2011.00847
文献标志码:
A
摘要:
为了解盐生杜氏藻（Dunaliella salina）主要光捕获蛋白LHCB蛋白的功能，将已获得的盐藻Lhcb3基因构建到原核表达载体pET32a-DsLhcb3，通过优化表达条件，建立了高效的重组系统. pET32a-DsLhcb3在大肠杆菌中的优化表达条件为1 mmol/L IPTG在37 ℃下诱导4 h. 采用镍离子亲合层析纯化获得LHCB3蛋白，并以此为抗原制备了多克隆抗体，经琼脂糖扩散检测效价，在1 : 16处有明显沉淀. 提取盐藻总蛋白，经过制备的LHCB3抗体杂交，在29 000处获得两条明显的杂交条带，为进一步研究盐藻LHCⅡ蛋白表达机理奠定了基础. 图4 参16
Abstract:
Light harvesting chlorophyll a/b-binding protein (LHCB) is the major component of antenna complex of photosystem Ⅱ. In order to investigate its functions, Lhcb3 gene in Dunaliella salina was expressed in Escherichia coli and the fusion protein was obtained. The recombinant vector pET32a-DsLhcb3 was transformed into E. coli, and the preparation for expression was optimized in order to construct a more effective recombinant system. This experiment suggested that the optimized expression procedure of pET32a-DsLhcb3 in E. coli was induced by 1 mmol/L IPTG at 37 ℃ for 4 hours. LHCB3 protein was purified by Ni2+ NTA agarose beads, which in turn the polyclonal antibody was achieved. Significant precipitation was detected at 1 : 16 by the agarose diffusion test. The LHCB3 antibody was then hybridized with total protein, and the test showed two hybridization bands at 29 000. This research laid a favorable foundation for further explanations of LHCⅡ mechanisms in D. salina. Fig 4, Ref 16

参考文献/References:

1 Sun QM (孙钦秒), Leng J (冷静), Li LB (李良璧), Kuang TY (匡廷云). Recent advances of studies on the structure and function of the light- harvesting chlorophyll A/B-protein complex. Chin Bull Bot (植物学通报), 2000, 17 (4): 289~301
2 Jansson S. The light-harvesting chlorophyll a/b-binding proteins. Biochim Biophys Acta, 1994, 1184: 1~19
3 Adamska I, Loppstech K. Low temperature increases the abundance of early light-inducible transcript under light stress conditions. J Biol Chem, 1994, 269: 30221~30226
4 Hobe S, Förster R, Klingler J, Paulsen H. N-proximal sequence motif in light-harvesting chlorophyll a/b-binding protein is essential for the trimerization of light-harvesting chlorophyll a/b complex. Biochemistry, 1995, 34: 10224~10228
5 Klimmer F, Sjödin A, Noutsos C, Leister D, Jansson S. Abundantly and rarely expressed Lhc protein genes exhibit distinct regulation patterns in plants. Plant Physiol, 2006, 140: 793~804
6 Bassi R, Rigoni F, Giacometti GM. Chlorophyll binding proteins with antenna function in higher plants and green algae. Photochem & Photobiol, 1990, 52 (6) : 1187~1206
7 Park S, Polle J, Melis A, Lee TK, Jin ES. Up-regulation of photoprotection and PSII-repair gene expression by irradiance in the unicellular green alga Dunaliella salina. Marine Biotechnology. 2006, 8: 120~128
8 Liu XD, Shen YG. NaCl induced phosphorylation of light harvesting chlorophyll a/b proteins in thylakoid membranes from the halotolerant green alga Dunaliella salina. FEBS Lett, 2004, 569: 337~340
9 Barzda V, Peterman EJ, Grondelle RV, Amerogen HV. The influence of aggregation on the triple formation in light-harvesting chlorophyll a/b pigment-protein complex of green plants. Biochemistry, 1998, 37: 546~551
10 Huang Q (黄秦), Cao Y (曹瑜), Wu P (吴鹏), Shi Y (史岩), Zhang S (张书), Qiao DR (乔代荣), Cao Y (曹毅). Purification and antibody preparation and analysis of different domains of GPDH proteins from Dunaliella salina. Chin J Appl Environ Biol (应用与环境生物学报), 2009, 15 (2): 202~206
11 Coligan JE, Kruisbeek AM, Margulies DH. Current Protocols in Immunology. New York, USA: John Wiley, 2002
12 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227 (5259): 680~685
13 Towbin H, Staehelin T, Gordon J. Elect rophoret ic transfer of proteins from polyacrylamide gels to nitrocellulose sheet s: procedure and some applications. Proc Natl Acad Sci USA, 1979, 76 (9): 4350~4354
14 Sun QM (孙钦秒), Li LB (李良璧), Mao DZ (毛大璋), Kuang TY (匡廷云). 豌豆Lhcb2基因在大肠杆菌中的高效表达及其表达产物与色素的重组. Sci China Life Sci (中国科学C辑), 2000, 30 (8): 363~369
15 Tang B (唐彬), Tong Z (童哲). A fast staining-destaining method for SDS-PAGE which favours the recovery of protein. Prog Biochem Biophys (生物化学与生物物理进展), 2000, 27 (2): 210~211
16 Zhang YJ (张亚杰). Stability of the major light-harvesting complex of photosystem II of higher plant: [Master Degree Dissertation]. Beijing, China: Institute of Botany, Chinese Academy of Science (北京: 中国科学院植物研究所), 2006. 99

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备注/Memo

备注/Memo:
国家自然科学基金项目（No. 30871321）和四川省“十一五”科技支撑计划项目（Nos. 2008GZ0020，2008GZ0021）资助
更新日期/Last Update: 2011-12-31