Bioluminescent engineered bacterium composition, and preparation method and application thereof

A bioluminescent, engineering technology, applied in biochemical equipment and methods, microbial-based methods, drug combinations, etc., can solve problems such as inability to effectively eliminate tumors, reduce cell anti-tumor effects, and toxic and side effects, and achieve monotherapy bad effect

Active Publication Date: 2021-03-26
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology uses engineered bacterial strains that quickly multiply within cancer tissue environments with labeled cytoskeletons (called chlorophores) attached to them for use as a way to stimulate photosensitizers like porphyrines or other molecular structures called quenchers. These photoactive agents are used during treatments designed specifically against certain types of neoplasms without affecting healthy ones. They have been shown effective even when administered systemically due to their ability to target specific areas inside the affected area while minimizing damage from radiation exposure.

Problems solved by technology

This patented technical problem addressed in this patents relates to developing an effective way to administer photo dynamic therapies specifically targeted towards certain types of neoplasms called BPTI, including brain glioma, lung carcinemia, liver metastasis, cervix uteriis, bladder cancer, stomach cancer, colon cancer, lymphatic system disease, choriocarcinoma, malignant mesothelioma, headaches caused by chemotherapeutic agents like anthracycline, fluorescein, interferon, laser radioscopy, and quantum luminosity techniques. These approaches also involve injecting artificially generated substances containing natural antigens from live organisms, leading to severe harmful sideeffects when delivered inside humans without affecting their own health status.

Method used

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  • Bioluminescent engineered bacterium composition, and preparation method and application thereof
  • Bioluminescent engineered bacterium composition, and preparation method and application thereof
  • Bioluminescent engineered bacterium composition, and preparation method and application thereof

Examples

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Embodiment 1

[0038] Embodiment 1: Preparation of bioluminescent engineered bacterial composition

[0039] Construction of bioluminescent engineered bacteria: Attenuated Salmonella was treated with ice-cold calcium chloride solution to prepare competent bacteria, and the plasmid pGL4.13[luc2 / SV40] with firefly luciferin was passed through standard steps Transfer to competent bacteria, spread the competent bacteria on the agar plate containing ampicillin, incubate at 37°C for 16 hours, add the luminescent substrate D-luciferin, and select the successfully transformed bioluminescence A single colony of engineered bacteria was propagated and cultured for subsequent experiments.

[0040] Mix the components in the composition according to the volume ratio, wherein component two is the luminescence substrate D-fluorescein dissolved in phosphate buffer solution, the concentration is 30mg / ml; component three is the photosensitive molecule dissolved in ammonium bicarbonate solution Chlorin e6, the ...

Embodiment 2

[0041]Example 2: Detection of Luminescence Properties of Bioluminescent Engineered Bacteria

[0042] The bioluminescent engineered bacterial composition prepared in Example 1 was tested for its luminescent properties, and the results were as follows: figure 1 shown. Such as figure 1 As shown in b, the fluorescence spectrum produced by the reaction of the bioluminescent bacteria with the substrate fluorescein has good compatibility with the absorption spectrum of the photosensitive molecule chlorin e6. figure 1 As shown in c and 1d, the engineered bacteria can produce bioluminescence up to 4 hours after reacting with their substrates as detected by small animal imaging. It shows that the present invention can utilize the fluorescence produced by the bioluminescent engineered bacteria and the substrate as an excitation light source.

Embodiment 3

[0043] Example 3: The engineered bacterium produces singlet oxygen when it mixes with its substrate and photosensitive molecule

[0044] The bioluminescent engineered bacteria prepared in Example 1 were mixed with their substrates and photosensitive molecules to detect the production of singlet oxygen with a SOSG kit. The result is as figure 2 As shown, when the bioluminescent engineered bacteria are mixed with their substrates and photosensitive molecules, the fluorescence of SOSG gradually increases with time, indicating that the excitation light generated by the bioluminescent engineered bacteria catalyzing the substrate can excite the photosensitive molecules to produce singlet states. Oxygen can be used for photodynamic therapy.

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Abstract

The invention discloses a bioluminescent engineered bacterium composition, and a preparation method and application thereof. The bioluminescent engineered bacterium composition disclosed by the invention comprises bioluminescent engineered bacteria, a luminescent substrate, photosensitizer molecules and biomacromolecules capable of being gelatinized in situ. The invention also discloses application of the bioluminescent engineered bacterium composition in tumour photodynamic therapy and immunotherapy. According to the invention, plasmids expressing firefly luciferase are transfected into attenuated salmonella to construct bioluminescent engineered bacteria; the bacteria can generate yellow-green light with the wavelength of 540-600 nm when being co-incubated with a substrate D-fluorescein,so as to excite Ce6 to generate singlet oxygen to realize photodynamic therapy of tumours; and meanwhile, the engineered bacteria can be used as an immunologic adjuvant to activate anti-tumour immuneresponse of an organism and enhance tumour photodynamic therapy, so that efficient synergistic therapy of tumours is realized.

Description

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Claims

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Application Information

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Owner SUZHOU UNIV
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