噬菌体基因Ⅴ蛋白基因的合成、重组表达及功能分析

食品科学 ›› 2012, Vol. 33 ›› Issue (1): 191-194.

噬菌体基因Ⅴ蛋白基因的合成、重组表达及功能分析

张大川，房国梁，李琦，陈江源，王岚，李睿，刘烈炬，刘志国*

武汉工业学院生物与制药工程学院

收稿日期:2011-07-02 修回日期:2012-01-09 出版日期:2012-01-15 发布日期:2012-01-12
通讯作者: 张大川 E-mail:zhangdachuan1985@163.com
基金资助:
武汉市科技局对外科技合作与交流计划项目（201070934341）与现代农业技术创新平台项目（201120637175-5）资助。

Synthesis, Recombinant Expression and Functional Analysis of Filamentousphage Gene V Protein

Da-Chuan ZHANG

Received:2011-07-02 Revised:2012-01-09 Online:2012-01-15 Published:2012-01-12
Contact: Da-Chuan ZHANG E-mail:zhangdachuan1985@163.com

摘要/Abstract

摘要： 目的：研究丝状噬菌体基因V蛋白(gene V protein，GVP)基因的合成、重组表达及其功能。方法：根据GVP的基因序列，选择大肠杆菌偏爱的密码子，设计合成了8个寡核苷酸片段，利用重叠延伸PCR合成GVP基因序列，将其与原核表达载体pET-28a-c(＋)质粒重组，转化大肠杆菌，获得GVP蛋白阳性表达菌株，异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达，产物经Ni+-NTA琼脂糖凝胶层析纯化，获得目的蛋白GVP，DNA结合实验检测其功能。结果：成功合成出GVP基因，重组体在大肠杆菌BL21(DE3)中诱导获得高效表达，DNA结合实验表明GVP与单链DNA间解离平衡常数Kd=7.27×10-5mol/L。结论：重组构建并高效表达的GVP蛋白具有较高单链DNA结合能力，可用于食品病原微生物特定单链DNA分子的浓缩和分离。

关键词: 基因Ⅴ蛋白(GⅤP), 大肠杆菌BL21(DE3), 重组, 表达

Abstract: Objective: To explore the gene synthesis, recombinant expression and functions of filamentousphage gene V protein (GVP). Methods: According to the gene sequence of GVP, eight oligonucleotide fragments with the selected E. coli-preferred codon were designed, and the GVP gene sequence was synthesized by overlap extension PCR. Then the synthesized sequence was inserted into pET-28a-c(+) plasmid. The recombinant plasmids obtained were transformed into E. coli to screen positive isolates of GVP. GVP expression in the positive strains was induced with IPTG. The recombinant proteins were purified by Ni+-NTA affinity chromatography. Results: GVP gene was successfully synthesized and highly expressed in E. coli BL21 (DE3) under the induction of IPTG, and the equilibrium dissociation constant was 7.27 × 10-5 mol/L between GVP and ssDNA. Conclusion: The recombinant GVP has high affinity with ssDNA and therefore can be used for the ssDNA detection of some pathogenic microorganisms in food.

Key words: gene V protein, E. coli BL21 (DE3), recombination, expression

中图分类号:

张大川，房国梁，李琦，陈江源，王岚，李睿，刘烈炬，刘志国*. 噬菌体基因Ⅴ蛋白基因的合成、重组表达及功能分析[J]. 食品科学, 2012, 33(1): 191-194.

Da-Chuan ZHANG. Synthesis, Recombinant Expression and Functional Analysis of Filamentousphage Gene V Protein[J]. FOOD SCIENCE, 2012, 33(1): 191-194.

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