烟草CSN4基因的原核表达及纯化
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CSN4cDNA序列，基因登录号为 KC866360。序列分析表明，CSN4基因ORF长为 1194bp，编码398个氨基酸，蛋白分子质量为4378kDa。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：025mM IPTG，15℃诱导16h获得浓度为0476mg/mL、纯度高达95%的CSN蛋白，为后续CSN4基因功能研究奠定了基础。
关键词：COP9信号复合体；基因克隆；原核表达；亲和纯化
中图分类号：Q78
文献标识码：A
文章编号：1008-0457（2017）02-0019-06国际DOI编码：10.15958/j.cnki.sdnyswxb.2017.02.004
Abstract：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. Sequence analysis showed that the length of open reading frame of N. tabacum CSN4 gene was 1194bp， encoding 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 0.25mM IPTG 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.
Key words：COP9 signalosome； gene clone； prokaryotic expression； affinity purification
COP9（constitutively photomorphogennic 9）信號复合体（CSN）最早是在拟南芥光形态建成中发现，是植物光形态发生的一个负调节子[1]，CSN复合体由 8 个不同亚基组成，可依次命名为 CSN1-CSN8[2]。这一复合体亚基从多种生物中被克隆及分析，如家蚕、菊花、甘...
|
== 试读已结束，如需继续阅读敬请充值会员 ==
|
|
本站文章均为原创投稿，仅供下载参考，付费用户可查看完整且有格式内容！
(费用标准：38元/2月，98元/2年，微信支付秒开通！) |
| 升级为会员即可查阅全文 。如需要查阅全文，请 免费注册 或 登录会员 |
|
