利用接头插入技术构建环型T载体(pYCQ1)

人工设计接头序列,其特点为含有3′突出A末端,同时人工接头序列内部含有XcmⅠ、BspM901、AceⅡ酶切位点。利用人工接头3′突出A与线性的pMD18-T载体的3′突出T连接,将线性的pMD18-T载体改造成环形具有自身复制功能的pYCQ1载体,降低了实验室使用T载体的成本,同时增加了载体的克隆位点。利用pYCQ1作为T载体时,用XcmⅠ酶切(XcmⅠ限制性片段具有3′T末端)pYCQ1载体可以得到线性的3′T粘性末端的pYCQ1-T载体。实验验证,pYCQ1-T载体可以在受体菌E.coliDH5α中进行大量自我复制,并能够克隆1 800 bp的外源PCR产物。

Construction of circular T vector (pYCQ1) by plunging an adaptor

The sequence of adaptor was artificially formed. The adaptor is characterized by 3' end with protruding A and it also contains restriction sites for enzymes Xcm Ⅰ ,Bsp M901 and Ace Ⅱ. With the ligation of adaptor 3' end protruding A with linear pMD18-T vector protruding T, linear pMDI8-T vector was transformed into circular pYCQ1 vector which had ability of self replication, thus reducing the costs of using T-vector, while adding more cloning sites. When the pYCQ1 vector was used as T-vector, it could be digested by Xcm Ⅰ, which an enzyme that has 3' end of T. The pYCQ1 vector was resulted in linear pYCQ-T-vector with 3' T sticky ends. In this experiment it was verified that 1 800 bp PCR product could be successfully cloned using pYCQ1-T vector. It was also verified that the pYCQ1 vector could be replicated by itself in E. coli DH5α.