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A kind of preparation method and application of universal bacterial vaccine

A technology for bacteria and vaccines, applied in the field of molecular biology, can solve problems such as difficult, difficult biofilm assembly, and technical difficulties, and achieve the effect of high yield

Active Publication Date: 2022-01-28
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(3) EV / OMV contains a large amount of bacterial nucleic acid, which has the risk of transmitting drug resistance genes
(4) EV / OMV is derived from the natural secretion of bacteria, and its quality control is difficult
(5) The process of EV / OMV preparation is difficult in large-scale scale-up
[0007] At present, biofilm self-assembly technology has been widely used in eukaryotes, including red blood cell membranes, immune cell membranes, tumor cell membranes, etc., but biofilm assembly based on prokaryotic biofilms still relies on bacterial autocrine EV / OMVs are not artificially assembled vesicles. This is because the bacterial cell membrane has a special structure of a large number of porins, glycoproteins, and peptidoglycans, which brings difficulties to the technology of artificially driving the formation of biomembrane assembled vesicles, especially those with thick For Gram-positive bacteria with cell walls, biofilm assembly is more difficult

Method used

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  • A kind of preparation method and application of universal bacterial vaccine
  • A kind of preparation method and application of universal bacterial vaccine
  • A kind of preparation method and application of universal bacterial vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] The present embodiment provides a kind of preparation method of general bacterial vaccine, comprises the following steps:

[0061] Step 1, the softening of bacterial biofilm: cultivate drug-resistant bacteria in the culture medium, when OD600 reaches 1.0, put C 10 h 14 N 2 Na 2 o 8 (EDTA·2Na) was slowly added to the culture medium until the concentration reached 10mM, incubated with shaking at 37°C for 30 minutes, then centrifuged the culture medium at 14,000×g for 20 minutes, removed the supernatant, and used HBSS (without Ca 2+ , Mg 2+ and phenol red, Servicebio) and resuspended in 300ml HBSS to obtain a bacterial suspension.

[0062] The drug-resistant bacteria include carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa, carbapenem-resistant Escherichia coli, and methicillin-resistant aureus Staphylococci, and vancomycin-resistant Enterococcus faecalis. The above bacteria were provided by the Second Affiliated Hospital of ...

Embodiment 2

[0072] In order to test the influence of different pressure intensities on the preparation of BBV, the method described in Example 1 was used in this example to prepare the BBV of Klebsiella pneumoniae, which were prepared under pressures of 200 bar, 400 bar, 800 bar, and 1200 bar respectively. The results proved that under the pressure of 1200bar, the bacteria can be driven to form a large number of complete BBVs, while the vesicles can also be formed under the conditions of 200bar, 400bar, and 800bar, but the structure is not complete.

[0073] Such as image 3 , showing representative electron micrographs of BBV driven by 800bar and 1200bar pressures. The left figure shows that after 1200bar pressure drives Klebsiella pneumoniae before purification, a large amount of complete BBV can be seen. The 1200bar and 800bar pressure-driven electron micrographs of purified bacteria in the right figure show that a large number of complete vesicles can be formed under the drive of 120...

Embodiment 3

[0076] Embodiment 3——BBV generation mechanism

[0077] In order to clarify the generation mechanism of BBV, the following experiments were carried out in this embodiment:

[0078] The Kp-BBV samples purified in Example 1, and the Kp whole bacteria, Kp lysate, and Kp-EV described in Comparative Example 1 were analyzed on 10% SDS-PAGE electrophoresis. Such as Figure 4 The SDS-PAGE analysis shown shows that Kp-BBV has the least protein composition.

[0079] The ITRAQ proteomics analysis of the Kp-BBV and the whole bacterial body proteins showed that 291 proteins were increased and 117 proteins were decreased in BBV compared with the whole bacterial body of Kp. Cluster analysis proved that the up-regulated proteins of Kp-BBV compared with Kp whole cells were mostly proteins on the biofilm, and most of the reduced proteins were intracellular proteins; GO enrichment analysis proved that Kp-BBV and Kp whole cells were up-regulated or down-regulated Most of the proteins in Kp-BBV ...

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Abstract

The present invention provides a preparation method and application of a general bacterial vaccine. The preparation method of the present invention is to use ultra-high pressure to drive bacteria through cracks to form stable bacterial biofilm vesicles (BBV). BBV is produced by a variety of Gram-negative and Gram-positive resistant bacteria, as well as non-resistant bacteria. BBV is artificially produced by bacteria, which releases bacterial intracellular proteins and nucleic acids. Compared with extracellular vesicles (EVs) naturally secreted by bacteria, BBV has higher yield and safety; BBV can be efficiently taken up and stimulated by DC cells It is mature, and as a vaccine, it has dual functions of inducing bacterial-specific humoral and cellular immune responses in vivo. Tests have shown that BBV vaccines from different bacterial sources all exhibit characteristics of resisting respective bacterial infections and good biocompatibility. The establishment of this BBV universal vaccine preparation platform breaks through the limitations of EV vaccines and is of great significance to the development of bacterial vaccines.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to a preparation method and application of a general bacterial vaccine. Background technique [0002] The global infection of drug-resistant bacteria is becoming more and more serious, and it has become a huge problem that threatens human life and health. It is reported that 700,000 people in the world die from drug-resistant bacterial infection every year. The number of deaths will reach 10 million and exceed the number of cancer deaths. During the period of COVID-19, about 50% of the dead patients had secondary bacterial infections, and the main infectious bacteria were Klebsiella pneumoniae and Acinetobacter baumannii. Drug-resistant bacteria are resistant to antibiotics, so once the infection is difficult to control, it causes a high fatality rate. Currently, the most serious drug-resistant bacteria are carbapenem-resistant Klebsiella pneumoniae, Escherichia coli, Acinetobacte...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/085A61K39/104A61K39/108A61K39/39A61K47/46A61P31/04A61P37/04C12N1/20
CPCA61K39/085A61K39/104A61K39/0258A61P31/04A61K39/39A61P37/04A61K47/46C12N1/20A61K2039/54A61K2039/542A61K2039/544A61K2039/55594A61K2039/55555Y02A50/30
Inventor 黄惟巍马雁冰李维冉杨忠倩华良群张启书龙琼白红妹杨旭孙文佳
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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