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  

Purification and Antibody Preparation and Analysis of Different Domains of GPDH Proteins from Dunaliella salina

作　　者：黄秦[1] 曹瑜[1] 吴鹏[1] 史岩[1] 张书[1] 乔代蓉[1] 曹毅[1] 

机构地区：[1]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

出　　处：《应用与环境生物学报》2009年第2期202-206,共5页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(Nos.30740055;30871321;30771312);教育部新世纪优秀人才支持计划(No.NCET-05-0785)资助~~

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.WesternBlot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.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

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 

分 类 号：Q946[生物学—植物学]