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植物研究 ›› 2014, Vol. 34 ›› Issue (4): 492-497.doi: 10.7525/j.issn.1673-5102.2014.04.011

• 论文 • 上一篇    下一篇

水稻锌指蛋白OsRZ3基因克隆及融合蛋白的原核表达、纯化

孙宇1;王金麟2*   

  1. 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
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2014-07-20 发布日期:2014-07-20
  • 通讯作者: 王金麟
  • 基金资助:
     

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

SUN Yu;WANG Jin-Lin*   

  1. 1.The Department of Ecological Engineering,Heilongjiang Vocational Institute of of Ecological Engineering,Harbin 150040;2.Graduate school,Northeast Forestry University,Harbin 150040
  • Received:1900-01-01 Revised:1900-01-01 Online:2014-07-20 Published:2014-07-20
  • Contact: WANG Jin-Lin
  • Supported by:
     

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。

关键词: 水稻, 锌指蛋白, 原核蛋白表达与纯化

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.

Key words: rice, zinc finger domain, Prokaryotic Protein Expression and Purification

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