牙龈卟啉单胞菌PG0839基因突变株的构建

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[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、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。

[关键词] 牙龈卟啉单胞菌； 毒力岛； 基因打靶

[中图分类号] R 781.5 [文献标志码] A [doi] 10.3969/j.issn.1000-1182.2012.02.020

Construction PG0839 gene-defective mutant of Porphyromonas gingivalis Liu Jingbo1, Pan Yaping1, Li Chen1, Lin Li1, Zhong Ming2，3. （1. Dept. of Periodontology, School of Stomatology, China Medical University, Shenyang 110002, China; 2. Central Laboratory, School of Stomatology, China Medical University, Shenyang 110002, China; 3. Dept. of Oral Pathology, School of Stomatology, China Medical University, Shenyang 110002, China）

[Abstract] Objective In order to determine the function of PG0839 gene from Porphyromonas gingivalis（P.gin-

givalis） 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 restric-

tion 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.

[Key words] Porphyromonas gingivalis； pathological islands； gene targeting

病原菌毒力岛是指编码细菌毒力基因簇的相对分子质量较大的染色体DN段。通过基因水平转移，细菌获得毒力岛基因。毒力岛编码不同功能的毒力相关基因，影响细菌的毒力变化。PG0839基因是牙龈卟啉单胞菌（Porphyromonas gingivalis，P.gin-

givalis）W83菌株PG0819—0844毒力岛中的重要部分。在慢性牙周炎患者P.gingivalis阳性的龈下菌斑标本中，毒力岛基因PG0839检出与牙周探诊深度和探诊出血指数之间的关系存在统计学意义，提示该基因

可能是慢性牙周炎的致病性基因[1]。目前许多学者通

过反向遗传学方法进行基因功能研究，即构建目的基因敲除菌株后，通过对突变株表型变化的研究，获得目的基因与细菌表型之间的相关性，从而获知基因的功能[2]。本研究拟遵循反向遗传技术路线，运用

等位基因重组技术构建PG0839基因突变菌株，为研究毒力岛基因PG0839的功能奠定实验基础。