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论文摘要

牙龈卟啉单胞菌PG0839基因突变株的构建

Construction PG0839 gene-defective mutant of Porphyromonas gingivalis

作者:刘静波 潘亚萍 李琛 林莉 钟鸣

Author:Liu Jingbo1, Pan Yaping1, Li Chen1, Lin Li1, Zhong Ming2,

收稿日期:2012-04-25          年卷(期)页码:2012,30(2):192-192-196

期刊名称:华西口腔医学杂志

Journal Name:West China Journal of Stomatology

关键字:牙龈卟啉单胞菌,毒力岛,基因打靶,

Key words:Porphyromonas gingivalis,pathological islands,gene targeting,

基金项目:

国家自然科学基金资助项目(81070834)

中文摘要

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BamH Ⅰ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、PR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。

英文摘要

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 electroporation 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, sequencing, polymerase chain reaction(PCR) and reverse transcription PCR. Conclusion A PG0839 gene-defective mutant was created successfully.

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