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作 者 ：孙宇;王金麟*

期 刊 ：植物研究 2014年 34卷 4期 页码：492-497

关键词：水稻；锌指蛋白；原核蛋白表达与纯化；

Keywords:rice, zinc finger domain, Prokaryotic Protein Expression and Purification,

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34 kD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^-1 IPTG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^-1 IPTG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^-1 IPTG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。

Abstract: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 BL21(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.1L 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 IPTG 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.

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