高效定点突变肠道病毒71 VP1基因的研究
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摘要
[目的]肠道病毒71(Enterovirus 71,EV71)是引发手足口病(Hand-foot-mouth Disease,HFMD)的主要病原体之一,为了筛选EV71抗原表位,开发新型EV71疫苗,利用优化后的PCR点突变技术定点突变EV71 VP1基因。[方法]设计含有突变位点的互补引物,模板使用量为10 ng,进行单引物PCR,将PCR产物混合后复性,转化至大肠杆菌BL21,筛选阳性克隆并测序。[结果]测序结果表明VP1基因突变率为40%～45%。[结论]该方法对传统的PCR点突变技术进行改进,降低模板使用量,使用单引物PCR,省略DpnⅠ酶解过程,可达到40%～45%的定点突变率,从而降低实验成本、简化操作流程。
[Objective] Enterovirus 71( EV71) is major causative agents of hand,foot,and mouth diseases( HFMDs). On the basis of predecessors,the modified site-directed mutation technique was performed to screen the epitopes of EV71. [Methods]Firstly,the complementary primers containing mutation sites was designed and single primer PCR was performed,mixing the PCR products followed by renaturation. After transformation,the positive clones were screened and sequenced. [Results]The mutation rate was 40%-45%.[Conclusion]With the modification of traditional site-directed mutation which included the reduction of reduction of the template volumn and single primer PCR without the digestion of DpnⅠ,40%-45% efficiency was achieved. Therefore,this method was low cost and easy to be operated for the site-directed mutation of EV71 VP1.
[Objective] Enterovirus 71( EV71) is major causative agents of hand,foot,and mouth diseases( HFMDs). On the basis of predecessors,the modified site-directed mutation technique was performed to screen the epitopes of EV71. [Methods]Firstly,the complementary primers containing mutation sites was designed and single primer PCR was performed,mixing the PCR products followed by renaturation. After transformation,the positive clones were screened and sequenced. [Results]The mutation rate was 40%-45%.[Conclusion]With the modification of traditional site-directed mutation which included the reduction of reduction of the template volumn and single primer PCR without the digestion of DpnⅠ,40%-45% efficiency was achieved. Therefore,this method was low cost and easy to be operated for the site-directed mutation of EV71 VP1.
引文
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[12]王文波,聂建辉,宋爱京,等. HIV-1膜蛋白抗原表位突变对感染性以及中和特性的影响[J].中国病毒病杂志,2012,2(5):341-346.
[13]余兴龙,涂长春,徐兴然,等.猪瘟病毒E2基因的定点突变、在大肠杆菌中的高效表达及表达产物的免疫原性[J].生物工程学报,2003,19(4):439-443.
[14] Michael P. Weiner,Gina L. Costa,Warren Schoettlin. Site-directed mutagenesis of double stranded DNA by the polymerase chain reaction[J]. Gene,1994,151(1-2):119-123.
[15]裴智勇,侯仙慧,桂小柯,等. Primer Spanner:一个高效的定点突变PCR引物设计在线工具[J].中国生物工程杂志,2015,35(10):53-58.
[16]高瑞平,程隆斌,李振秋.一种简便快速的单引物PCR定点突变方法[J].中国生物工程杂志,2015,35(5):61-65.
[17] Liu Y,Wu T,Song J,et al. A mutant screening method by critical annealing temperature PCR for site-directed mutagenesis[J].BMC Biotechnology,2013,13(1):21.
[18]李国亮,钱伟,张淑江,等.利用改良SOE-PCR技术定点突变TuMVC4-VPg基因[J].中国蔬菜,2017,(10):39-44.
[19]黄建华,王晓姣,魏照辉,等.大肠杆菌中顺式-3-羟脯氨酸羟化酶的定向改造[J].食品与发酵工业,2017,43(01):7-11.
[20] Zheng L,Baumann U,Reymond JL:An efficient one-step sitedirected and site-saturation mutagenesis protocol[J]. Nucleic Acids Res.,2004,32(14):e115.
[21] Chapnik N,Sherman H,Froy O:A one-tube site-directed mutagenesis method using PCR and primer extension[J]. Anal Biochem.,2008,372(2):255-257.
[2] HO M,Chen E R,HSU K H,et al. An epidemic of enterovirus 71infection in Taiwan. Taiwan Enterovirus Epidemic Working Group[J]. The New England Journal of Medicine,1999,341(13):929
[3] Komatsu H,Shimizu Y,Takeuchi Y,et al. Outbreak of severe neurologic involvement associated with enterovirus 71 infection[J]. Pediatr Neurol,1999,20(1):17-23.
[4] Xu J,Qian Y,Wang SX,et al. EV71:An emerging infectious disease vaccine target in the Far East[J]. Vaccine,2010,28(20):3516-3521.
[5] Lee Minshi,Tseng F C,Wang J R,et al. Challenges to licensure of enterovirus 71 vaccines[J]. PLo S Negl Trop Dis,2012,6(8):e1737.
[6] Chang,L Y. Enterovirus 71 in Taiwan[J]. Pediatrics&Neonatology,2008,49(4):103-112.
[7] Lin W,Su Y,Jiang M,et al. Clinical features for 89 deaths of hand,foot and mouth disease in Guangxi,China,2014[J]. Int J Infect Dis.,2017,64(10):15-19.
[8] Sarma N,Chakraborty S,Dutta A,et al. Hand,foot and mouth disease in West Bengal,India:A preliminary report on clinicovirological trend over 3 successive years(2013-2015)[J]. Indian J Dermatol,2017,62(5):486-490.
[9] Oem J K,Lee K N,Cho I S,et al. Identification and antigenic site analysis of foot-and-mouth disease virus from pigs and cattle in korea[J]. J Vet Sci,2005,6(2):117-124.
[10] M. R. Ma,L. Hui,M. L. Wang,et al. Overall codon usage pattern of enterovirus 71[J]. Genetics and Molecular Research,2014,13(1):336-343.
[11] Ghochikyan A,Petrushina I,Davtyan H,Immunoge nicety of epitope vaccines targeting different B cell antigenic determinants of human alpha synuclein:feasibility study[J]. Neurosci Lett,2014,560(1):86-91.
[12]王文波,聂建辉,宋爱京,等. HIV-1膜蛋白抗原表位突变对感染性以及中和特性的影响[J].中国病毒病杂志,2012,2(5):341-346.
[13]余兴龙,涂长春,徐兴然,等.猪瘟病毒E2基因的定点突变、在大肠杆菌中的高效表达及表达产物的免疫原性[J].生物工程学报,2003,19(4):439-443.
[14] Michael P. Weiner,Gina L. Costa,Warren Schoettlin. Site-directed mutagenesis of double stranded DNA by the polymerase chain reaction[J]. Gene,1994,151(1-2):119-123.
[15]裴智勇,侯仙慧,桂小柯,等. Primer Spanner:一个高效的定点突变PCR引物设计在线工具[J].中国生物工程杂志,2015,35(10):53-58.
[16]高瑞平,程隆斌,李振秋.一种简便快速的单引物PCR定点突变方法[J].中国生物工程杂志,2015,35(5):61-65.
[17] Liu Y,Wu T,Song J,et al. A mutant screening method by critical annealing temperature PCR for site-directed mutagenesis[J].BMC Biotechnology,2013,13(1):21.
[18]李国亮,钱伟,张淑江,等.利用改良SOE-PCR技术定点突变TuMVC4-VPg基因[J].中国蔬菜,2017,(10):39-44.
[19]黄建华,王晓姣,魏照辉,等.大肠杆菌中顺式-3-羟脯氨酸羟化酶的定向改造[J].食品与发酵工业,2017,43(01):7-11.
[20] Zheng L,Baumann U,Reymond JL:An efficient one-step sitedirected and site-saturation mutagenesis protocol[J]. Nucleic Acids Res.,2004,32(14):e115.
[21] Chapnik N,Sherman H,Froy O:A one-tube site-directed mutagenesis method using PCR and primer extension[J]. Anal Biochem.,2008,372(2):255-257.