噬菌体基因Ⅴ蛋白基因的合成、重组表达及功能分析

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

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。

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.