文档介绍：
重组七鳃鳗 PHB2 蛋白的原核表达及纯化
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王颖,高杨,李铁松,李庆伟
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(辽宁师范大学生命科学学院,大连,116081)
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摘要:以课题组前期构建的 pMD18-T-Lm-PHB2 质粒为模板,PCR 扩增 Lm-PHB2 基因全长 CDS 区,PCR 产物经
HindⅢ和 EcoRI 双酶切后连接至表达载体 pET-32a,构建重组质粒 pET-32a-Lm-PHB2。将鉴定正确的重组质
粒转化入大肠杆菌 Rosetta Blue,经 IPTG 诱导表达后,表达产物通过 SDS-PAGE 和 Western blotting 进
行检测,利用镍柱亲和层析纯化得到纯度较高的目的蛋白 rLm-PHB2。结果表明,经 PCR、双酶切和测序鉴
定,所构建的重组质粒 pET-32a-Lm-PHB2 序列正确,表达产物于裂解菌液的上清和沉淀中均存在,经
SDS-PAGE 和 Western blotting 证实在约 47KD 处有目的蛋白 rLm-PHB2 的表达条带。构建重组七鳃鳗 PHB2
蛋白的原核表达载体,并大量表达和纯化目的蛋白,为该蛋白后续的抗体制备及生物活性研究奠定了基
础。
关键词:海洋生物学;七鳃鳗;PHB2;原核表达;蛋白纯化
中图分类号:R915
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Prokaryotic expression and purification of bined lamprey PHB2 protein
WANG Ying, GAO Yang, LI Tiesong, LI Qingwei
(Life Science College of Liaoning Normal University,Dalian,116081)
Abstract: Objective: We aimed to construct the soluble prokaryotic expression vector of PHB2,
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obtain adequate amount of purified rLm-PHB2 protein for further study of its biological activity in
vivo. Methods: The PHB2 gene was amplified from the pMD18-T-Lm-PHB2 plasmid template
which was previously constructed. The PCR products were subjected to HindⅢ and EcoRI
digestion and then linked to the soluble expression vector pET-32a. The identified binant
plasmid pET-32a-Lm-PHB2 was transformed into Rosetta Blue, and the IPTG induced expression
of rLm-PHB2 was confirmed by SDS-PAGE and Western blotting assay. The