牙龈卟啉单胞菌PG0839基因突变株的构建

Construction PG0839 gene-defective mutant of Porphyromonas gingivalis

目的构建牙龈卟啉单胞菌毒力岛基因PG0839突变菌株,为研究PG0839基因功能提供实验基础。方法扩增1 584 bp PG0839基因片段,对聚合酶链反应(PCR)产物和pUC19载体进行BamHⅠ和EcoRⅠ双酶切,连接酶切产物得到质粒pPG0839-1。将2 101 bp erm基因产物插入到pPG0839-1中PG0839基因的EcoRⅤ位点,构建质粒pPG0839-2,作为电穿孔的供体质粒。电穿孔转化于受体菌牙龈卟啉单胞菌W83菌株,红霉素抗性培养基筛选阳性克隆,命名为PG0839基因突变菌株。结果运用插入失活方法构建PG0839基因突变菌株,进而通过酶切、测序、PCR和反转录PCR对PG0839基因突变菌株进行验证,证实PG0839基因突变菌株构建成功。结论本实验成功构建PG0839基因突变菌株。

Objective In order to determine the function of PG0839 gene from Porphyromonas gingivalis（P.gingivalis） W83 strains,we intended to create a mutant in the PG0839 gene by homologous recombination.Methods 1 584 bp PG0839 gene fragment was amplified,digested by BamH Ⅰ and EcoR Ⅰ,purified and ligated to pUC19.The recombinant plasmid was designated as pPG0839-1.The erm cassette（2 101 bp） was inserted into the EcoR Ⅴ restriction site of the PG0839 gene.The resultant recombinant plasmid,pPG0839-2,was used as a donor in the electropo-ration of P.gingivalis W83.After electroporated and selected on erythromycin brain heart infusion plates,a single colony was collected and designated as PG0839 gene-defective mutant.Results A mutant in PG0839 gene was created by insertional inactivation,and inactivation of PG0839 gene was confirmed by restriction endonuclease digestive,sequen-cing,polymerase chain reaction（PCR） and reverse transcription PCR.Conclusion A PG0839 gene-defective mutant was created successfully.