Method for increasing yield of outer membrane vesicles (OMVs) of bacteria and application of OMVs of bacteria

A technology of outer membrane vesicles and bacteria, applied in the biological field, can solve the problems of small secretion, high cost, limited use and promotion, etc., and achieve the effect of enhancing immune effect, reducing production cost, and good immune enhancing function

Active Publication Date: 2020-07-28
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, outer membrane vesicles are carriers for bacteria to release substances to the outside world, exchange signal molecules, and transfer genetic materials. Under normal circumstances, their secretion is small
If outer membrane vesicles are collected for biological products, the cost is extremely high, which limits the use and promotion of this substance

Method used

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  • Method for increasing yield of outer membrane vesicles (OMVs) of bacteria and application of OMVs of bacteria
  • Method for increasing yield of outer membrane vesicles (OMVs) of bacteria and application of OMVs of bacteria
  • Method for increasing yield of outer membrane vesicles (OMVs) of bacteria and application of OMVs of bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Deletion of Escherichia coli J5 strain yjeE gene

[0028] (1) Material

[0029] Escherichia coli J5 strain was purchased from ATCC in the United States with the strain code ATCC43745.

[0030] Preparation method of LB medium: Weigh 10 g of tryptone, 5 g of yeast powder and 10 g of sodium chloride, dissolve in distilled water and set the volume to 1000 mL, adjust the pH to 7.4, and autoclave at 121 °C for 15 min. If it is necessary to prepare a solid plate, add another 16g of agar before autoclaving.

[0031] Ampicillin, chloramphenicol and L-arabinose were purchased from Shanghai Sangong.

[0032] rTaq PCR MIX was purchased from Nanjing Nuoweizan Biological Co., Ltd.

[0033] The pKD3 plasmid, pKD46, and pCP20 plasmids were donated by Professor Zhu Guoqiang of Yangzhou University, but they can all be purchased commercially.

[0034] (2) Primer design

[0035] According to the upstream and downstream outer wings of the Escherichia coli K-12MG1655 strain (a...

Embodiment 2

[0058] Example 2 The cultivation of recombinant bacteria J5△yjeE and the preparation method of outer membrane vesicles

[0059] (1) Medium and buffer

[0060] LB medium: Take 10 g of tryptone, 5 g of yeast powder, and 10 g of sodium chloride, dissolve it in distilled water, and adjust the volume to 1000 mL, adjust the pH to 7.4, and autoclave at 121 °C for 15 min. If it is necessary to prepare a solid plate, add another 16g of agar before autoclaving.

[0061] HPO medium: Take 5g of soybean peptone, 15g of tryptone, 5g of yeast powder, 3g of glucose, 0.4g of disodium hydrogen phosphate, add 10mL of 250mM potassium chloride solution, 5mL of 2M magnesium chloride solution, dissolve in distilled water and dilute to 1000mL, Adjust the pH to 7.4. Autoclave at 121°C for 15 minutes. Before use, another 10 mL of filter-sterilized fetal bovine serum was added.

[0062] PBS buffer: an aqueous solution containing 8mM disodium hydrogen phosphate dodecahydrate, 1.5mM potassium dihydroge...

Embodiment 3

[0069] Example 3 Safety Evaluation of Outer Membrane Vesicles

[0070] 20 female mice of clean grade BALB / c were randomly divided into 2 groups, 10 in each group. One group of mice (OMV injection group) used the outer membrane vesicles prepared by J5ΔyjeE in Example 2, and injected them into leg muscles at a dose of 100ug / only; another group of mice (control group) was inoculated with outer membrane vesicles. Membrane vesicles with the same volume of PBS buffer.

[0071] After inoculation, mice were observed every day and body weight changes were recorded. After 42 days, the mice were sacrificed, and the mice were dissected to observe whether the internal organs (heart, liver, spleen, lung and kidney) had pathological changes, and the leg muscle tissue was taken for HE staining section observation.

[0072] There was no significant difference in the body weight change of the mice injected with OMV compared with the control group (e.g. Figure 4 ), indicating that the prepared...

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Abstract

The invention provides a method for increasing the yield of outer membrane vesicles (OMVs) of bacteria and application of the OMVs of bacteria, and belongs to the technical field of biology. The method comprises the step of culturing a gram-negative bacteria mutant strain with an yjeE gene knocked out. The invention further provides application of the OMVs, prepared by the method, of the bacteriain preparation of a vaccine and an immunoenhancement. By use of the method, the yield of the OMVs of Escherichia coli reaches 701 mg / L, is increased by about 100 times than the culture yield of an original strain in an LB culture medium, the method is application to mass production of the OMVs in the pharmaceutical industry, and the production cost is significantly reduced. The OMVs, prepared by the method, of the bacteria have better immunoenhancement functions, can be added to common vaccines and can significantly enhance the immune effects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for increasing the yield of bacterial outer membrane vesicles and an application thereof. Background technique [0002] Outer membrane vesicles (Outer membrane vesicles, OMVs) are the products secreted by budding on the surface of Gram-negative bacteria. -300nm range. This structure includes lipopolysaccharide (LPS), outer membrane proteins, phospholipids, bacterial toxins, adhesins, DNA, and periplasmic components that are enveloped by the outer membrane during formation. Because outer membrane vesicles cannot replicate and contain a large number of bacterial antigens, they have good immunogenicity and immune enhancement effects, and can activate the host cell immune system. Therefore, outer membrane vesicles can be used as a new type of bacterial vaccine or immune enhancer with great potential. [0003] However, outer membrane vesicles are carriers for bacte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21A61K39/39A61P37/04C12R1/19
CPCC12N15/70C12N1/20C07K14/245A61K39/39A61P37/04A61K2039/55594Y02W10/10
Inventor 周明旭张金秋邓碧华马芳徐悦卢宇侯继波
Owner JIANGSU ACAD OF AGRI SCI
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