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Bacterial ghost preparation method of independent lysis gene E

A gene-splitting, independent technology, applied in the field of bioengineering, can solve the problems of inability to import, difficult bacteria sloughing, and consumption of a certain time, and achieve the effects of good protection efficiency, good immunogenicity and simple operation.

Active Publication Date: 2015-07-29
山东山科酵爵生物制品有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still significant restrictive genetic barriers in many pathogenic bacteria, and the expression regulation system of the cleavage gene E cannot be introduced into these bacteria or cannot be recognized and started to express, so it is difficult to successfully prepare sloughs
In addition, when preparing the slough, the expression regulation system of the cleavage gene E is introduced into the bacteria, and the antibiotic resistance gene is also introduced, which will easily lead to the lateral spread of the antibiotic resistance gene, and the expression regulation system of the lysis gene E is introduced into the bacteria and bacteria. It takes a certain amount of time to induce expression

Method used

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  • Bacterial ghost preparation method of independent lysis gene E
  • Bacterial ghost preparation method of independent lysis gene E

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1, the mensuration of chemical substance minimum inhibitory concentration (MIC) and minimum bacterial growth concentration (MGC)

[0025] 1.1 Culture of Escherichia coli O138

[0026] Escherichia coli O138 was inoculated with LB liquid medium, and placed in a constant temperature incubator at 37°C for static culture for 72 hours.

[0027] 1.2 Determination of chemical substance MIC

[0028] Determination of NaOH, Triton X-100, Na by Micro Broth Dilution 2 EDTA and H 2 o 2the MIC. Take a 96-well cell culture plate, dilute a certain concentration of the above-mentioned chemical substances with a 2-fold ratio of LB liquid medium, and then add an equal amount of O138 bacterial solution (about 1.0×l0 6 CFU / ml), so that the final bacterial concentration in each hole is about 5×10 5 CFU / ml. Place the culture plate in a 37°C incubator and incubate for about 16h to 20h to judge the results, and the test is performed 3 times in parallel. The MIC is defined as t...

Embodiment 2

[0031] Embodiment 2, the optimization of the preparation method of Escherichia coli O138 slough

[0032] 2.1 Plackett–Burman experimental design

[0033] The Plackett-Burman experimental design is an experimental design based on the principle of incomplete balance blocks, which can quickly and effectively screen out some of the most important factors from many variables for further research, and has the advantages of simple data processing, applicable to multiple These factors are usually used in screening experiments in the early stages of projects.

[0034] The Plackett-Burman experimental design with the number of experiments N=12 was selected to investigate 5 factors, each factor was taken at two levels of +1 and -1, and the mass of the slough was taken as the response value. Four chemical substances NaOH, Triton X-100, Na 2 EDTA and H 2 o 2 4 factors respectively, +1 for MIC and -1 for MGC. The fifth factor represents a factor formed by the interaction of two differe...

Embodiment 3

[0055] Embodiment 3, preparation and application of Escherichia coli O138 slough

[0056] 3.1 Preparation of Escherichia coli O138 slough

[0057] According to the analysis results of the Plackett-Burman test design, three batches of E. coli O138 sloughs were designed and prepared according to experiments 1, 4, and 9, and the quality of the sloughs reached 100%. Take a small amount of sloughs and dilute them with 0.01M PBS, spread them on LB plates, and incubate at 37°C for 5 days. No bacterial growth was found, indicating that the Escherichia coli O138 slants prepared by this method do not contain live bacteria. The prepared Escherichia coli O138 slough was freeze-dried and stored.

[0058] 3.2 Specific targeted application of slough

[0059] Hela cells were inoculated in DMEM complete medium (containing 10% newborn calf serum, penicillin 100U / ml, streptomycin 100U / ml), at 37°C, saturated humidity, 5% CO 2 cultured in an incubator. Hela cells in the logarithmic growth pha...

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Abstract

The invention discloses a bacterial ghost preparation method of an independent lysis gene E. According to the preparation method, a bacterium suspension solution is added with four chemical substances including NaOH, Triton X-100, Na2EDTA and H2O2 to prepare the bacterial ghost in order to satisfy the requirements of complete release of bacterium content and completeness of a bacterium ghost. The method is simple and rapid to operate; except lysis holes, the cell shape and surface structure of the prepared bacterial ghost are not obviously changed. The bacterial ghost prepared by the method disclosed by the invention can enter a specific cell in a targeted manner, is free of toxicity to the cell and can be used as a novel transferring carrier for a drug. The bacterial ghost prepared by the method disclosed by the invention can also be used for immunizing a BALB / c mouse by intraperitoneal injection or oral gavage, also has quite good protection efficiency to homologous counteracting toxic substances and can be used as a novel vaccine.

Description

technical field [0001] The invention relates to a method for preparing a slough, in particular to a method for preparing a slough that does not depend on a cleavage gene E, and also relates to the application of the slough prepared by the method, which belongs to the field of bioengineering. Background technique [0002] Bacterial ghosts are empty bacterial shells that do not contain nucleic acid and cytoplasm, and most of the current strategies and technologies for preparing bacterial ghosts are based on the expression of cleavage proteins from the cleavage gene E of bacteriophage PhiX174, thereby lysing Gram-negative cells. formed by bacteria. The slough retains the same bacterial cell membrane structure and related antigenic components as live bacteria, which can induce the body's humoral and cellular immune responses. The natural highly conserved structure PAMP (pathogen-associated molecular patterns) of the decidual membrane, such as lipopolysaccharide, peptidoglycan, ...

Claims

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Application Information

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IPC IPC(8): C12N1/20A61K39/108A61K47/46A61P31/04C12R1/19
CPCY02A50/30
Inventor 祝文兴刘新利
Owner 山东山科酵爵生物制品有限公司
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