斑点叉尾鮰BPI抗菌肽的cDNA 克隆及原核表达载体的构建

生物技术通报 ›› 2012, Vol. 0 ›› Issue (12): 131-138.

斑点叉尾鮰BPI抗菌肽的cDNA 克隆及原核表达载体的构建

高北, 陶妍

上海海洋大学食品学院，上海 201306

收稿日期:2012-05-29 修回日期:2013-01-25 出版日期:2012-12-26 发布日期:2013-02-06
作者简介:高北, 女, 硕士研究生, 研究方向:水产生物分子生物学;E-mail:wintergb@hotmail.com
基金资助:
: 上海市教育委员会重点创新基金项目（11ZZ148）, 上海市科促会联盟计划项目（4319）, 上海市宝山区科学技术委员会科研基金项目（CXY-2010-31）

Cloning and Construction of Prokaryotic Expression Vectors forChannel Catfish BPI Antibacterial Peptide

Gao Bei, Tao Yan

College of Food Science and Technology，Shanghai Ocean University，Shanghai 201306

Received:2012-05-29 Revised:2013-01-25 Published:2012-12-26 Online:2013-02-06

摘要/Abstract

摘要： 杀菌/ 渗透增强蛋白（bactericidal /permeability increasing protein，BPI）是生物机体细胞中表达的一种能够特异性杀灭革兰氏阴性菌的抗菌类蛋白，该蛋白包括氨基端和羧基端两个结构域，其中氨基端结构域主要承担杀菌和中和内毒素的功能。参考已经报道的对人类BPI 进行重组DNA 表达的研究结果，首先通过RT-PCR 和巢式PCR 从斑点叉尾鮰（Ictalurus punctatus）鳃中克隆了编码BPI 氨基端活性结构域的cDNA 片段“BPI_Ntd”，该片段由175 个氨基酸残基组成，其中63 个氨基酸残基在空间上形成一个非极性的脂质结合袋，与革兰氏阴性菌外膜上脂多糖（LPS）结合，从而改变细菌膜的通透性，达到杀菌的效果。根据斑点叉尾鮰与其他生物之间BPI 氨基酸序列的比对结果，发现氨基端存在40 个保守的氨基酸残基，其中9 个高度保守的残基位于脂多糖结合区域内。为了进一步建立原核表达系统，根据pET-32_a（+）和pET-28_a（+）两种质粒的不同特点，选择其作为原核表达质粒，将“BPI_Ntd”片段分别插入两种质粒，通过菌落PCR、EcoR I 和Hind III 双酶切反应以及DNA 测序等方法，证明了重组表达质粒“pET-32_a-BPI_Ntd”和“pET-28_a-BPI_Ntd”已被成功构建。

关键词: 斑点叉尾鮰, BPI结构域, cDNA, 克隆, 原核表达

Abstract: Bactericidal/permeability increasing protein（BPI）, an antimicrobial protein expressed in organism cells, shows particularantimicrobial activity against gram-negative bacteria. BPI contains two domains, N-terminal domain and C-terminal domain. The antibiotic andendotoxin-neutralizing functions of BPI are attributed to the N-terminal domain. The present study was performed with reference to the reportfor the recombinant DNA expression of human BPI. A cDNA fragment “BPI_Ntd” encoding the N-terminal active domain of BPI was clonedfrom the gill of channel catfish（Ictalurus punctatus）by RT-PCR and nested PCR. This fragment encodes a peptide consisting of 175 aminoacid residues, in which 63 residues can form a three-dimensional apolar lipid-binding pocket which is used to bind lipopolysaccharide（LPS）located in outer membrane of gram-negative bacteria. The comparison of the channel catfish BPI with the BPIs from other organisms showed that40 conserved amino acid residues appear at the N-terminal domain, and among them 9 highly conserved residues located in the LPS-bindingregion. The pET-32_a（+）and pET-28_a（+）with different characteristics were chosen as the prokaryotic expression vectors to constructthe prokaryotic expression system. “BPI_Ntd” fragment was inserted into these two plasmids respectively. The colony PCR, EcoR I and Hind IIIrestriction endonuclease digestion and DNA sequencing demonstrated that the “pET-32_a-BPI_Ntd” and “pET-28_a-BPI_Ntd” recombinant expressionplasmids were constructed successfully.

Key words: Channel catfish, BPI Domain, cDNA cloning, Prokaryotic expression

高北, 陶妍. 斑点叉尾鮰BPI抗菌肽的cDNA 克隆及原核表达载体的构建[J]. 生物技术通报, 2012, 0(12): 131-138.

Gao Bei, Tao Yan. Cloning and Construction of Prokaryotic Expression Vectors forChannel Catfish BPI Antibacterial Peptide[J]. Biotechnology Bulletin, 2012, 0(12): 131-138.

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