|本期目录/Table of Contents|

[1]黄卫东,郭嘉义.铜绿假单胞菌含Pilz结构域蛋白基因的克隆及原核表达[J].宁夏医科大学学报,2010,(05):564-567.
 HUANG Wei-dong,GUO Jia-yi.Cloning and Prokaryotic Expression of the Genes Containing Pilz Domain in Pseudomonas Aeruginosa[J].Ningxia Medical University,2010,(05):564-567.
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铜绿假单胞菌含Pilz结构域蛋白基因的克隆及原核表达(PDF)
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《宁夏医科大学学报》[ISSN:1005-8486/CN:64-1029/R]

卷:
期数:
2010年05期
页码:
564-567
栏目:
论著
出版日期:
2011-12-30

文章信息/Info

Title:
Cloning and Prokaryotic Expression of the Genes Containing Pilz Domain in Pseudomonas Aeruginosa
作者:
黄卫东1 郭嘉义2
1.宁夏医科大学基础医学院生化与分子生物学教研室,银川 750004; 2.宁夏医科大学医学科学研究所,银川 750004
Author(s):
HUANG Wei-dong GUO Jia-yi
1. Dept. of Biochemistry and Molecule Biology, Ningxia Med. Univ., Yinchuan 750004; 2. Institute of Medical Science, Ningxia Med. Univ., Yinchuan 750004
关键词:
铜绿假单胞菌 c-di-GMP第二信使 Pilz结构域蛋白 基因克隆 原核表达载体构建
Keywords:
Pseudomonas aeruginosa second messenger c-di-GMP Pilz domain-containing protein Gene cloning Prokaryotic expression
分类号:
R378.99+1
DOI:
-
文献标志码:
A
摘要:
目的 克隆铜绿假单胞菌含Pilz结构域蛋白基因并构建其原核表达载体,为进一步研究这七种含Pilz结构域蛋白是否为第二信使c-di-GMP 潜在的受体分子奠定实验基础。方法 提取铜绿假单胞菌PAO1基因组DNA, 用PCR方法从中扩增出七种功能未知的含Pilz结构域蛋白基因, 将其克隆到原核表达载体pET32a中, 在大肠杆 菌BL21(DE3)中用IPTG诱导表达,表达产物通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行鉴定。结果 成功扩增出七种含Pilz结构域蛋白基因并构建其原核表达载体, 测序结果与NCBI数据库收录序列相一致。SDS-PAGE检验证明目的基因在大肠杆菌BL21中获得高效表达。结论 从铜绿假单胞菌基因组中成功地克隆了七种含Pilz结构域蛋白基因,其构建的原核表达载体在大肠杆菌BL21中获得了高效表达。
Abstract:
Objective To clone genes containing Pilz domains in Pseudomonas aeruginosa and to construct their prokaryotic expressing vectors. Methods Genomic DNA was extracted from P. aeruginosa PAO1 strain and seven genes of unknown function which containing Pilz domains were amplified by PCR. PCR products were cloned into pET32a vector and then expressed in E.Coli BL21 by IPTG induction. Expressed products were determined by SDS-PAGE analysis. Results The genes containing Pilz domain were amplified by PCR and the DNA sequences of these genes were consistent with that published by NCBI. SDS-PAGE analysis indicated that these fusion proteins could be highly expressed in the prokaryotic system. Conclusion Seven genes containing Pilz domain from Pseudomonas aeruginosa had been successfully cloned and highly expressed in E.Coli BL21, which therefore provided experimental foundation for further functional analysis of these proteins, especially their roles in the novel secondary messenger c-di-GMP signaling transduction.

参考文献/References:

[1] CK Stover, XQ Pham, AL Erwin, et al. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen [J]. Nature, 2000,406:959-964.
[2] Kumar M, Chatterji D. Cyclic di-GMP: a second messenger required for long-term survival, but not for biofilm formation, in Mycobacterium smegmatis[J]. Microbiology, 2008, 154:2942-2955.
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[5] RomLing U, Amikam D. Cyclic di-GMP as a second messenger [J]. Curr Opin Microbiol, 2006,9:218-228.
[6] Tamayo R, Pratt JT, Camilli A. Roles of cyclic diguanylate in the regulation of bacterial pathogenesis [J]. Annu Rev Microbiol, 2007, 61: 131-148.
[7] Amikam D, Galperin MY. Pilz domain is part of the bacterial cdi-GMP binding protein [J]. Bioinformatics, 2006, 22:3-6.
[8] Ryjenkov DA, Simm R, RomLing U, et al. The Pilz domain is a receptor for the second messenger c-di-GMP: the Pilz domain protein YcgR controls motility in enterobacteria [J]. J Biol Chem, 2006, 281: 30310-30314.
[9] Merighi M, Lee VT, Hyodo M, et al. The second messenger bis-(30-50)-cyclic-GMP and its Pilz domain containing receptor Alg44 are required for alginate biosynthesis in Pseudomonas aeruginosa [J]. Mol Microbiol, 2007, 65:876-895.

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备注/Memo

备注/Memo:
收稿日期:2009-12-30 作者简介:黄卫东(1969-), 女, 博士后, 主要从事细胞和分子生物学研究工作。
更新日期/Last Update: 2010-05-20