重组flap核酸内切酶1的表达及活性测定方法的建立

Expression and activity assay of recombinant flap endonuclease 1

摘要:

Flap核酸内切酶1 (Flap endonuclease 1, FEN1) 是一种能催化核酸侵入反应的核酸内切酶，可应用于信号放大检测方法，但该酶详细的表达纯化工艺尚无报道，并且活性难以准确测定，限制了其应用。通过合成嗜热古球菌Archaeoglobus fulgidus来源的FEN1基因序列，构建了pET24a(+)-FEN1-His重组质粒，并通过优化表达条件，得到了FEN1最优表达条件为：37 ℃、200 r/min振荡培养8 h后，加入诱导剂IPTG至终浓度为0.05 mmol/L，再于37 ℃、200 r/min诱导表达11 h，最终经镍亲和层析成功纯化得到了分子量约为38 kDa的重组FEN1。同时建立了基于荧光标记探针的FEN1活性测定方法，准确测定了重组FEN1的活性，为建立基于该酶的核酸检测方法提供了可靠的酶活力依据。最终将重组FEN1用于实时荧光PCR偶联高特异核酸侵入信号扩增法检测了乙醛脱氢酶2基因 (aldh2) 的基因型，得到了准确的分型结果，表明重组FEN1能用于基因多态性的分型检测中，为发展基于核酸侵入反应的核酸检测方法提供了可靠的工具酶。

Abstract:

Flap endonuclease 1 (FEN1) is an endonuclease that catalyzes invasive reaction. It can be used in signal-amplification reaction-based nucleic acid assay. However, the application of FEN1 is hampered due to the lack of detailed protocols to express and purify the enzyme, and to quantify the enzyme activity. In this paper, the DNA fragment coding the gene of FEN1 from Archaeoglobus fulgidus was synthesized, and inserted into the plasmid of pET24a(+) to express recombinant FEN1 with His-tag. After optimizing the expression, detailed expression protocol of FEN1 was obtained by culturing the recombinant E. coli at 37 ℃ with 200 r/min of shaking for 8 h, followed by inducing with 0.05 mmol/L IPTG at 37 ℃ for 11 h. The purified recombinant FEN1 with the molecular mass of 38 kDa was obtained by Ni-affinity chromatography. Moreover, we developed a accurate quantification method with fluorescence-labelled probes. Finally, the recombinant FEN1 was used in real-time PCR coupled with high specific invader assay for aldh2 gene genotyping to obtain the correct typing results, indicating that the recombinant FEN1 can be used in gene polymorphism detection. We provide a reliable enzyme for developing invasive reaction-based nucleic acid assay.