EV713D聚合酶基因体外重组质粒的构建、表达及活性检测
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摘要
目的构建编码肠道病毒71型(EV71)3D聚合酶基因的重组质粒,表达和纯化3D聚合酶并检测其活性。方法将编码3D聚合酶的基因片段克隆至pGEX-4T-1原核表达载体,重组质粒转化入大肠埃希菌BL21(DE3),IPTG诱导表达,经谷胱甘肽琼脂糖亲和层析、TEV酶酶切、Ni-NTA柱亲和纯化后获得高纯度3D聚合酶蛋白,放射测量法是体外酶活力测定方法中较常见的一种方法,本研究通过测定插入poly(U) RNA放射性标志UMP的量确定3D聚合酶的活性。结果经酶切、PCR、测序鉴定pGEX-4T-3D重组质粒构建成功,3D聚合酶基因片段大小为1 386 bp。与未诱导相比,经IPTG诱导后带有GST标签的3D聚合酶成功表达,表达产物的相对分子质量约为79×10~3,TEV酶酶切掉GST标签后的相对分子质量约为55×10~3。纯化的3D聚合酶经体外延伸反应后跑变性尿素聚丙烯酰胺凝胶电泳得知,3D聚合酶溶解液DMSO组相比于3D聚合酶强抑制剂EDTA组,在DMSO组中,可以观察到强电泳条带,表明放射性同位素标志程度强,表明反应速率快,即3D聚合酶酶活较好。结论构建了EV71 3D聚合酶基因重组质粒,其表达产物3D聚合酶为以EV71 3D聚合酶为靶点的抗EV71药物筛选奠定基础。
Objective To construct a recombinant plasmid encoding the 3 D polymerase gene of enterovirus 71(EV71) in order to express and purify 3 D polymerase and evaluate its activity. Methods A DNA fragment encoding 3 D polymerase was cloned into a pGEX-4 T-1 prokaryotic expression vector. The recombinant plasmid was transformed into E. coli BL21(DE3), and protein expression was induced with IPTG. The expressed protein was purified using affinity chromatography with glutathione agarose, enzyme digestion with TEV, and affinity chromatography(Ni-NTA). Highly pure 3 D polymerase was obtained, and its functional activity was evaluated by measuring the amount of UMP inserted into poly(U) RNA. Radiometry is a common method for the determination of enzyme activity in vitro. Results Restriction analysis and sequencing verified that the recombinant plasmid pGEX-4 T-3 D was successfully constructed. The 3 D polymerase gene fragment was 1,386 bp in length. After expression was induced with IPTG, 3 D polymerase with a GST tag was successfully expressed in comparison to the untreated group. The expressed product was about 79×10~3 in size. When the GST tag was removed with the TEV enzyme, the purified 3 C polymerase was about 55×10~3 in size. An extension reaction in vitro and denatured urea polyacrylamide gel electrophoresis revealed that 3 D polymerase in the presence of the solvent DMSO produced strong electrophoresis bands in comparison to 3 D polymerase in the presence of the strong inhibitor EDTA, indicating that the amount of radioisotopes was high and the reaction rate was fast, that is, 3 D polymerase had better enzyme activity. Experimental results indicated that purified 3 D polymerase has polymerase activity. Conclusion The recombinant plasmid pGEX-4 T-3 D was created and 3 D polymerase with a high level of functional activity was successfully prepared. This polymerase can be used not only to study the function and mechanism of action of EV71 and also to screen and design anti-EV71 drugs targeting 3 D polymerase.
Objective To construct a recombinant plasmid encoding the 3 D polymerase gene of enterovirus 71(EV71) in order to express and purify 3 D polymerase and evaluate its activity. Methods A DNA fragment encoding 3 D polymerase was cloned into a pGEX-4 T-1 prokaryotic expression vector. The recombinant plasmid was transformed into E. coli BL21(DE3), and protein expression was induced with IPTG. The expressed protein was purified using affinity chromatography with glutathione agarose, enzyme digestion with TEV, and affinity chromatography(Ni-NTA). Highly pure 3 D polymerase was obtained, and its functional activity was evaluated by measuring the amount of UMP inserted into poly(U) RNA. Radiometry is a common method for the determination of enzyme activity in vitro. Results Restriction analysis and sequencing verified that the recombinant plasmid pGEX-4 T-3 D was successfully constructed. The 3 D polymerase gene fragment was 1,386 bp in length. After expression was induced with IPTG, 3 D polymerase with a GST tag was successfully expressed in comparison to the untreated group. The expressed product was about 79×10~3 in size. When the GST tag was removed with the TEV enzyme, the purified 3 C polymerase was about 55×10~3 in size. An extension reaction in vitro and denatured urea polyacrylamide gel electrophoresis revealed that 3 D polymerase in the presence of the solvent DMSO produced strong electrophoresis bands in comparison to 3 D polymerase in the presence of the strong inhibitor EDTA, indicating that the amount of radioisotopes was high and the reaction rate was fast, that is, 3 D polymerase had better enzyme activity. Experimental results indicated that purified 3 D polymerase has polymerase activity. Conclusion The recombinant plasmid pGEX-4 T-3 D was created and 3 D polymerase with a high level of functional activity was successfully prepared. This polymerase can be used not only to study the function and mechanism of action of EV71 and also to screen and design anti-EV71 drugs targeting 3 D polymerase.
引文
[1] Yan L,Zheng Z,Bo S,et al.SUMO modification stabilizes enterovirus 71 polymerase 3D to facilitate viral replication[J].J Virol,2016,90(23):10472.
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[7] Chen TC,Chang HY,Lin PF,et al.Novel antiviral agent DTriP-22 targets RNA-dependent RNA polymerase of enterovirus 71[J].Antimicrob Agents Chemother,2009,53(7):2740-7.
[8] Li X,Liu Y,Wu T,et al.The antiviral effect of baicalin on enterovirus 71 in vitro[J].Viruses,2015,7(8):4756-71.
[9] Sun Y,Wang Y,Shan C,et al.Enterovirus 71 VPg uridylation uses a two-molecular mechanism of 3D polymerase[J].J Virol,2012,86(24):13662-71.
[10] 张玲莉,张星亮.线粒体自噬受体Bnip3的载体构建及蛋白表达纯化[J].生物技术,2015,25(4):315-9.
[11] 肖亚飞.肿瘤抑制蛋白APC与Amer1的结合机理研究[D].上海:上海交通大学,2014.
[12] 安贤惠,周秀芬,汪志军,等.沙门氏菌硫修饰基因dptC的克隆表达与分离纯化[J].微生物学报,2013,53(10):1111-6.
[13] 张爱宏,刘国庆.放射性同位素标记法检测脂蛋白脂肪酶活性及其应用[J].中国动脉硬化杂志,2003,11(6):573-5.
[14] Zahner D,Sener A,Malaisse WJ.Radioisotopic measurement of femtomolar amounts of NAD(P)H in the assay of enzymatic activity at a single cell level[J].BBA Molecul Cell Res,1990,1053(2):125-9.
[15] 王喜萍,刘望夷,王鸣歧.放射性同位素标记测定RNA N-糖苷酶活性的新方法[J].生物化学与生物物理进展,1990,17(6):55.
[16] 李玥,李会娟,高芳,等.蛋白激酶的生化活性检测方法研究进展[J].生命的化学,2014,34(5):636-8.
[2] Victorio CB,Xu Y,Ng Q,et al.Phenotypic and genotypic characteristics of novel mouse cell line (NIH/3T3)-adapted human enterovirus 71 Strains (EV71:TLLm and EV71:TLLmv)[J].Plos One,2014,9(3):e92719.
[3] Xing WJ,Liao QH,Cecile V,et al.Hand,foot,and mouth disease in China,2008-12:an epidemiological study[J].Lancet Infect Dis,2014,14(4):308-18.
[4] Velu AB,Chen GW,Hsieh PT,et al.BPR-3P0128 inhibits RNA-dependent RNA polymerase elongation and VPg uridylylation activities of Enterovirus 71[J].Antiviral Res,2014(112):18-25.
[5] Xiao XU,Yao Y,Jing LI,et al.Identification of the transcriptional activity domain of EV71 3D(pol)[J].Chin J Virol,2016:560-5.
[6] Hung HC,Chen TC,Fang MY,et al.Inhibition of enterovirus 71 replication and the viral 3D polymerase by aurintricarboxylic acid[J].J Antimicrob Chemother,2010,65(4):676-83.
[7] Chen TC,Chang HY,Lin PF,et al.Novel antiviral agent DTriP-22 targets RNA-dependent RNA polymerase of enterovirus 71[J].Antimicrob Agents Chemother,2009,53(7):2740-7.
[8] Li X,Liu Y,Wu T,et al.The antiviral effect of baicalin on enterovirus 71 in vitro[J].Viruses,2015,7(8):4756-71.
[9] Sun Y,Wang Y,Shan C,et al.Enterovirus 71 VPg uridylation uses a two-molecular mechanism of 3D polymerase[J].J Virol,2012,86(24):13662-71.
[10] 张玲莉,张星亮.线粒体自噬受体Bnip3的载体构建及蛋白表达纯化[J].生物技术,2015,25(4):315-9.
[11] 肖亚飞.肿瘤抑制蛋白APC与Amer1的结合机理研究[D].上海:上海交通大学,2014.
[12] 安贤惠,周秀芬,汪志军,等.沙门氏菌硫修饰基因dptC的克隆表达与分离纯化[J].微生物学报,2013,53(10):1111-6.
[13] 张爱宏,刘国庆.放射性同位素标记法检测脂蛋白脂肪酶活性及其应用[J].中国动脉硬化杂志,2003,11(6):573-5.
[14] Zahner D,Sener A,Malaisse WJ.Radioisotopic measurement of femtomolar amounts of NAD(P)H in the assay of enzymatic activity at a single cell level[J].BBA Molecul Cell Res,1990,1053(2):125-9.
[15] 王喜萍,刘望夷,王鸣歧.放射性同位素标记测定RNA N-糖苷酶活性的新方法[J].生物化学与生物物理进展,1990,17(6):55.
[16] 李玥,李会娟,高芳,等.蛋白激酶的生化活性检测方法研究进展[J].生命的化学,2014,34(5):636-8.