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Tuberculosis mucosal gene vaccine assembled with mannosylated chitosan delivery system

A technology of saccharified chitosan and genetic vaccine, applied in the field of biogenetic engineering, can solve the problems of increased tuberculosis concurrent infection and poor tuberculosis effect, and achieve the effect of promoting intake

Active Publication Date: 2020-07-31
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention effectively solves the technical problem in the prior art that due to the ineffectiveness of the conventional BCG vaccine in preventing adult pulmonary tuberculosis, the proliferation of tuberculosis drug-resistant strains and the increase in the concurrent infection of tuberculosis caused by AIDS, the existing BCG vaccine is not effective in preventing and treating tuberculosis.

Method used

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  • Tuberculosis mucosal gene vaccine assembled with mannosylated chitosan delivery system
  • Tuberculosis mucosal gene vaccine assembled with mannosylated chitosan delivery system
  • Tuberculosis mucosal gene vaccine assembled with mannosylated chitosan delivery system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The preparation of embodiment 1 mannosylated chitosan-pPES tuberculosis mucosa gene vaccine

[0052] 1) Mass preparation of plasmid DNA: The above-mentioned tuberculosis antigen-encoding plasmid pPES was carried out according to QIAGEN PlasmidMegaKit.

[0053] 2) prepare man-chitosan solution and pcDNA3.1-pPES solution respectively, method is as follows: first prepare the man-chitosan solution of 0.02%, pH5.5, weigh 0.02g mannosylated chitosan man-chitosan (M W About 400000g / mol, deacetylation degree 75%-85%, mannose substitution degree 7.5%), slowly dissolve it with 1ml 1% HAC (acetic acid), and then use 10mM PBS (pH7.4) to dilute to 100ml, Place at 37°C for 1 hour, then filter through a 0.22 μm filter membrane and store for later use. pcDNA3.1-pPES plasmid with 5mM Na 2 SO 4 Dissolved, the DNA concentration in the prepared solution was 800 μg / ml, and stored at -20°C for later use.

[0054] 3) Man-chitosan-DNA nanoparticles were prepared by co-precipitation method ...

Embodiment 2

[0055] Example 2 Mannosylated Chitosan-pPES Tuberculosis Mucosa Gene Vaccine Intranasally Immunized C57BL / 6 Mice

[0056]C57BL / 6 female mice at 6-8 weeks were divided into 6 groups: chitosan-pcDNA3, man-chitosan-pcDNA3, pPES, chitosan-pPES, man-chitosan-pPES, BCG groups, 21 mice in each group. The mice were injected intraperitoneally with 80-120 μl of 0.75% pentobarbital sodium to make them slightly anesthetized. The uniform nanoparticle turbid solution freshly prepared in Example 1 was used to immunize mice with 50 μg DNA / 125ul nasal drop. Vaccine solutions such as pPES, chitosan-pPES and man-chitosan-pPES containing 50 μg of plasmid DNA were instilled dropwise into the nasal cavity on both sides of the mouse. Intranasal immunization every 2 weeks, a total of 4 times. BCG is used for subcutaneous immunization: with BCG (1×10 7 CFU) of PBS was inoculated subcutaneously at multiple points on the back of the mouse, with a total amount of 100 μl, and immunized once. Blood was...

Embodiment 3

[0057] Example 3 Mannosylated chitosan-pPES nasal immunization can enhance tuberculosis-specific alveolar lavage fluid SIgA antibody response

[0058] The level of specific IgG in serum of immunized mice was detected by indirect ELISA. Polystyrene microwell plates (co-star) were coated with 5 μg / ml HSP65 protein (coating solution 0.1M Na 2 CO 3 , pH9.6) overnight at 4°C. Block the plate with 5% milk-PBS, 200 μl / well, and incubate at 37°C for 1h. Add each mouse serum (1:100, 1:1000, 1:10000, 1:100000 dilution) in turn, 100 μl / well, 37°C for 1h, after washing the plate, add HRP-goat anti-mouse IgG and incubate at 37°C for 1h, After washing the plate, develop color with TMB for 15min, 2N H 2 SO 4 Test A after terminating the reaction 450 value.

[0059] Such as image 3 A shows: the man-chitosan-pPES tuberculosis mucosal gene vaccine immunized mice by intranasal dripping induced HSP65-specific serum IgG after 4 weeks, and its level continued to increase with time, reachin...

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Abstract

The invention relates to a mannosylated chitosan delivery system assembled tuberculosis mucosa gene vaccine. The mannosylated chitosan delivery system assembled tuberculosis mucosa gene vaccine is a nanoparticle complex compounded through copolymerization and crosslinking of mannosylated chitosan and a tuberculosis antigen encoding plasmid pPES; the tuberculosis antigen encoding plasmid pPES is amulti-T cell epitope tuberculosis gene vaccine with heat shock protein 65 as an epitope scaffold, which is disclosed in the invention with the application number of 201310682146.4. The invention further discloses a preparation method of the mannosylated chitosan delivery system assembled tuberculosis mucosa gene vaccine. The preparation method comprises the following steps: preparing a mannosylated chitosan solution with the concentration of 0.015-0.025% and the pH of 5.2-5.5; dissolving the tuberculosis antigen encoding plasmid pPES into a buffer solution to obtain a tuberculosis antigen encoding plasmid solution with the concentration of 800 [mu]g / ml-1 mg / ml; and mixing the mannosylated chitosan solution and the tuberculosis antigen encoding plasmid solution at 54-57 DEG C at a high speed of 15000-17000 rpm, and performing a copolymerization and chemical crosslinking reaction. The invention further discloses application of the mannosylated chitosan delivery system assembled tuberculosis mucosa gene vaccine to preparation of medicines for preventing or treating tuberculosis. The tuberculosis mucosa gene vaccine has the potential as a novel preventive and therapeutic tuberculosis vaccine.

Description

technical field [0001] The invention relates to the field of biological genetic engineering, in particular to a tuberculosis mucosa gene vaccine assembled with a mannosylated chitosan delivery system. Background technique [0002] The resurgence of pulmonary tuberculosis caused by Mycobacterium tuberculosis infection is a major global health problem today, due to the increasing population density and mobility, the ineffectiveness of conventional BCG vaccine for adult tuberculosis prevention, the emergence of drug-resistant strains of tuberculosis, and AIDS. Tuberculosis is concurrently infected. At present, the incidence and death of tuberculosis are becoming more and more serious. There are 8 million active tuberculosis patients and 3 million deaths in the world every year; about 550 million people in my country have been infected with tuberculosis, and 130,000 people die of tuberculosis every year. 120,000 new patients. In 2006, pulmonary tuberculosis has become the first i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/385A61K39/04A61P31/06
Inventor 徐薇吴曼丽
Owner SUZHOU UNIV
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