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Method for marking acinetobacter baumannii by green fluorescent protein gene

A technology of Acinetobacter baumannii and green fluorescent protein, applied in the biological field, can solve the problems of life-threatening patients

Inactive Publication Date: 2021-02-26
HANGZHOU FIRST PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As one of the important pathogens of nosocomial infection in China, Acinetobacter baumannii mainly causes upper respiratory tract infection, urethritis, catheter-associated bacteremia, post-burn wound infection, meningitis, etc. in intensive care units and immunocompromised populations. At the same time, it is also easy to colonize and adhere to the environmental surface of medical devices, which poses a serious threat to the lives of patients.
Especially in recent years, the number of multidrug-resistant and pan-drug-resistant Acinetobacter baumannii strains has increased year by year, and the emergence of all-drug-resistant bacteria has brought serious challenges to clinical treatment.

Method used

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  • Method for marking acinetobacter baumannii by green fluorescent protein gene
  • Method for marking acinetobacter baumannii by green fluorescent protein gene
  • Method for marking acinetobacter baumannii by green fluorescent protein gene

Examples

Experimental program
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Effect test

Embodiment 1A

[0022] Example 1Ab-GFP strain construction

[0023] 1. Experimental materials

[0024] Human alveolar epithelial cells (HPAEpiC) cell line was purchased from CELLBIO (No.: CBR130583), RPMI 1640 (Gino, GNM31800), RPMI 1640 double antibody (Gino, GNM-31800-S), PBS buffer 1× (Gino, GNM20012), 0.25% trypsin + EDTA (Gino, GNM-25260), fetal bovine serum (CAPRICORNSCIENTIFIC, FBS-11A), agar powder (Best, BS1068), peptone (Best, BS7006 ), yeast powder (Best, BS1069), sodium chloride (Sinopharm Chemical Reagent Co., Ltd.), Triton X-100 (polyethylene glycol octylphenyl ether, Dalian Meilun Biotechnology Co., Ltd., MB2486), iFluor TM 647phalloidin (phalloidin-iFluor647 conjugate, Dalian Meilun Biotechnology Co., Ltd., MB5939), DAPI (4', 6-diamidino-2-phenylindole, Thermo, 62247), paraformaldehyde ( McLean, P804536-500g), bovine serum albumin (Shanghai Yuanye Biotechnology Co., Ltd., S25762), 10× polylysine (Dalian Meilun Biotechnology Co., Ltd., MA0174). The strains, plasmids and pri...

Embodiment 2

[0063] 1. Construction of fluorescently labeled Acinetobacter baumannii infection cell model

[0064] Acinetobacter baumannii mainly infects the respiratory tract and causes the occurrence and development of related respiratory infection diseases. Therefore, in the present invention, human alveolar epithelial cells (Human alveolar epithelial cells, HPAEpiC, cell strain purchased from CELLBIO, number: CBR130583) are used as A cellular model for studying bacteria-host interactions. The result is as Figure 4 As shown, by fluorescence microscope observation, the model of infection of host cells by the GFP-labeled strain of Acinetobacter baumannii can be successfully constructed, and the relative position, adhesion or invasion of bacteria and host cells can be accurately located. Provide basic experimental techniques for dynamic real-time observation of bacterial-cell interaction research, as well as biological functions of Acinetobacter baumannii-related pathogenic factors.

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Abstract

The invention provides a method for marking acinetobacter baumannii by a green fluorescent protein gene. The method comprises the following steps: constructing a pET-RA-Y escherichia coli-acinetobacter baumannii shuttle plasmid (including a promoter sequence, a GFP sequence, an acinetobacter baumannii gene replication origin sequence and a rifampicin resistance selection marker), and introducing into an acinetobacter baumannii standard strain ATCC17978; and observing that the thallus shows green fluorescence under a fluorescence microscope. The invention also establishes a fluorescently-labeled acinetobacter baumannii infected cell model for the stability and reliability of the fluorescent-labeled acinetobacter baumannii constructed by the method, provides technical support for studying the biological action of bacterial pathogenic factors and the interaction condition of bacteria and hosts, provides a simple and intuitive method for studying the relationship between the acinetobacterbaumannii and host due to introduction of the green fluorescent protein gene.

Description

technical field [0001] The invention belongs to the field of biological technology, and specifically relates to a method for marking the green fluorescent protein gene of Acinetobacter baumannii, using a shuttle plasmid to obtain a fluorescently labeled strain of Acinetobacter baumannii capable of stably expressing green fluorescent protein, and its construction method and application . Background technique [0002] Acinetobacter baumannii (Acinetobacterbanumannii, Ab) is a Gram-negative bacillus, ranking first in the pathogenic Acinetobacter genus (Acinetobacterspp.). As one of the important pathogens of nosocomial infection in China, Acinetobacter baumannii mainly causes upper respiratory tract infection, urethritis, catheter-associated bacteremia, post-burn wound infection, meningitis, etc. in intensive care units and immunocompromised populations. At the same time, it is also easy to colonize and adhere to environmental surfaces such as medical devices, which poses a se...

Claims

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Application Information

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IPC IPC(8): C12N15/65C12N15/74G01N21/64C12R1/01
CPCC12N15/65C12N15/74G01N21/6486
Inventor 余道军潘平
Owner HANGZHOU FIRST PEOPLES HOSPITAL
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