烟草CSN4基因的原核表达及纯化  

The Prokaryotic Expression and Purification of Tobacco CSN4 Gene

作　　者：龚露[1] 钟杰[1] 高洁[1] 佟硕秋 吴拥军[1] 

机构地区：[1]贵州大学生命科学学院,贵州贵阳550025

出　　处：《山地农业生物学报》2017年第2期19-24,共6页Journal of Mountain Agriculture and Biology

基　　金：烟草行业重点实验室开放课题(中烟办[2015]-06号)

摘　　要：以野生型烟草(Nicotiana tabacum)为试验材料,采用RT-PCR克隆获得CSN复合体亚基CSN4c DNA序列,基因登录号为KC866360。序列分析表明,CSN4基因ORF长为1194 bp,编码398个氨基酸,蛋白分子质量为43.78 k Da。将CSN4构建至原核表达载体,获得重组子pET28a-CSN4/BL21(DE3),通过Ni-IDA亲和层析纯化目的蛋白,SDS-PAGE和Western Blotting检测,结果显示:0.25 mM IPTG,15℃诱导16 h获得浓度为0.476 mg/m L、纯度高达95%的CSN蛋白,为后续CSN4基因功能研究奠定了基础。In this study, the COP9 signal complex subunit CSN4 cDNA sequence was cloned by RT - PCR amplification from the wild type Nicotiana tabacum, accession number KC866360 in GenBank database. Se- quence analysis showed that the length of open reading frame of N. tabacum CSN4 gene was 1194bp, enco- ding 398 amino acids, and the molecular weight of the protein is 43.78kDa. The recombinant pET28a - CSN4/BL21 （ DE3 ） had been obtained by cloning CSN4 gene into the prokaryotic expression vector pET28a. The target protein was purified by Ni - IDA affinity chromatography, and the results of SDS - PAGE and Western Blotting indicated that the recombinant strain was induced with 0.25raM IIYFG at 15℃ for 16h, the target protein concentration was 0. 476mg/mL and its purity was up to 95%, which was laid the foundation for further study protein - function.

关 键 词：COP9信号复合体 基因克隆 原核表达 亲和纯化 

分 类 号：Q78[生物学—分子生物学]