Development of cloning vectors and estabiishment of transformation system for the periodontitis- pathogenic anaerobes

牙周炎致病性厌氧菌克隆载体的研制及转化体系的建立

• 批准号: 03670857
• 负责人: YOSHIMOTO Hisashi
• 金额: $ 1.15万
• 依托单位: Kanagawa Dental college
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03670857/
• 关键词: Porphyromonas gingivalis; Transformation; Electroporation; Plasmid; Restriction

Genetic transformation, by electroporation, of Porphyromonas gingivalis was accomplished with DNA of plasmid pE5-2, or its derivative pYT7. Prior to transformation, pE5-2 was transferred from E. coli to P. gingivalis strains by conjugation (mobilization with R751), and plasmid DNA was purified from the transconjugants thus obtained. Transformation was possible when the recipient strain and the donor from which the plasmid DNA was purified were homologous. If they were heterologous each other, transformation did not occur at all, or only occured at a very low frequency. This evidence suggests presence of the restriction-modification system in most of P. gingivalis strains. pYT7, a derivative of pE5-2, was obtained by self-ligation of the 8.0 kb fragment of AvaI-digested pE5-2 from P. gingivalis, which has several single-cutting restriction sites such as EcoRI, AvaI, ClaI and BstEII. However, it was not stable enough, though is much more stable than its parent, pE5-2, in P. gingivalis cells probably because its rep gene was originated in a relatively distant species, Bacteroides eggerthii.

用质粒pE5-2或其衍生物pYT7的DNA通过电穿孔完成牙龈卟啉单胞菌的遗传转化。在转化之前，通过接合(用R751动员)将pE5-2从大肠杆菌转移至牙龈卟啉单胞菌菌株，并从由此获得的转接合子中纯化质粒DNA。当受体菌株和从中纯化质粒DNA的供体是同源的时，转化是可能的。如果它们彼此异源，则根本不会发生转化，或者仅以非常低的频率发生转化。该证据表明大多数牙龈卟啉单胞菌菌株中都存在限制性修饰系统。 pYT7 是 pE5-2 的衍生物，通过自连接来自牙龈卟啉单胞菌的 AvaI 消化的 pE5-2 的 8.0 kb 片段而获得，该片段具有多个单切割限制性位点，例如 EcoRI、AvaI、ClaI 和 BstEII。然而，在牙龈卟啉单胞菌细胞中，它虽然比其亲本pE5-2稳定得多，但还不够稳定，可能是因为它的rep基因起源于相对遥远的物种，埃格氏拟杆菌。

项目成果

Hisashi Yoshimoto 他3名: "Genetic transformation of Porphyromonas gingivalis by Electroporation" Oral Microbiology and Immunology. 8. (1993)

Hisashi Yoshimoto 和其他 3 人：“通过电穿孔对牙龈卟啉单胞菌进行遗传转化”口腔微生物学和免疫学 8。（1993）。

Hisashi Yoshimoto他3名: "Genetic transformation of Porphyromonas gingivalis by Electroporation" Oral Microbiology and Immunology. 8. (1993)

Hisashi Yoshimoto 和其他 3 人：“通过电穿孔对牙龈卟啉单胞菌进行遗传转化”口腔微生物学和免疫学 8。（1993）。

Yoshimoto,Hisashi: "Genetic Transformation of Porphyromonas gingivalis by Electroporation" Microbiology and Immunology. Vol.8. (1993)

Yoshimoto，Hisashi：“通过电穿孔对牙龈卟啉单胞菌进行遗传转化”微生物学和免疫学。