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Tuberculosis gene vaccine assembled by chitosan delivery system and preparation method and use thereof

A technology of deacetylated chitosan and delivery system, which can be applied in application, gene therapy, genetic engineering and other directions, and can solve problems such as poor prevention and treatment of tuberculosis

Inactive Publication Date: 2009-06-17
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The object of the present invention is to provide a tuberculosis gene vaccine assembled by a chitosan delivery system and its preparation and application. The tuberculosis gene vaccine assembled by this chitosan delivery system will solve the problems in the prior art The ineffectiveness of conventional BCG vaccines, the emergence of drug-resistant strains of tuberculosis and the concomitant infection of tuberculosis caused by AIDS make the existing vaccines ineffective in preventing and treating tuberculosis.

Method used

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  • Tuberculosis gene vaccine assembled by chitosan delivery system and preparation method and use thereof
  • Tuberculosis gene vaccine assembled by chitosan delivery system and preparation method and use thereof
  • Tuberculosis gene vaccine assembled by chitosan delivery system and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0145] Embodiment 1: Construction of pcDNA3.1-ECANS tuberculosis gene vaccine

[0146] 1. Prediction of target T cell epitopes of ECANS tuberculosis gene vaccine

[0147] Through the analysis of BLAST network database, DNAstar biological software and network database (http: / / www.syfpeithi.com / scripts / MHCServer.dll / home.htm), comprehensive hydrophilicity and hydrophobicity, softness, antigen index, surface accessibility, and HLAI, class II molecular binding, and other parameters, predicted four T cell epitopes obtained: the 189-228 gene of Mycobacterium tuberculosis ESAT-6 protein (EAST-6 189-228 ); the 369-405 gene of Mycobacterium tuberculosis Ag85A protein (Ag85A 369-405 ); the 162-207 gene of Mycobacterium tuberculosis CFP-10 protein (CFP10 162-207 ); the 420-459 gene of Mycobacterium tuberculosis Ag85B protein (Ag85B 420-459 ).

[0148] Using pcDNA3.1 or pVAX as the plasmid vector and the Mycobacterium tuberculosis HSP65 gene as the chimeric epitope gene carrier, on th...

Embodiment 2

[0149] Example 2 Construction of pcDNA3.1-ECANS tuberculosis gene vaccine

[0150] In order not to introduce a restriction site between the HSP65 gene and the T cell epitope gene, the present invention utilizes the method of direct synthesis of DNA primers and PCR to sequentially amplify three sections of partially overlapping HSP65 gene from the 5' and 3' ends The gene fragments of T cell epitopes and T cell epitopes were denatured and connected by overlapping complementary sequences, and finally the HSP65 full-length gene chimerized with 4 T cell epitopes was amplified by PCR with 5' and 3' HSP65 primers. At the same time, EcoR I and Hind III restriction sites are respectively placed at both ends of the gene, and can be connected into vector pcDNA3.1(-) or prokaryotic expression vector pET32a after double digestion.

[0151]First extract the DNA of Mycobacterium tuberculosis H37Rv strain (Shanghai Center for Disease Control and Prevention, Department of Tuberculosis) as a te...

Embodiment 3

[0220] Example 3 Construction of pET32a-ECANS prokaryotic expression vector and protein expression and purification

[0221] The amplified ECANS coding gene fragment or HSP65 gene was double-digested with EcoR I and Hind III, and connected with the corresponding prokaryotic expression vector pET32a to construct pET32a-ECANS and pET32a-HSP65 prokaryotic expression plasmids.

[0222] Escherichia coli BL21(DE3) competent cells were transformed with pET32a-ECANS, cultured overnight at 37°C, and positive clones were screened. Shake culture in LB (Amp100μg / ml) liquid medium until A600 reaches about 0.75, and add isopropylthiosemiglucoside (IPTG) to a final concentration of 0.5mM. Continue shaking and culturing for 3 hours, collect the bacteria by centrifugation at 4000r / min for 20min, resuspend in 1×PBS, sonicate, centrifuge at 12000r / min for 20min at 4°C, and harvest the supernatant and precipitate respectively. The supernatant was passed through an affinity chromatography column,...

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Abstract

The invention relates to a tuberculosis gene vaccine assembled by a chitosan delivery system, which consists of chitosan and tuberculosis antigen encoding plasmids, wherein full-length genes of tubercle bacillus heat shock proteins HSP65 are inserted into the tuberculosis antigen encoding plasmids; and four T cell epitope genes EAST-6[189-228], Ag85A[369-405], CFP10[162-207] and Ag85B[420-459] from tubercle bacillus antigens are inserted into the HSP65 full-length genes. The invention also discloses application of the tuberculosis gene vaccine. By performing nasal drip of the gene vaccine on an immune mouse, the gene vaccine is proved to be capable of inducing response to special antibodies of a plurality of tuberculosis antigens, inducing killing response of locally strong tuberculosis special T cells of the whole body and lung mucous membranes, simultaneously inducing immunological response of Th1 which secretes high-level IFN gamma and have the effect which is obviously superior to the prior BCG vaccine, and is a superior vaccine for preventing and treating tuberculosis.

Description

technical field [0001] The invention relates to the field of biological genetic engineering, in particular to a vaccine for preventing or treating tuberculosis and its preparation method and application, in particular to a tuberculosis gene vaccine assembled with a deacetylated chitosan delivery system and its preparation and application. Background technique [0002] The resurgence of 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, the emergence of drug-resistant strains of tuberculosis, and the co-infection of tuberculosis caused by AIDS disease At present, the incidence and death of tuberculosis are very serious. There are 8 million active tuberculosis patients in the world every year and 3 million deaths; about 550 million people in my country have been infected with tuberculosis, 130,000 people die of tuberculosis ever...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K39/04C12N15/31C12N15/79A61P31/06
Inventor 徐薇熊思东岳艳高海峰
Owner FUDAN UNIV
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