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Preparation method of porphyromonas gingivalis rgpa gene knockout mutant strain

A technology of porphyrin gingivalis and gene knockout, applied in the field of stomatology, can solve problems such as low work efficiency, and achieve the effects of high recombination efficiency, simple and flexible operation

Inactive Publication Date: 2018-09-07
NANJING STOMATOLOGICAL HOSPITAL
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  • Application Information

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Problems solved by technology

However, using conventional methods to knock out gingival protease requires a lot of screening work, and the work efficiency is low

Method used

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  • Preparation method of porphyromonas gingivalis rgpa gene knockout mutant strain
  • Preparation method of porphyromonas gingivalis rgpa gene knockout mutant strain
  • Preparation method of porphyromonas gingivalis rgpa gene knockout mutant strain

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Embodiment Construction

[0017] Adopt the following method to prepare the Porphyromonas gingivalis RgpA gene knockout mutant strain, the principle is as follows figure 1 shown.

[0018] 1. Preparation of plasmid pPHU281_up_erm_down

[0019] (1) Amplification and purification of the upper and lower homology arms of the RgpA gene

[0020] Select the start codon of the RgpA gene coding region to start the sequence of 818bp upstream as the upstream homology arm (sequence as shown in SEQ ID NO: 1), and the stop codon of the RgpA coding region to start the sequence of 686bp downstream as the downstream homology arm ( The sequence is shown in SEQ ID NO: 2).

[0021] According to the RgpA gene sequence (GenBank: 6330747) of P.gATCC33277 strain (wild strain), primers for amplifying the upper and lower homology arms of the RgpA gene were designed by using Oligo6 software, and after homology analysis of the primers by BLAST, Nanjing Jin Synthesized by Sirui Biotechnology Co., Ltd.

[0022] Wherein, the prime...

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Abstract

The invention provides a preparation method of a porphyromonas gingivalis RgpA gene knock-out mutant strain, and relates to the field of oral medicine. The method includes the steps that upstream and downstream homologous arms of porphyromonas gingivalis RgpA genes are amplified; erythrocin resistance gene segments are amplified; homologous recombination fragments with the upstream and downstream homologous arms of the RgpA genes at the two ends and the erythrocin resistance gene segments in the middle positions are constructed; the homologous recombination fragments are converted into porphyromonas gingivalis, homologous recombination is performed, an erythrocin resistance plate is adopted for screening, and then the porphyromonas gingivalis RgpA gene knock-out mutant strain is obtained. According to the preparation method of the porphyromonas gingivalis RgpA gene knock-out mutant strain, operation is easy, convenient and flexible, no complex enzyme-cut and link-up or screening is needed, no specificity requirement exists in selection of mutation sites, and recombination efficiency is high.

Description

technical field [0001] The invention relates to the field of stomatology, in particular to a method for preparing a Porphyromonas gingivalis RgpA gene knockout mutant. Background technique [0002] Porphyromonas gingivalis (abbreviated as P.g) is a non-glycolytic gram-negative anaerobic coccus, which is one of the important periodontal pathogens with extensive research and sufficient evidence. Some foreign scholars have found that P.g can be detected in 37% of individuals aged 0-18. Gingivalin (Gingipains) is a trypsin-like cysteine ​​protease synthesized in P.g cells and secreted outside cells, which plays an important role in its pathogenicity. It includes gingivalin protease K (gingipainK, Kgp) with specific binding activity to lysine residues and gingipain protease R (gingipainR, Rgp) with specific binding activity to arginine residues. Among them, Rgp includes two forms of RgpA and RgpB. In order to study the pathogenic mechanism of Porphyromonas gingivalis and find ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74
Inventor 孙卫斌张瑞杨洁
Owner NANJING STOMATOLOGICAL HOSPITAL
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