基于Eam1105Ⅰ酶切的p18K-T载体的构建

Construction of p18K-T Vector Based on Digestion by Eam1105Ⅰ

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,NheⅠand AclⅠ,were added to facilitate the subsequent digestion and ligation of cloned fragments.Furthermore,two Eam1105Ⅰrestriction endonuclease sites were inserted between XbaⅠand BglⅡto 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 Eam1105Ⅰenzyme.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.