Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Multi-T cell epitope tuberculosis gene vaccine with HSP65 as epitope scaffold

A gene vaccine and gene technology, applied in gene therapy, genetic engineering, plant genetic improvement, etc., can solve problems such as poor prevention and treatment of tuberculosis

Inactive Publication Date: 2014-11-05
SUZHOU UNIV
View PDF5 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The object of the present invention is to provide a multi-T epitope tuberculosis gene vaccine using HSP65 derived from Mycobacterium tuberculosis as an epitope scaffold and its preparation method and application. The multi-T epitope using HSP65 as an epitope scaffold The tuberculosis gene needs to solve the technical problems that the existing vaccines are not effective in preventing and treating tuberculosis due to the ineffectiveness of the conventional BCG vaccine, the emergence of tuberculosis drug-resistant strains and tuberculosis infection caused by AIDS

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Multi-T cell epitope tuberculosis gene vaccine with HSP65 as epitope scaffold
  • Multi-T cell epitope tuberculosis gene vaccine with HSP65 as epitope scaffold
  • Multi-T cell epitope tuberculosis gene vaccine with HSP65 as epitope scaffold

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0187] Embodiment 1: Construction of pcDNA3-PES tuberculosis gene vaccine

[0188] By consulting relevant literature, five T cell epitopes derived from Mycobacterium tuberculosis H37Rv strain were selected: the 1-60 gene of ESAT-6 protein (EAST-6 1-60 ); the 721-765 gene of Ag85B protein (Ag85B 721-765 ); 7-33 gene of MTB protein (MTB10.4 7-33 ); the 721-765 gene of PPE25 protein (PPE25 721-765 ); the 10-54 gene of PE19 protein (PE19 10-54 ). Such as figure 1 As shown, using pcDNA3.1 or pVAX as the plasmid vector and HSP65 gene as the chimeric epitope gene vector, based on the computer prediction of its secondary structure, the position that does not affect the key structure: the 438th base After inserting the 1-60th gene of ESAT-6, after the 453rd base, insert the 721-765th gene of Ag85B, after the 471st base, insert the 7th-33rd gene of MTB10.4, after the 1185th base The 721-765 gene of PPE25 is inserted into the 10-54 gene of PE19, and the 7-33 gene of MTB10.4 prote...

Embodiment 2

[0325] Example 2 Construction of pET32a-HSP65 prokaryotic expression vector and protein expression and purification

[0326] In order to obtain a large amount of HSP65 protein antigen, the amplified HSP65 coding gene was used EcoR I with Hind III Double enzyme digestion, and the prokaryotic expression vector pET32a that has been digested by the corresponding enzymes, are connected to construct the pET32a-HSP65 prokaryotic expression plasmid.

[0327]Escherichia coli BL21 (DE3) competent cells were transformed with pET32a-HSP65, cultured overnight at 37°C, and positive clones were screened. Shake culture in LB liquid medium until A600 reaches 0.6-1.0, and add isopropylthiosemiglucoside (IPTG) to a final concentration of 1mM. Continue shaking culture for 4 hours, collect bacteria by centrifugation at 4000r / min for 20min, resuspend in 1×PBS, sonicate, centrifuge at 12000r / min at 4°C for 20min, and harvest supernatant and precipitate respectively. The supernatant was passed...

Embodiment 3

[0328] Example 3 Preparation of chitosan-pES tuberculosis gene vaccine

[0329] 1) Mass preparation of plasmid DNA: according to QIAGEN Plasmid Mega Kit.

[0330] 2) Prepare chitosan solution and pcDNA3.1-PES solution respectively, the method is as follows: first prepare 0.02% chitosan solution with pH 5.7, weigh 0.02g chitosan (Fluka, MW about 400000, deacetylation degree 75%-85%) , first dissolve it slowly with 500μl-1ml 1% HAc solution, and place it at 37°C for 1hr. Then weigh 0.042g NaAc, with 80ml H 2 O is dissolved, add the above-mentioned completely dissolved chitosan solution, mix well, and adjust the pH value to 5.7 with 1N NaOH, which is 0.02% chitosan solution with pH 5.7. pcDNA3.1-PES plasmid with 5mM Na 2 SO 4 Dissolve, DNA concentration is 1mg / ml, -20 0 C save it for later use.

[0331] 3) Prepare chitosan-DNA nanoparticles by co-precipitation method (co-aggregation method), as follows: in a 55°C water bath, drop 0.02% chitosan solution with pH 5.7 into 8...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
antibody titeraaaaaaaaaa
Login to View More

Abstract

The invention discloses a multi-T cell epitope tuberculosis gene vaccine with HSP65 as an epitope scaffold. The full-length gene of HSP65 protein in which 5 T cell epitope polypeptide genes originated from mycobacterium tuberculosis antigen are embedded is inserted into a vector. A preparation method for the vaccine comprises a step of inserting the 5 T cell epitope genes originated from mycobacterium tuberculosis antigen into the HSP65 full-length gene, wherein the 5 T cell epitope genes comprise EAST-61-60, Ag85B721-765, MTB10.47-33, PPE25721-765 and PE1910-54. It is proved that the tuberculosis gene vaccine can induce response of serum IgG and lung SIgA to the HSP65 vector and induce cellular immunologic response of specific strong secretion of high-level IFN gamma to multiple tuberculosis antigen epitopes through intranasal immunization of mice with a nanometer granular compound formed by the gene vaccine and deacetylated chitosan, so the tuberculosis gene vaccine is a potential vaccine for prevention and treatment of tuberculosis.

Description

technical field [0001] The invention belongs to the field of biological genetic engineering, and relates to a gene vaccine for tuberculosis and its preparation method and application, in particular to a multi-T cell epitope tuberculosis with Mycobacterium tuberculosis heat shock protein 65 (HSP65) as an epitope scaffold Gene vaccine and its preparation method and application. Background technique [0002] The resurgence of tuberculosis is a major global health problem. Due to the ineffectiveness of conventional BCG vaccine for tuberculosis prevention, the emergence of tuberculosis drug-resistant strains, and the co-infection of tuberculosis caused by HIV infection, the incidence and death of tuberculosis are currently high and the situation is very serious. . There are 8 million active tuberculosis patients in the world and 3 million deaths every year; about 550 million people in my country have been infected with tuberculosis, and there are about 120,000 new cases and 130,...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K39/04A61P31/06C12N15/31C12N15/79
Inventor 徐薇吴曼丽熊飞
Owner SUZHOU UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com