水稻锌指蛋白OsRZ3基因克隆及融合蛋白的原核表达、纯化  

The Gene Cloning,Prokaryotic Expression and Purification of Rice Zinc Finger Protein OsRZ3

作　　者：孙宇[1] 王金麟[2] 

机构地区：[1]黑龙江生态工程职业学院生态工程系,哈尔滨150040 [2]东北林业大学研究生院,哈尔滨150040

出　　处：《植物研究》2014年第4期492-497,共6页Bulletin of Botanical Research

基　　金：<哈尔滨地区治污性湿地景观的营造>;黑龙江省教育厅科研项目(12515219)

摘　　要：根据NCBI登录的水稻锌指蛋白基因(NO.AK062094,OsRZ3 gene)设计特异引物,通过RT-PCR克隆该基因cDNA全长序列,核苷酸测序并比对氨基酸同源性表明具有C2HC-锌指结构域、分析预测蛋白质分子量为34kD和等电点为9.05;拟南芥原生质体亚细胞定位检测表明其在细胞核。构建pGEX6P-3::OsRZ3融合载体,电转化的大肠杆菌BL21(DE3)菌株经1 mmol·L-1IPTG小量诱导表达,SDS-pAGEX蛋白电泳表明60 kD融合蛋白4 h最大;在LB液体培养中0.5 mmol·L-1IPTG过夜大量诱导表达,通过GST吸附柱层析获得大量包涵体纯化蛋白,1L菌液可诱导纯化到浓度1.58 mg·mL-1包涵体融合蛋白。所构建的融合蛋白原核表达系统能有效表达pGEX6P-3::OsRZ3融合蛋白,0.5 mmol·L-1IPTG大量诱导,通过GST柱吸附获得包涵体纯化蛋白,为作进一步的抗体制备和染色质免疫共沉淀等DNA结合的特性研究准备了基础。This research is based on the NCBI＇ s NO. AK062094, OsRZ3 gene, and uses it to design specific primer. Then through adding the cDNA＇ s full length genome by RT-PCR, nucleotide sequencing, and compare the homology of nucleotide to show C2HC-zinc finger domain. Analyze and forecast protein molecular mass-34 kD and isoelectric point is 9.05; Arabidopsis protoplast the cytoplasm location detect indicates it is in the nucleus. Structure pGEX6P-3 ： ： OsRZ3 fusion vector, transformation of electrical Escherichia coli BI2.1 （ DE3 ） bacterial strain is induced by 1 mmol~ L-1 IPTG , SDS-pAGEX protein electrophoresis to show it can reach the maximum 60 kD fusion protein through 4 hours; in LB liquid culture, 0.5 mmol ~ L-1 IPTG is induced and express. We can get a lots of fusion protein through GST adsorption column. 1 L bacterial suspension can induce and purify to concentration 1.58 mg· mL-1 inclusion body fusion protein. The system of fusion protein pronucleus can express pGEX6P-3 ： ： OsRZ3 fusion protein, 0.5 mmol· L- 1 ItrrG is induced , we get a lots of fusion protein through GST adsorption column. This is the basis of further antibody preparation and chromatin co-immunoprecipitation and the DNA combine.

关 键 词：水稻 锌指蛋白 原核蛋白表达与纯化 

分 类 号：S511[农业科学—作物学]