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Method of constructing selection-marker-free self-luminescent mycobacterium abscessus and establishment of corresponding in-vitro high-flux medicine-screening model

A mycobacterium, self-luminescence technology, applied in the field of Mycobacterium abscessus, can solve the problem of not finding the resistance screening marker of Mycobacterium abscessus, and achieve the effect of convenient detection of drug activity, reduced workload, and saving time for drug screening

Inactive Publication Date: 2015-07-08
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, this patent for the first time will April The resistance gene serves as a resistance selection marker for M. abscessus, and no resistance selection markers for M. abscessus have been found before

Method used

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  • Method of constructing selection-marker-free self-luminescent mycobacterium abscessus and establishment of corresponding in-vitro high-flux medicine-screening model
  • Method of constructing selection-marker-free self-luminescent mycobacterium abscessus and establishment of corresponding in-vitro high-flux medicine-screening model
  • Method of constructing selection-marker-free self-luminescent mycobacterium abscessus and establishment of corresponding in-vitro high-flux medicine-screening model

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Experimental program
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Effect test

Embodiment 1

[0049] Construct the integrated plasmid pOPAI1B, see the construction procedure Figure 1~3 , for a map of plasmid pOPAI1B see Figure 4 . Such as Figure 4 As shown, the plasmid pOPAI1B contains the mycobacterial strong promoter in the counterclockwise direction ( Hsp60 ), the enzyme gene required for luminescence ( LuxCDABE ), apramycin resistance gene ( April ) (SEQ ID NO:1) and integrase gene ( Int ) (SEQ ID NO:6) fusion gene, a direct repeat sequence located at both ends of the fusion gene DifR (SEQ ID NO:2) and Dif L (SEQ ID NO:3), phage integration site ( attP ) (SEQ ID NO:4), and the ampicillin resistance gene ( Ampr ) and replicators ( rep ). The functions of each component are as follows:

[0050] Hsp60 : A mycobacterial strong promoter, which can promote the strong expression of subsequent genes.

[0051] LuxCDABE : Enzyme gene required for luminescence, the expression of this gene allows the host bacteria to emit light autonomously [5] . ...

Embodiment 2

[0087] Example 2 A preparation method of self-luminescent Mycobacterium abscessus without resistance selection marker

[0088] The 7H9 medium, 7H11 medium, and Tween 80 involved in this example were all purchased from Guangzhou Huaqisheng Biological Company.

[0089] Biorad electroporation instrument (Biorad GenePulser Xcell) and electroporation cup were purchased from Biorad Company.

[0090] 1. Materials to be prepared:

[0091] 1) Mycobacterium abscessus ( Mycobacterium.abscessus )

[0092] 2) 10% glycerol + 0.05% Tween 80 + 20mL glycerin + 180 mL water + 100μL Tween 80, and filter with a 0.2μm filter. It is required to be freshly prepared and not older than 1 week.

[0093] 3) Sterilized glass beads, 50ml centrifuge tube, Biorad 0.2cm electric cup, 25ml pipette.

[0094] 4) 50ml of well-grown bacteria solution in the logarithmic phase [the OD value of the bacteria solution reaches 0.6-1.3]. 1ml tip with filter.

[0095] 2. Electroconversion:

[0096] 1) The ...

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Abstract

The invention discloses a method of constructing selection-marker-free self-luminescent mycobacterium abscessus and establishment of a corresponding in-vitro high-flux medicine-screening model. During the construction process, a key factor is the construction of a recombinant plasmid pOPAI1B, which contains a fused gene including a luminescent required enzyme gene LuxCDABE, a resistance screening gene Apr and an integrase gene Int; direct repeat sequences DifR and DifL at the two ends of the fused gene respectively; and a phage integration site attP and a replicon. The recombinant plasmid pOPAI1B is transformed into the mycobacterium abscessus and then is screened to obtain the selection-marker-free self-luminescent mycobacterium abscessus of which a resistance gene and the integrase gene are both lost. The self-luminescent mycobacterium abscessus can screen drugs in a high flux, can determine sterilizing or bacteria-resistant effects of the drug by means of detection of luminescent situation of bacteria, can be used for in-vitro drug activity detection, is free of any substrates, can continuously detect the same sample, is high in sensitivity and is strong in repeatability.

Description

technical field [0001] The present invention relates to a kind of Mycobacterium abscessus ( Mycobacterium abcessus ), specifically an autoluminescent M. abscessus without a selectable marker. In addition, the present invention also applies the apramycin resistance gene as a resistance selection marker to Mycobacterium abscessus for the first time. Background technique [0002] Mycobacterium (Mycobacterium) is a class of slender slightly curved microorganisms, sometimes branched or filamentous. At present, mycobacteria have been classified into actinomycetes in taxonomy. Actinomycetes that are pathogenic to humans can be divided into two categories: those containing and those not containing mycolic acids. Mycobacteria belong to the group containing mycolic acids. The main characteristic of this genus of bacteria is that the cell wall contains a large amount of lipids, mainly mycolic acids. This is closely related to its dyeability, growth characteristics, pathogenicity, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/53C12N1/21C12R1/32
Inventor 张天宇曹元元伍甜谭守勇谭耀驹蔡杏珊刘春平
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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