三角帆蚌（Hyriopsis cumingii）Perlucin蛋白原核表达条件的优化及表达产物的鉴定

生物技术通报 ›› 2013, Vol. 0 ›› Issue (7): 114-118.

三角帆蚌（Hyriopsis cumingii）Perlucin蛋白原核表达条件的优化及表达产物的鉴定

林静云,白志毅,李家乐

（上海海洋大学水产与生命学院农业部淡水水产种质资源重点实验室，上海 201306）

收稿日期:2013-01-02 修回日期:2013-07-19 出版日期:2013-07-19 发布日期:2013-09-02
作者简介:林静云，女，硕士研究生，研究方向：水产动物遗传育种；E-mail：ljy19890910@163.com
基金资助:
国家科技支撑计划课题（2012BAD26B04），上海市高校知识服务平台项目（ZF1206）

Purification and Optimization of Prokaryotic Expression of Perlucin Protein in the Freshwater Pearl Mussel，Hyriopsis cumingii

Lin Jingyun, Bai Zhiyi, Li Jiale

（Key Laboratory of Freshwater Aquatic Genetic Resources Certificated by Ministry of Agriculture，College of Fisheries and Life Science，Shanghai Ocean University，Shanghai 201306）

Received:2013-01-02 Revised:2013-07-19 Published:2013-07-19 Online:2013-09-02

摘要/Abstract

摘要： 采用PCR方法从三角帆蚌外套膜cDNA中扩增perlucin基因的开放阅读框，连接到pET-28a表达载体，转化大肠杆菌BL21（DE3），经IPTG诱导表达重组融合蛋白pET-28a-Hcperlucin。通过优化培养温度、IPTG 浓度以及诱导时间，得出重组融合蛋白的最佳表达条件分别为：37℃、0.1 mmol/L IPTG、6 h。重组融合蛋白主要以包涵体形式存在。SDS-PAGE和Western blot分析结果表明，表达重组融合蛋白的相对分子量与预测值相符，约为21 kD，并能与鼠抗His-tag单克隆抗体进行特异性结合，表明重组融合蛋白构建成功。

关键词: 三角帆蚌, Perlucin, 原核表达, Western, blot分析

Abstract: Perlucin was originally isolated from nacre of abalone inner shell and improved to promote calcium carbonate precipitation at ambient conditions, nucleate the calcium carbonate crystallization and modify the morphology of calcium carbonate crystals. The open reading frame（ORF）of the perlucin gene was amplified from the mantle cDNA of the freshwater pearl mussel, Hyriopsis cumingii. The ORF from this species was ligated into the expression plasmid pET-28a. Following transfer of the recombinant plasmid into host bacteria, Escherichia coli BL21（DE3）cells, the recombinant pET-28a-Hcperlucin protein was expressed by induction with isopropyl-β-thiogalactopyranoside（IPTG）. SDS-PAGE analysis showed that inducing the cells at 37℃ in 0.1 mmol/L of IPTG for 6 h was the optimal conditions for expression of the recombinant pET-28a-Hcperlucin protein. Inclusion body was the main existence form of the recombinant protein. The expression products were detected by SDS-PAGE and Western blot. The results showed that the recombinant expression plasmid of pET-28a-Hcperlucin, expressing a 21 kD protein that could be recognized by mouse anti-His-tag Mab, was successfully constructed.

Key words: Hyriopsis cumingii, Perlucin, Prokaryotic expression Western blot analysis

林静云,白志毅,李家乐. 三角帆蚌（Hyriopsis cumingii）Perlucin蛋白原核表达条件的优化及表达产物的鉴定[J]. 生物技术通报, 2013, 0(7): 114-118.

Lin Jingyun, Bai Zhiyi, Li Jiale. Purification and Optimization of Prokaryotic Expression of Perlucin Protein in the Freshwater Pearl Mussel，Hyriopsis cumingii[J]. Biotechnology Bulletin, 2013, 0(7): 114-118.

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