基于Eam1105Ⅰ酶切的p18K-T载体的构建
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摘要
TA克隆是PCR产物克隆最简单的方法,而T载体的改良对提高TA克隆效率、简化克隆片段后续的酶切、连接等实验步骤有着十分重要的意义。本研究以pMD18-T载体为基础,将原有的氨苄青霉素抗性筛选标记基因替换成卡那霉素抗性筛选标记基因,克服了原载体筛选抗性(氨苄青霉素)容易失活的问题,并在原有酶切位点的基础上增加了NsiⅠ、BsaⅠ、BglⅡ、NheⅠ和AclⅠ五个酶切位点,方便了克隆片段后续的酶切和连接操作,同时在XbaⅠ和BglⅡ之间引入了两个Eam1105I限制性核酸内切酶位点,构建成一个环状中间载体p18KT-M。利用Eam1105I酶切中间载体p18KT-M可以制备3'端带有T尾的线性化p18K-T载体。经TA克隆验证,p18K-T载体连接PCR产物的效率与原pMD18-T载体的效率相当。
TA cloning is the simplest method for PCR product cloning, and the improvement of T-Vector is of great significance for improving TA cloning efficiency and simplifying the experimental steps, such as subsequent digestion and ligation of cloned fragments. In this research, based on pMD18-T vector, the ampicin resistance screening marker gene was replaced by kanamycin resistance screening marker gene, which solved the problem of inactivation of the original vector in screening resistance(ampicillin). Meanwhile, on the basis of the original digestion sites, five restriction sites, NsiⅠ, BsaⅠ, BglⅡ, NheⅠ and AclⅠ, were added to facilitate the subsequent digestion and ligation of cloned fragments. Furthermore, two Eam1105Ⅰ restriction endonuclease sites were inserted between XbaⅠ and BglⅡ to construct a circular intermediate vector p18KT-M. The linearized p18K-T vector with a T-tail at 3' end could be prepared by digesting intermediate vector p18KT-M with Eam1105Ⅰ enzyme. The results of TA cloning indicated that the efficiency of p18K-T vector to connect PCR products was equal to that of pMD18-T vector.
TA cloning is the simplest method for PCR product cloning, and the improvement of T-Vector is of great significance for improving TA cloning efficiency and simplifying the experimental steps, such as subsequent digestion and ligation of cloned fragments. In this research, based on pMD18-T vector, the ampicin resistance screening marker gene was replaced by kanamycin resistance screening marker gene, which solved the problem of inactivation of the original vector in screening resistance(ampicillin). Meanwhile, on the basis of the original digestion sites, five restriction sites, NsiⅠ, BsaⅠ, BglⅡ, NheⅠ and AclⅠ, were added to facilitate the subsequent digestion and ligation of cloned fragments. Furthermore, two Eam1105Ⅰ restriction endonuclease sites were inserted between XbaⅠ and BglⅡ to construct a circular intermediate vector p18KT-M. The linearized p18K-T vector with a T-tail at 3' end could be prepared by digesting intermediate vector p18KT-M with Eam1105Ⅰ enzyme. The results of TA cloning indicated that the efficiency of p18K-T vector to connect PCR products was equal to that of pMD18-T vector.
引文
Arashi-Heese N.,Miwa M.,and Shibata H.,1999,XcmⅠsite-containing vector for direct cloning and in vitro transcription of PCR product,Mol.Biotechnol.,12(3):281-283
de Vries E.,1998,p UCPCR1.A vector for direct cloning of PCRproducts in a double Xcm1 restriction site offering compatible single 3'-overhanging T residues,Mol.Biotechnol.,10(3):273-274
Holton T.A.,and Graham M.W.,1991,A simple and efficient method for direct cloning of PCR products using dd T-tailed vectors,Nucleic Acids Res.,19(5):1156
Ichihara Y.,and Kurosawa Y.,1993,Construction of new T vectors for direct cloning of PCR products,Gene,130(1):153-154
Jo C.,and Jo S.A.,2001,A simple method to construct T-vectors using XcmⅠcassettes amplified by nonspecific PCR,Plasmid,45(1):37-40
Lin C.S.,Wu S.W.,and Yang D.Y.,2009,Development of T-vectors,Zhejiang Gongye Daxue Xuebao(Journal of Zhejiang University of Technology),37(6):623-628(林陈水,武胜伟,杨丹燕,2009,T载体研究进展,浙江工业大学学报,37(6):623-628)
Li M.T.,Tan W.Y.,Sun M.Y.,Li Y.G.,and Li W.B.,2015,Construction of TA cloning plant expression vector with herbicide resistance,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(11):2436-2440(李梦婷,谭文雍,孙铭阳,李永光,李文滨,2015,具有除草剂抗性的TA克隆植物表达载体的构建,基因组学与应用生物学,34(11):2436-2440)
Marchuk D.,Drumm M.,Saulino A.,and Collins F.S.,1991,Construction of T-vectors,a rapid and general system for direct cloning of unmodified PCR products,Nucleic Acids Res.,19(5):1154
Mead D.A.,Pey N.K.,Herrnstadt C.,Marcil R.A.,and Smith L.M.,1991,A universal method for the direct cloning of PCRamplified nucleic acid,Biotechnology(NY),9(7):657-663
Sun C.L.,Li Y.W.,Wang Y.,Gong T.,and Yi R.L.,2011,Construction of a p UC19-T vector based on XcmⅠ,Anhui Nongye Kexue(Journal of Anhui Agricultural Sciences),39(17):10182-10184(孙程龙,李业伟,王颖,宫婷,扈良,2011,基于XcmⅠ酶切的p UC19-T载体的构建,安徽农业科学,39(17):10182-10184)
Zhou M.Y.,and Gomez-Sanchez C.E.,2000,Universal TA cloning,Curr.Issues Mol.Biol.,2(1):1-7
de Vries E.,1998,p UCPCR1.A vector for direct cloning of PCRproducts in a double Xcm1 restriction site offering compatible single 3'-overhanging T residues,Mol.Biotechnol.,10(3):273-274
Holton T.A.,and Graham M.W.,1991,A simple and efficient method for direct cloning of PCR products using dd T-tailed vectors,Nucleic Acids Res.,19(5):1156
Ichihara Y.,and Kurosawa Y.,1993,Construction of new T vectors for direct cloning of PCR products,Gene,130(1):153-154
Jo C.,and Jo S.A.,2001,A simple method to construct T-vectors using XcmⅠcassettes amplified by nonspecific PCR,Plasmid,45(1):37-40
Lin C.S.,Wu S.W.,and Yang D.Y.,2009,Development of T-vectors,Zhejiang Gongye Daxue Xuebao(Journal of Zhejiang University of Technology),37(6):623-628(林陈水,武胜伟,杨丹燕,2009,T载体研究进展,浙江工业大学学报,37(6):623-628)
Li M.T.,Tan W.Y.,Sun M.Y.,Li Y.G.,and Li W.B.,2015,Construction of TA cloning plant expression vector with herbicide resistance,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(11):2436-2440(李梦婷,谭文雍,孙铭阳,李永光,李文滨,2015,具有除草剂抗性的TA克隆植物表达载体的构建,基因组学与应用生物学,34(11):2436-2440)
Marchuk D.,Drumm M.,Saulino A.,and Collins F.S.,1991,Construction of T-vectors,a rapid and general system for direct cloning of unmodified PCR products,Nucleic Acids Res.,19(5):1154
Mead D.A.,Pey N.K.,Herrnstadt C.,Marcil R.A.,and Smith L.M.,1991,A universal method for the direct cloning of PCRamplified nucleic acid,Biotechnology(NY),9(7):657-663
Sun C.L.,Li Y.W.,Wang Y.,Gong T.,and Yi R.L.,2011,Construction of a p UC19-T vector based on XcmⅠ,Anhui Nongye Kexue(Journal of Anhui Agricultural Sciences),39(17):10182-10184(孙程龙,李业伟,王颖,宫婷,扈良,2011,基于XcmⅠ酶切的p UC19-T载体的构建,安徽农业科学,39(17):10182-10184)
Zhou M.Y.,and Gomez-Sanchez C.E.,2000,Universal TA cloning,Curr.Issues Mol.Biol.,2(1):1-7
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