应用体外重组PCR技术构建Ppdc-ldh融合基因

Construction of Ppdc-ldh funsion gene by recombinant PCR in vitro

利用重组PCR技术连接分别从干酪乳杆菌(Lactobacillus casei)ATCC334中扩增的乳酸脱氢酶基因(ldh)和运动发酵单胞菌(Zymomonas mobilis)CP4中扩增的丙酮酸脱羧酶基因启动子(Ppdc)片段,构建了Ppdc-ldh融合基因。并将其克隆到pMD19-T载体上,再转化到大肠杆菌DH5α中,得到阳性克隆质粒pT-Ppdc-ldh。序列分析表明该克隆具有正确的基因序列和阅读框,具有起始密码子GTG和终止密码子TAA,启动子的-35区和-10区序列与起始密码子之间的距离没有改变,可以插入到原核表达载体中进行表达。

The 5′ terminus of ldh gene from Lactobacillus casei ATCC334 was fused to the 3′terminus of pdc gene promoter （Ppdc） locus from Zymomonas mobilis CP4 by recombinant PCR to construct Ppdc-ldh funsion gene. The Ppdc-ldh gene was cloned into pMD19-T vector to obtain pT- Ppdc-ldh plasmid, which was transformed into Escherichia coli DH5α for screening. According to sequence analysis, the Ppdc-ldh fragment was successful inserted into pMD19-T with correct reading frame. The initiation codon and termination codon of Ppdc-ldh gene were GTG and TAA, respectively. The distance from-35 or-10 sequences of Ppdc promoter to initiation codon was unchanging. Therefore, the plasmid pT-Ppdc-ldh can be expressed in prokaryotic hosts.