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Escherichia coli VO outer membrane protein nanoparticle vaccine modified plga with chitosan and its preparation method and application

A technology of Escherichia coli and outer membrane protein, which is applied in the directions of non-active ingredients medical preparations, medical preparations containing active ingredients, antibacterial drugs, etc., can solve the lack of functional groups that can be covalently modified on the surface of nanoparticles, Short residence time, limited application, etc.

Active Publication Date: 2022-04-29
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the lack of functional groups that can be covalently modified on the surface of its nanoparticles and the short residence time in the body limit its application in the field of vaccine vectors.

Method used

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  • Escherichia coli VO outer membrane protein nanoparticle vaccine modified plga with chitosan and its preparation method and application
  • Escherichia coli VO outer membrane protein nanoparticle vaccine modified plga with chitosan and its preparation method and application
  • Escherichia coli VO outer membrane protein nanoparticle vaccine modified plga with chitosan and its preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, the preparation of different prescription nanoparticles

[0037] In order to determine the optimal formulation of nanoparticles, five groups of schemes were designed in this project: CS nanoparticles without PLGA (S1); PLGA nanoparticles with dichloromethane as solvent (S2); PLGA nanoparticles with acetone as solvent. particles (S3); PLGA-modified CS nanoparticles (S4); CS-modified PLGA nanoparticles (S5). The particle size, dispersion and three-dimensional shape of nanoparticles are detected by nanometer particle size analyzer and scanning electron microscope, and the optimal formula is obtained by screening.

[0038] Recipe 1: CS nanoparticles without PLGA (S1)

[0039] 1) Weigh 0.02g of chitosan (CS), add 20ml of glacial acetic acid with a volume fraction of 1%, and stir until completely dissolved to prepare a chitosan solution.

[0040] 2) Weigh 5 mg of sodium polyphosphate, add it into 5 ml of pure water, stir until completely dissolved, and prepare...

Embodiment 2

[0086] Embodiment 2, the determination of the optimum wrapping amount of Vo protein

[0087] In order to clarify the maximum Vo protein encapsulation capacity of this nanoparticle preparation process, the protein Vo was first purified and diluted to different concentrations (0.25mg / ml, 0.50mg / ml, 0.75mg / ml, 1.0mg / ml, 1.25 mg / ml, 1.5mg / ml), and prepared CS-PLGA nanoparticles wrapped with different concentrations of Vo protein. The particle size and PdI were analyzed by a nanometer particle size potentiometer, and the effect of loading different concentrations of Vo protein on the particle size and PDI value of nanoparticles was evaluated to determine the optimal wrapping amount of CS-PLGA nanoparticles. The specific method is as follows:

[0088] (1) Purification of Vo protein

[0089] 1) Take out the pGEX-Vo / X-blue strain (Gu, H.; Liao, Y.; Zhang, J.; Wang, Y.; Liu, Z.; Cheng, P.; Wang , X.; Zou, Q.; Gu, J., Rational Design and Evaluation of an Artificial Escherichia coli K1Pr...

Embodiment 3

[0119] Embodiment 3, optimization of nanoparticle preparation process

[0120] In order to further improve the stability of VoNP, this study attempted to optimize the preparation process of nanoparticles by freeze-drying.

[0121] (1) Nanoparticles made by non-freeze-drying process

[0122] 1) Weigh 0.05g PLGA, add 5ml acetone, stir until completely dissolved.

[0123] 2) Weigh 0.02g CS, add it into 20ml 1% glacial acetic acid, stir until completely dissolved.

[0124] 3) Weigh 0.4g of PVA and 0.04g of sodium polyphosphate, add them into 15ml of pure water, and stir well.

[0125] 4) Add Vo protein 1.0mg / 5ml to the liquid in 3) (the blank control group only adds 5ml pure water).

[0126] 5) Under magnetic stirring at 800 rpm, add the liquid in 1) dropwise to 3).

[0127] 6) Under magnetic stirring at 800 rpm, add the liquid in 2) to 5) dropwise.

[0128] 7) Stir at 800 r for 8 hours in a fume hood (room temperature, wind power 60%).

[0129] 8) The sample is filtered wit...

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Abstract

The invention discloses an Escherichia coli Vo outer membrane protein nanoparticle vaccine modified with chitosan to PLGA and its preparation method and application. The particle size of the nanoparticle vaccine is 200nm-300nm, and the PdI value is between 0.02-0.3; the preparation method thereof As follows: Add the Vo protein solution to the solution containing polyvinyl alcohol and sodium polyphosphate, then add the polylactic acid-glycolic acid copolymer solution dissolved in acetone under stirring, then add the chitosan solution dissolved in glacial acetic acid under stirring, and fully disperse, Filtration, centrifugation and washing to obtain the nanoparticle vaccine; the prepared vaccine has a good protective effect on Escherichia coli K1 infection, and it is compatible with Al(OH) 3佐剂 The adsorbed Vo vaccines were equivalent without statistically significant difference, and could be used as a vaccine to prevent Escherichia coli K1 infection-related diseases, which was of great significance for the prevention and treatment of Escherichia coli K1 infection-related diseases.

Description

technical field [0001] The invention relates to the field of nano vaccines, in particular to an Escherichia coli Vo outer membrane protein nanoparticle vaccine modified with chitosan to modify PLGA, and also relates to a preparation method and application of the vaccine. Background technique [0002] Escherichia coli K1 (E.coli K1) is the second most common pathogen causing bacterial meningitis in newborns. It has a high mortality rate after infection, and cured children often have various neurological sequelae, such as hydrocephalus, epilepsy, and mental retardation. etc., causing a serious burden to the family and society. It has been reported in the literature that the fatality rate of neonatal meningitis caused by Ecoli.K1 infection is 5-40%, and 30% of recovered children are accompanied by neurological sequelae. Outer membrane protein A (Outer membrane A, OmpA) is the key pathogenic factor of E.coliK1, and its extracellular Loops play an important role in E.coli K1 inf...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/108A61K9/51A61K47/34A61K47/36A61P31/04A61P25/00
CPCA61K39/0258A61K9/5161A61K9/5153A61P31/04A61P25/00Y02A50/30
Inventor 孙红武顾江张瑾赵莉群杨赟王颖高晨韦金佞程新张卫军邹全明
Owner ARMY MEDICAL UNIV
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