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Tuberculosis mucosa gene vaccine assembled by using chitosan oligosaccharide delivery system and preparation and application of tuberculosis mucosa gene vaccine

A delivery system and gene vaccine technology are applied to the tuberculosis mucosal gene vaccine assembled by the chitosan oligosaccharide delivery system and the fields of preparation and application thereof, which can solve the problems of poor prevention and treatment of tuberculosis and the like

Inactive Publication Date: 2014-03-05
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 mucosal gene vaccine assembled by a chitosan oligosaccharide delivery system and its preparation and application. The ineffectiveness of the vaccine, the emergence of drug-resistant strains of tuberculosis and the concurrent infection of tuberculosis caused by AIDS make the existing vaccines ineffective in preventing and treating tuberculosis.

Method used

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  • Tuberculosis mucosa gene vaccine assembled by using chitosan oligosaccharide delivery system and preparation and application of tuberculosis mucosa gene vaccine
  • Tuberculosis mucosa gene vaccine assembled by using chitosan oligosaccharide delivery system and preparation and application of tuberculosis mucosa gene vaccine
  • Tuberculosis mucosa gene vaccine assembled by using chitosan oligosaccharide delivery system and preparation and application of tuberculosis mucosa gene vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0138] Embodiment 1: Construction of pcDNA3.1-ECANS tuberculosis gene vaccine

[0139] 1. Prediction of target T cell epitopes of ECANS tuberculosis gene vaccine

[0140] 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: 189-228 genes of Mycobacterium tuberculosis ESAT-6 protein (ESAT-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 ).

[0141]Using pcDNA3.1 or pVAX as the plasmid vector and the Mycobacterium tuberculosis HSP65 gene as the chimeric epitope gene carrier, on the ba...

Embodiment 2

[0142] The construction of embodiment 2pcDNA3.1-ECANS tuberculosis gene vaccine

[0143] 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 chimeric with four T cell epitopes was amplified by PCR with 5' and 3' HSP65 primers. At the same time, EcoRI and Hind III enzyme cutting sites were respectively placed at both ends of the gene, which can be connected into the vector pcDNA3.1(-) or the prokaryotic expression vector pET32a after double digestion.

[0144] Firstly, the DNA of Mycobacterium tuberculosis H37Rv strain was extracted, used as a template, and the HSP65 fragment was amplified ...

Embodiment 3

[0214] Example 3 Isolation of mouse lung lymphocytes

[0215] Take the mouse lung tissue 10 days after the last immunization, weigh about 100mg / mouse, mix the lungs of 3 mice in each group, cut them into pieces and place them in 10ml of digestive solution (collagenase (1mg / ml), DNase (5U / ml) ), 1640-10% fetal bovine serum), shake at 180rpm on a shaker at 37°C for 120min. Pass the suspension through nylon finger cots to remove fragments, centrifuge at 1500rpm for 5min, resuspend the cells in 4ml 40% Percoll, gently add to an equal volume of 70%Percoll, centrifuge at 2400rpm for 30min, absorb lymphocytes, wash once with 10ml PBS, Resuspend in 1640-10% fetal bovine serum culture medium, adjust the concentration to 5×10 6 / ml spare.

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Abstract

The invention discloses a tuberculosis mucosa gene vaccine assembled by using a chitosan oligosaccharide delivery system. The vaccine is formed by copolymerizing, crosslinking and compounding a chitosan oligosaccharide and a tuberculosis antigen encoding plasmid; a full-length gene of a heat shock protein HSP65 derived from a mycobacterium tuberculosis H37Rv strain is inserted into the tuberculosis antigen encoding plasmid; and four T cell epitope genes, namely, ESAT-6189-228, Ag85A369-405, CFP10162-207 and Ag85B420-459 derived from the mycobacterium tuberculosis H37Rv strain are inserted into the HSP65 full-length gene. The invention further discloses a preparation method and an application of the tuberculosis mucosa gene vaccine. The tuberculosis mucosa gene vaccine disclosed by the invention can be used for inducing tuberculosis specific serum antibodies and systemic Th1 immune response, can be used for inducing lung mucosa locally-enhanced tuberculosis specific secretion type SIgA and IFNgama+Th1 response, and has a remarkably superior effect to that of a naked pECANS gene vaccine.

Description

technical field [0001] The invention relates to the field of biogenetic engineering, in particular to a vaccine for preventing or treating tuberculosis and its preparation method and application, in particular to a tuberculosis mucosa gene vaccine assembled with a chitosan oligosaccharide 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 ev...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K47/48A61K39/04C07K14/35C12N15/79A61P31/06
Inventor 徐薇熊思东艾文清
Owner FUDAN UNIV
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