(2.南京军区南京总医院药理科, 江苏南京 210002)
(3.南京军区南京总医院药理科, 江苏南京 210009)
(4.Department of Pharmacology,Nanjing General Hospital, Nanjing ,Jiangsu,China 210002)
【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p ET24a(+)-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 k Da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。
【基金资助】 国家科技重大专项(No.2013ZX10004103); 江苏省临床科技专项(No.BL2012061); 国家自然科学基金(Nos.31300704,21475151,21405176,21275161); 中国博士后基金特别资助项目基金(Nos.2013T60938,2014T71011); 江苏省六大人才高峰项目(No.2015-WSN-085)资助;
Expression and activity assay of recombinant flap endonuclease 1
(2.Department of Pharmacology, Nanjing General Hospital, Nanjing, Jiangsu, China 210002)
(3.Department of Pharmacology, Nanjing General Hospital, Nanjing, Jiangsu, China 210009)
【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 °C with 200 r/min of shaking for 8 h, followed by inducing with 0.05 mmol/L IPTG at 37 °C 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.
【Keywords】 flap endonuclease 1; signal amplification; activity assay;
【Funds】 National Science and Technology Major Project of the Ministry of Science and Technology of China (No. 2013ZX10004103); Jiangsu Province's Clinical Science & Technology Special Project (No. BL2012061); National Natural Science Foundation of China (Nos. 31300704, 21475151, 21405176, 21275161); China Postdoctoral Science Special Foundation (Nos. 2013T60938, 2014T71011); Six Talent Peaks Project in Jiangsu Province (No. 2015-WSN-085);
 Kaiser MW, Lyamicheva N, Ma W, et al. A comparison of eubacterial and archaeal structure-specific 5'-exonucleases. J Biol Chem, 1999, 274 (30): 21387–21394.
 Feng M, Patel D, Dervan JJ, et al. Roles of divalent metal ions in flap endonuclease-substrate interactions. Nat Struct Mol Biol, 2004, 11 (5): 450–456.
 Oh H, Smith CL. Evolving methods for single nucleotide polymorphism detection: factor V leiden mutation detection. J Clin Lab Anal, 2011, 25 (4): 259–288.
 Ginocchio CC, Barth D, Zhang F. Comparison of the third wave invader human papillomavirus (HPV) assay and the digene HPV hybrid capture 2 assay for detection of high-risk HPV DNA. J Clin Microbiol, 2008, 46 (5): 1641–1646.
 Hall JG, Eis PS, Law SM, et al. Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction. Proc Natl Acad Sci USA, 2000, 97 (15): 8272–8277.
 Takanashi M, Ito S, Kaneto H, et al. Development and clinical application of an Invader Plus® assay for the detection of genital mycoplasmas. J Infect Chemother, 2015, 21 (7): 157–164.
 Tang YW, Allawi HT, De Leon-Carnes M, et al. Detection and differentiation of wild-type and vaccine mutant varicella-zoster viruses using an Invader Plus® method. J Clin Virol, 2007, 40 (2): 129–134.
 Kan T, Hashimoto S, Kawabe N, et al. The clinical features of patients with a Y93H variant of hepatitis C virus detected by a PCR invader assay. J Gastroenterol, 2016, 51 (1): 63–70.
 Nie B, Shortreed MR, Smith LM. Scoring single-nucleotide polymorphisms at the single-molecule level by counting individual DNA cleavage events on surfaces. Anal Chem, 2005, 77 (20): 6594–6600.
 Zou BJ, Ma YJ, Wu HP, et al. Ultrasensitive DNA detection by cascade enzymatic signal amplification based on Afu flap endonuclease coupled with nicking endonuclease. Angew Chem Int Ed, 2011, 50 (32): 7395–7398.
 Zou BJ, Song QX, Wang JP, et al. Invasive reaction assisted strand-displacement signal amplification for sensitive DNA detection. Chem Commun (Camb), 2014, 50 (89): 13722–13724.
 Zou BJ, Cao XM, Wu HP, et al. Sensitive and specific colorimetric DNA detection by invasive reaction coupled with nicking endonucleaseassisted nanoparticles amplification. Biosens Bioelectron, 2015, 66: 50–54.
 Kani S, Tanaka Y, Matsuura K, et al. Development of new IL28B genotyping method using invader plus assay. Microbiol Immunol, 2012, 56 (5): 318–323.
 Oler AT, Attie AD. A rapid, microplate SNP genotype assay for the leptinob allele. J Lipid Res, 2008, 49 (5): 1126–1129.
 Boonyarit H, Mahasirimongkol S, Chavalvechakul N, et al. Development of a SNP set for human identification: a set with high powers of discrimination which yields high genetic information from naturally degraded DNA samples in the Thai population. Forensic Sci Int Genet, 2014, 11 (4): 166–173.
 Tadokoro K, Kobayashi M, Suzuki F, et al. Comparative quantitative analysis of hepatitis C mutations at amino acids 70 and 91 in the core region by the Q-Invader assay. J Virol Methods, 2013, 189 (1): 221–227.
 Tadokoro K, Suzuki F, Kobayashi M, et al. Rapid detection of drug-resistant mutations in hepatitis B virus by the PCR-Invader assay. J Virol Methods, 2011, 171 (1): 67–73.
 Fujikura J, Nakao K, Sone M, et al. Induced pluripotent stem cells generated from diabetic patients with mitochondrial DNA A3243G mutation. Diabetologia, 2012, 55 (6): 1689–1698.
 Mabuchi H, Nohara A, Noguchi T, et al. Genotypic and phenotypic features in homozygous familial hypercholesterolemia caused by proprotein convertase subtilisin/kexin type 9 (PCSK9) gain-of-function mutation. Atherosclerosis, 2014, 236 (1): 54–61.
 Wong AK, Chan RC, Nichols WS, et al. Invader human papillomavirus (HPV) type 16 and 18 assays as adjuncts to HPV screening of cervical papanicolaou smears with atypical squamous cells of undetermined significance. Cancer, 2009, 115 (4): 823–832.
 Wong DK, Yuen MF, Yuan H, et al. Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients. Hepatology, 2004, 40 (3): 727–737.
 Hjertner B, Meehan B, Mc Killen J, et al. Adaptation of an Invader® assay for the detection of African swine fever virus DNA. J Virol Methods, 2005, 124 (1/2): 1–10.
 Allawi HT, Dahlberg JE, Olson S, et al. Quantitation of micro RNAs using a modified Invader assay. RNA, 2004, 10 (7): 1153–1161.
 Tian H, Cao L, Tan Y, et al. Multiplex m RNA assay using electrophoretic tags for highthroughput gene expression analysis. Nucl Acids Res, 2004, 32 (16): 219–231.
 Wang JP, Zou BJ, Chen ZY, et al. Characterization of recombinant single-stranded DNA-binding protein from Escherichia coli and its application in accurate pyrosequencing. Chin J Biotech, 2011, 27 (10): 1513–1520 (in Chinese with English abstract).
 Xu S, Zou BJ, Wang JP, et al. Expression of thermostable recombiant Luciola lateralis luciferase and development of heat-stable pyrosequencing system. Chin J Biotech, 2012, 28 (6): 763–771 (in Chinese with English abstract).
 Zheng ML, Qi XM, Tong H, et al. Detection of single nucleotide polymorphism genotyping by real-time polymerase chain reaction coupled with high specific invader assay in single tube. Chin J Anal Chem, 2015, 43 (7): 1001–1008 (in Chinese with English abstract).