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Spontaneously lighting kansas mycobacterium and construction method thereof

A technology of mycobacteria and self-luminescence, applied in the field of genetic engineering, can solve the problems of long treatment cycle and large side effects of drugs, and achieve the effect of convenient detection of drug activity

Active Publication Date: 2019-01-18
湖北景瑞天恒生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the treatment cycle for patients with Mycobacterium kansasii is long, and the side effects of drugs are large, so there is an urgent need to develop new antibiotics

Method used

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  • Spontaneously lighting kansas mycobacterium and construction method thereof
  • Spontaneously lighting kansas mycobacterium and construction method thereof
  • Spontaneously lighting kansas mycobacterium and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] An autonomous luminescent Mycobacterium kansasii, the autonomous luminescent Mycobacterium kansasii is based on Mycobacterium kansasii, which is obtained by the electroporation method, inserting the recombinant plasmid pOPTIg, and subcultured and screened;

[0045] like figure 1 As shown, the recombinant plasmid pOPTIg contains the promoter (Hsp60promoter), the enzyme gene required for luminescence (luxCDABE), the resistance selection gene Tsr (SEQ ID NO: 4), the integrase gene Int and the integrase integration site in a clockwise direction The fusion gene of attP (SEQ ID NO: 1), the direct repeat sequences DifL (SEQ ID NO: 2) and DifR (SEQ ID NO: 3) located at both ends of the fusion gene, and the ampicillin resistance gene (Ampr) and replication Sub (Rep); the functions of each component are as follows:

[0046] Promoter (Hsp60promoter): a mycobacterial strong promoter, which can promote the strong expression of subsequent genes;

[0047] Enzyme gene required for lu...

Embodiment 2

[0062] The invention provides a preparation method of autonomous luminescent mycobacterium kansasii

[0063] The 7H9 medium, 7H11 medium, and Tween 80 involved in this example were all purchased from Guangzhou Huaqisheng Biological Company.

[0064] Biorad electroporation instrument (Biorad GenePulserXceII) and electroporation cup were purchased from Biorad Company.

[0065] 1. Materials to be prepared:

[0066] 1) Mycobacteria Kansasii

[0067] 2) 10% glycerin + 0.05% Tween 80 + 20mL glycerin + 180mL water + 100uL Tween 80, and filter with a 0.2um filter, fresh preparation is required, no more than 1 week;

[0068] 3) Sterilized glass beads, 50mL centrifuge tube, Biorad 0.2cm electric cup, 25mL pipette;

[0069] 4) 50mL of well-grown bacterial liquid in the logarithmic phase [OD value of the bacterial liquid reaches 0.6-0.8]. 1mL tip with filter.

[0070] 2. Electric conversion

[0071]1) Inoculate the bacteria to be transformed (Mycobacteria Kansasii) into a conical fl...

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Abstract

The invention discloses a spontaneously lighting kansas mycobacterium. The spontaneously lighting kansas mycobacterium is based on Mycobacterium Kansas, which is inserted into a recombinant plasmid pOPTIg by electroporation and screened by subculture. The autonomously illuminated Mycobacterium kansii provided by the present invention is more stable in subculture; the method for constructing autonomously luminescent Kansas vaginalis designed by the present invention, in the process of providing a recombinant plasmid pOPTIg, the recombinant plasmid pOPTIg is transferred Screening in Mycobacterium kansii to obtain autonomously luminescent Kansas bacillus with no resistance screening markers for loss of resistance genes, integrase genes, and integration sites.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a self-luminous Mycobacterium kansasii and a construction method thereof. Background technique [0002] Mycobacterium (Mycobacterium) is a class of slender, slightly curved microorganisms, sometimes branched or filamentous. At present, Mycobacterium has been classified into Actinomycetes in taxonomy. Actinomycetes that are pathogenic to humans can be divided into two categories with and without mycolic acid. Mycobacteria belong to the group containing mycolic acids. The main feature of bacteria of this genus is that the cell wall contains a large amount of lipids, mainly mycolic acids. This is closely related to its dyeability, growth characteristics, pathogenicity, and resistance. Generally, it is not easy to dye. If it is dyed by heating or prolonging the dyeing time, it can resist the decolorization of the strong deacidifying agent hydrochloric acid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/87C12R1/32
CPCC12N15/74C12N15/87
Inventor 王邦兴王余业郝建帮方坤刘晨程杰
Owner 湖北景瑞天恒生物科技有限公司
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