跨膜蛋白基因克隆载体pMBL的构建及验证

Construction and Verification of the Effectiveness of pMBL:A Cloning Vector of Exported Proteins Encoding Genes

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) ,以作为最终构建的载体克隆到带跨膜信号基因片段的报告基因。所设计的上游引物中依次带有分别处于 3个不同阅读框架、且形成匹配粘性末端的 3个酶切位点BglⅡ、BclⅠ、BamHⅠ ,以便最终构建的载体捕获基因时的对位融合和表达。pET 2 8经BglⅠ、Bst110 7Ⅰ双酶切 ,去除约 2 5kb的lacⅠ等基因的冗余片段 ,保留Kan抗性基因、复制原点、多克隆位点等结构 ,并消除BglⅡ位点 ,获得作为最终构建载体的抗性基因和基本骨架的过渡质粒pKan。△P△SPAmp基因经pGEM T EASY载体 ,克隆到pKan的EcoRⅠ和XbaⅠ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HindⅢ位点端EcoRⅠ位点的质粒 ,即得到用于跨膜蛋白信号基因片段捕获克隆的目的载体pMBL ,大小为 3 4 6kb。经酶切鉴定和测序 ,证明构建的载体与预期设计的一致。应用四环素抗性基因 (Tet)片段对构建的跨膜蛋白基因克隆载体pMBL的有效性进行了验证 ,在克隆入EcoRⅠ和BglⅡ双酶切 (0位 )的载体中 ,Kan和Amp双抗平板筛选到阳性克隆子 ,经酶切和测序均显示Tet基因已对位插入 ,并启动了 β

The β-lactamase was used as the reporter of expression and transmembrane secretion in this paper.A fragment of Amp resistance gene encoding the mature part of β-lactamase (△P△SP Amp,i.e.without promoter and signal peptide coding sequences) was amplified from pUC18 vector.The upstream primer has Bgl Ⅱ, Bcl Ⅰ, Bam HⅠ in three reading frame respectively,in order to in frame fuse and express target genes together with the downstream reporter in finally constructed vector.Meanwhile,pET-28 was digested with the restriction enzymes Bgl Ⅱ and Bst 1107Ⅰ.The 2.8 kb fragment with replication origin, Kan resistance gene and MCS was recovered,filled,self-ligated and resulted in a plasmid pKan-B.The Bgl Ⅱ site on pKan-B was then filled and the plasmid pKan was obtained.The △P△SP Amp gene,which was first cloned into pGEM-T-EASY vector,was inserted into pKan between Eco RⅠ and Xba Ⅰ sites.A plasmid pMBL-E was selected,with which the bacteria host could grew on Kan plate but not on plate with both Amp and Kan.An Eco RⅠ site beside Hin dⅢ on the plasmid pMBL-E was then filled,and the plasmid pMBL,a cloning vector of the exported proteins encoding genes was finally obtained.Both results of the restriction enzyme digestion and sequencing demonstrated the correctness of the construction.The Tet resistance gene,a transmemebrane protein encoding gene,was applied to verify the effectiveness of the reporter in the vector.Cut with Eco RⅠ and Bam HⅠ,a 375 bp fragment including promoter and 96 animo acids coding sequence (including signal peptide) of Tet was obtained from pBR322 vector.The fragment was then ligated to the vector pMBL which had been cut with both enzymes of Eco RⅠ and Bgl Ⅰ,or Eco RⅠ and Bcl Ⅰ,or Eco RⅠ and Bam HⅠ(as 0,+1,+2 respectively of the β-lactamase gene reading frame).Kan and Amp double resistant colonies only grew with the Eco RⅠ and Bgl Ⅱcombination (0 position).Restriction enzyme digestion and sequencing results of the recombinant plasmid showed that Tet resistance gene,which promoted the expression and transmembrane secretion of downstream β-lactamase,was inserted in a correct reading frame into the vector.Thus,the results verified the effectiveness of the constructed vector pMBL,which may be used effectively to clone genes encoding exported proteins with promoters and signal peptide sequences.