捕获输出蛋白编码基因的小分子量质粒载体的构建与验证

Construction of a low molecular weight plasmid vector for the capture of genes encoding exported proteins.

自行设计一对引物,从质粒pUC18上扩增一段无启动子和信号肽的β-内酰胺酶基因(△P△SPAmp),经pGEM-T-EASY载体克隆到pET-28衍生质粒pKan的EcoR 和Xba 间,得到在Kan平板中生长而在Amp和Kan双抗平板中不能生长的转化子pMBL-E质粒.经部分酶切补平自连,筛选得到消除Hind 位点端EcoR 位点的质粒,即为用于输出信号基因片段捕获的目的载体pMBL,大小为3.46kb.经酶切鉴定和测序分析,表明构建的载体与预期设计的一致,并应用四环素抗性基因(Tetr)对载体的有效性进行了验证,证明构建的载体pMBL能有效捕获含启动子和信号肽序列的输出蛋白编码基因.

A pair of primers were designed to amplify a fragment of AMP gene, encoding merely mature part of β-lactamase with no promoter and signal peptide (△P△SP Amp), from pUC18. The gene was cloned into pGEM-T-EASY vector, and cut with EcoRⅠ. The recovered △P△SP Amp gene was then cloned and inserted between EcoRⅠ and XbaⅠ sites of plasmid pKan, derived from plasmid pET-28. Thus it resulted in a plasmid pMBL-E, with which the host could grow on plate with Kan but not with both Amp and Kan. The plasmid pMBL-E was then partially digested with EcoRⅠ, filled and self-ligated. A vector pMBL, about 3.46 kb in size, for the capture of the genes encoding exported proteins was finally selected, in which the EcoRⅠ site beside HindⅢ site on pMBL-E was deleted. Both results of the restriction enzyme digestion and sequencing demonstrated the correctness of the construction. The effectiveness of the vector was verified, using a 375 bp Tet^r gene fragment of pBR322, which showed that the pMBL vector would effectively clone genes encoding exported proteins with promoters and signal peptide sequences.