DHA-1型β-内酰胺酶基因克隆及其结构

The Cloning of the DHA-1 Tape β-lactamase’s Gene and Its Structure

为了克隆多重耐药肺炎克雷伯菌质粒介导的DHA-1型β-内酰胺酶并探测其序列的结构特征,抽提接合菌的质粒,用限制性内切酶HindIII消化,酶切粘末端用Taq酶补平并在末端加单个的脱氧腺苷(A),与pGEM-TEasy载体进行T-A克隆.在含氨苄西林和头孢西丁的平板上筛选重组菌,抽提重组质粒,酶切分析,克隆片段用步移法测序.通过以上方法,克隆筛选到含5.2kb插入片段的重组质粒pT948,插入片段测序发现其含有一个DHA-1基因和一个ampR基因,依次在DHA-1结构基因的侧翼发现一个插入序列IS26.因此,可以认为成功克隆了质粒介导的DHA-1基因,并在其相邻序列中存在插入序列IS26.

In order to clone DHA - 1 gene from the plasmid of multiple - drug resistance Klebsiella pneumoniae producing plasmid- mediated β- lactamase and investigate its sequence structure characteristic, the plasmids of transconjugant is extracted and digested with restriction endonuclease HindIII, Taq DNA polymerase is applied to fill the recessed 3＇ termini, and a single deoxyadenosine is added to the 3 termimi of fragments. Then these fragments are ligated with pGEM - T Easy vector.E, coli DH5α containing cloning vectors are selected on Mac - Conkey agar plates with ampicillin and cefoxitin. Cloned fragments are Primer walking sequences. Recombinant plasmid pT948 containing a 5.2 - kb insert is obtained. The insert fragment contains a structural gene DHA - 1 and a regulatory gene ampR, insertion sequence （IS26） element. The plasmid -mediated ampC gene cloned was identified as blaDHA-1. IS26 observed on the flanks of the blaDHA-1 may be concerne（t with the translocation of blaDHA-1 gene region from the chromosome to plasmid.