【摘要】 目的保留重组质粒pMD18-T-ldh中变形链球菌ldh基因上下游同源区,用运动发酵单胞菌adhB基因置换ldh基因构建重组质粒。方法PCR法扩增adhB基因,克隆到PGEM-T载体,构建pGEMA重组质粒,以pMD18-T-ldh重组质粒为模板,反向PCR法缺失ldh,Nco I和Xho I酶产物片段和pGEMA质粒,连接得到重组质粒pMDLA。结果成功克隆adhB基因,并用该基因置换ldh基因构建重组质粒pMDLA。结论本实验成功将反向PCR法引入缺失突变诱导,丰富了运动发酵单胞菌在医学领域的应用。更多还原

【Abstract】 Objective To construct a recombinant plasmid through substituting the adhB gene of Zymomonas mobilis for the ldh gene of Streptococcus mutans with a reservation of upstream and downstream homology region of ldh in pMD18-T-ldh.Methods The adhB gene was amplified from total DNA of Zymomonas mobilis by PCR and was cloned in to pGEMT vector.The entire cloned ldh gene of Streptococcus mutans in pMD18-T-ldh was deleted by inverse PCR.Restriction enzymes NcoI and XhoI were used to digest the DNA fragment obtained from inverse PCR amplification.To acquire recombinant plasmid pMDLA,recombinant plasmid pGEMA and ligate the two fragments were performed.Results The adhB was successfully cloned and sequenced.The adh was replaced by adhB.Recombiant plasmids pGEMTA and pMDLA were constructed.Conclusion Results of the study expanded the possible therapeutic use of Zymomonas mobilis;and demonstrated that inverse PCR technique is a quickly and easily method in inducing gene-deletion mutation.更多还原