Klenow(exo-)蛋白的制备及其在RAA检测体系中的应用研究
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摘要
为了建立一种Klenow(exo-)蛋白的制备方法,并对其在RAA(recombinase-aid amplification)技术中的应用进行初步研究,通过Overlap点突变法构建获得目的基因片段,并构建Klenow(exo-)蛋白表达载体进而转化至大肠杆菌BL21(DE3)菌株进行诱导表达。表达产物通过Ni-NTA螯合层析进行初步纯化,然后用Heparine柱层析进一步纯化,通过SDS-PAGE鉴定纯化产物,获得了纯度较高的目的蛋白。用纯化后的目的蛋白配制RAA反应体系,针对肉源实际样本设计突变位点进行检测。实验结果显示该体系具有较强的特异性,在模板基因序列3′端出现双碱基差异时不会进行有效扩增,相较于常规的PCR检测方式该体系能够实现双碱基差异位点的有效区分。该技术未来有望大规模应用于SNPs的检测。
A preparation method of Klenow(exo-) protein was established and its application in RAA technology was preliminary studied. The target gene fragment was constructed by the Overlap point mutation method, and the Klenow(exo-) protein expression vector was constructed and transformed into Escherichia coli BL21(DE3) strain to induce expression. The expressed product was subjected to preliminary purification by Ni-NTA chelate chromatography, followed by Heparine column chromatography for further purification, and the purified product was identified by SDS-PAGE. A protein of higher purity was obtained. The purified RAA reaction system was prepared using the purified target protein, and the mutation site was designed for the actual sample of the meat source. The experimental results showed that the system has strong specificity and don't amplified when there is a double base difference at the 3′ end of the template gene sequence, compared with the conventional PCR detection method, the system could effectively distinguish double base mutation. The system was expected to applied in detection of SNPs in the future.
A preparation method of Klenow(exo-) protein was established and its application in RAA technology was preliminary studied. The target gene fragment was constructed by the Overlap point mutation method, and the Klenow(exo-) protein expression vector was constructed and transformed into Escherichia coli BL21(DE3) strain to induce expression. The expressed product was subjected to preliminary purification by Ni-NTA chelate chromatography, followed by Heparine column chromatography for further purification, and the purified product was identified by SDS-PAGE. A protein of higher purity was obtained. The purified RAA reaction system was prepared using the purified target protein, and the mutation site was designed for the actual sample of the meat source. The experimental results showed that the system has strong specificity and don't amplified when there is a double base difference at the 3′ end of the template gene sequence, compared with the conventional PCR detection method, the system could effectively distinguish double base mutation. The system was expected to applied in detection of SNPs in the future.
引文
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[19] 庞建虎,乔龙亮,黄海龙,等.重组酶聚合酶扩增技术快速检测鳗利斯顿氏菌[J].农业生物技术学报,2018,26(6):1056-1063.
[2] 吕蓓,程海荣,严庆丰,等.用重组酶介导扩增技术快速扩增核酸[J].中国科学:生命科学,2010,40(10):983-988.
[3] 于凌云,白俊杰,叶星,等.大口黑鲈MyoD基因结构和单核苷酸多态性位点的筛选[J].水产学报,2009,33(1):1-8.
[4] 许家磊,王宇,后猛,等.SNP检测方法的研究进展[J].分子植物育种,2015,13(2):475-482.
[5] 寇晓进.高保真DNA聚合酶分子开关用于TP53基因热点突变的检测[D].江苏苏州:苏州大学,硕士学位论文,2013.
[6] Freemont P S,Ollis D L,Steitz T A,et al..A domain of the Klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity [J].Proteins Struct.Funct.Bioinform.,1986,1(1):66-73.
[7] Horton R M,Zeling C,Ho S N,et al..Gene splicing by overlap extension:Tailor-made genes using the polymerase chain reaction[J].BioTechniques,1990,8(5) 528-535.
[8] Hardy C D,Martin P K.Isothermal SNP Detection Method:,US 20080118917 A1[P].2008.
[9] Sherry S T,Ward M,Sirotkin K.Use of molecμLar variation in the NCBI dbSNP database[J].Human Mutation,2015,15(1):68-75.
[10] 张佳,廖端芳,张旭,等.高保真DNA聚合酶在SNP检测中的应用[J].中南医学科学杂志,2003,31(2):128-131.
[11] 周翠兰.高保真DNA聚合酶介导的复合分子“开/关”技术平台在Y染色体识别中的应用[D].湖南衡阳:南华大学,硕士学位论文,2006.
[12] Beese L,Derbyshire V,Steitz T.Structure of DNA polymerase I Klenow fragment bound to duplex DNA[J].Science,1993,260(5106):352-355.
[13] Pandey V N,Kaushik N,Sanzgiri R P,et al..Site directed mutagenesis of DNA polymerase I (Klenow) from Escherichia coli[J].FEBS J.,1993,214(1):59-65.
[14] Derbyshire V,Freemont P S,Sanderson M R,et al..Genetic and crystallographic studies of the 3′,5′-exonucleolytic site of DNA polymerase I [J].Science,1988,240(4849):199-201.
[15] Joyce C M,Derbyshire V.Purification of Escherichia coli,DNA polymerase I and Klenow fragment[J].Methods Enzymol.,1995,262(95):3.
[16] 蒋宗英,邹秉杰,刘云龙,等.Klenow(exo-)的表达、活性测定及其在焦磷酸测序中的应用[J].药物生物技术,2016(3):201-207.
[17] Cargill M,Altsh L D,Ireland J,et al..Characterization of single-nucleotide polymorphisms in coding regions of human genes.[J].Nat.Genet.,1999,22(3):231.
[18] Ahmed F A,Larrea-Sarmiento A,Alvarez A M,et al..Genome-informed diagnostics for specific and rapid detection of Pectobacterium species using recombinase polymerase amplification coupled with a lateral flow device[J].Sci.Rep.,2018,8(1):1-11.
[19] 庞建虎,乔龙亮,黄海龙,等.重组酶聚合酶扩增技术快速检测鳗利斯顿氏菌[J].农业生物技术学报,2018,26(6):1056-1063.
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