[1]谷笑笑,王振华,潘康成,等.藏猪β-防御素2基因克隆及原核表达载体的构建[J].江苏农业学报,2017,(04):848-853.[doi:doi:10.3969/j.issn.1000-4440.2017.04.019]
　GU Xiao-xiao,WANG Zhen-hua,PAN Kang-cheng,et al.Cloning and construction of prokaryotic expression vector of porcine β-defensin-2 gene of Tibetan swine[J].,2017,(04):848-853.[doi:doi:10.3969/j.issn.1000-4440.2017.04.019]
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藏猪β-防御素2基因克隆及原核表达载体的构建()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2017年04期
页码:
848-853
栏目:
畜牧兽医·水产养殖
出版日期:
2017-08-30

文章信息/Info

Title:
Cloning and construction of prokaryotic expression vector of porcine β-defensin-2 gene of Tibetan swine
作者:
谷笑笑1; 王振华2; 潘康成1; 张立平3; 周尧3
（1.四川农业大学动物医学院动物微生态研究中心，四川成都611130；2.成都农业科技职业学院，四川成都611130；3.绵阳泰科生物技术有限公司，四川绵阳621000）
Author(s):
GU Xiao-xiao1; WANG Zhen-hua2; PAN Kang-cheng1; ZHANG Li-ping3; ZHOU Yao3
(1. Animal Microecology Institute, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China;2.Chengdu Agricultural College, Chengdu 611130, China; 3.Mianyang Taike Biotechnology Co. Ltd., Mianyang 621000, China)
关键词:
藏猪; β-防御素2; 克隆; 原核表达载体
Keywords:
Tibetan swine; β-defensin-2; cloning; prokaryotic expression vector
分类号:
S858.28
DOI:
doi:10.3969/j.issn.1000-4440.2017.04.019
文献标志码:
A
摘要:
为了构建藏猪β-防御素2（pBD-2）成熟肽原核表达载体，从藏猪肝组织中提取总RNA，通过RT-PCR获取pBD-2目的基因，并与pMD19-T载体连接构建重组质粒，命名为pMD19T-pBD-2；以pMD19T-pBD-2质粒为模板，克隆pBD-2成熟肽cDNA序列并构建成熟肽pET-32a-pBD-2表达质粒。结果显示，藏猪pBD-2基因全长为298 bp，与野猪和家猪的pBD-2基因同源性分别达1000%和989%；藏猪pBD-2基因具有1个完整的开放阅读框，编码69个氨基酸组成的多肽，包含21个氨基酸残基的信号肽、11个氨基酸残基的前导肽和37个氨基酸残基的成熟肽；编码的pBD-2成熟肽稳定性较好，分子量4 085.78，等电点889，具有6个半胱氨酸（Cys）残基，7个带正电荷和2个带负电荷的氨基酸残基，为亲脂性和亲水性多肽；从pMD19T-pBD-2质粒中亚克隆得到长度为114 bp藏猪pBD-2成熟肽cDNA序列，菌落PCR、酶切和测序鉴定证明成功构建了成熟肽pET-32a-pBD-2原核表达质粒。
Abstract:
To construct the prokaryotic expression vector of porcine β-defensin-2 (pBD-2) mature peptide of Tibetan pig, the pBD-2 gene amplified by RT-PCR was cloned into pMD19-T vector to construct pMD19T-pBD-2 plasmid which was used as a template to amplify pBD-2 mature peptide fragment. The Tibetan pig pBD-2 gene was 298 bp in length, and shared sequence similarities of 988% and 1000% with domestic pigs and wild boars respectively. The pBD-2 gene has a complete open reading frame encoding a 69-amino-acid polypeptide which contains a signal peptide consisting of 21 amino acid residues, a leader peptide consisting of 11 amino acid residues and a mature peptide consisting of 37 amino acid residues. The stable pBD-2 mature peptide is 4 085.78 in molecular weight, with isoelectric point of 8.89, and is a lipophilic and hydrophilic polypeptide containing six cysteine residues, seven positively charged amino acid residues and two negatively charged amino acid residues. A 114-bp Tibetan pig pBD-2 mature peptide cDNA sequence was subcloned from pMD19T-pBD-2 plasmid. Colony PCR, enzymatic digestion and sequencing verified the successful construction of prokaryotic expression vector pET-32a-pBD-2.

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备注/Memo

备注/Memo:
收稿日期：2017-03-02 基金项目：四川省高等学校科技创新团队资助项目(KM406183.1)；四川农业大学学科双支计划项目（03570434） 作者简介：谷笑笑（1994-），女，河北武邑人，硕士研究生，主要从事动物微生态学研究。 通讯作者：潘康成，(E-mail)pankangcheng71@126.com
更新日期/Last Update: 2017-09-01