Tuberculosis gene vaccine based on T cell epitope as well as preparation method and use thereof

A gene vaccine and epitope technology, applied in gene therapy, antibacterial drugs, pharmaceutical formulations, etc., can solve problems such as poor prevention and treatment of tuberculosis

Inactive Publication Date: 2009-06-10
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to provide a tuberculosis gene vaccine based on T cell epitope and its preparation method and application, said tuberculosis gene vaccine based on T cell epitope will solve the ineffectiveness of

Method used

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  • Tuberculosis gene vaccine based on T cell epitope as well as preparation method and use thereof
  • Tuberculosis gene vaccine based on T cell epitope as well as preparation method and use thereof
  • Tuberculosis gene vaccine based on T cell epitope as well as preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0129] Example 1: Prediction of the target antigen gene of pcDNA3-ECANS gene vaccine

[0130] 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 ).

[0131] Such as figure 1 As shown, it is planned to use pcDNA3.1 or pVAX as the plasmid vector, and use the Mycobacterium tuberculosis HSP65 gene as the gene carrier of the chimeric epitope. On the basis of computer pre...

Embodiment 2

[0132] Example 2 Construction of pcDNA3-ECANS tuberculosis gene vaccine

[0133] 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 segments of 17-base overlap from the 5' and 3' ends. Gene fragments containing part of the HSP65 gene and T cell epitopes are denatured and connected by overlapping complementary sequences, and finally the 5' and 3' HSP65 upper and lower primers are used to PCR amplify chimeric 4 T cell epitopes The full-length gene of HSP65. 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.

[0134] First extract the DNA of Mycobacterium tuberculosis H37Rv strain (Shanghai Center for Disease Control and Prevention, Department of Tuberculosis)...

Embodiment 3

[0204] Example 3 Construction of pET32a-ECANS prokaryotic expression vector and protein expression and purification

[0205] In order to obtain a large amount of HSP65 or ECANS protein antigens, the amplified ECANSHSP65 coding gene was double-digested with EcoRI and HindIII, and connected with the corresponding prokaryotic expression vector pET32a to construct pET32a-ECANS and pET32a-HSP65 prokaryotic expression plasmids .

[0206] 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 w...

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Abstract

The invention discloses a tuberculosis gene vaccine based on T cell epitopes, wherein a full-length gene, embedded with four T cell epitope polypeptide genes which come from mycobacterium tuberculosis antigen, of mycobacterium tuberculosis heat shock protein is inserted into a vector. The invention also discloses a method for preparing the vaccine, which comprises the following steps: four T cell epitope genes, namely EAST-6189-228, Ag85A369-405, CFP10162-207 and Ag85B420-459 which come from the mycobacterium tuberculosis antigen are inserted into an HSP65 full-length gene. The invention also discloses application of an ECANS tuberculosis gene vaccine. Through the intramuscular injection of the gene vaccine into an immune mouse, the experiment proves that the vaccine can induce a specific antibody which aims at a plurality of tuberculosis antigens to response, can induce stronger tuberculosis specific killing response, can induce Th1 immune response at the same time, secrete high-level IFN gamma, and is a good vaccine for preventing and treating tuberculosis.

Description

Technical field: [0001] The invention belongs to the field of biological genetic engineering, and in particular relates to a tuberculosis vaccine and its preparation method and application, in particular to a T cell epitope-based tuberculosis 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, the emergence of tuberculosis drug-resistant strains, and tuberculosis infection caused by AIDS, the incidence and death of tuberculosis are currently very serious. There are 8 million active tuberculosis patients worldwide every year And lead to 3 million deaths; about 550 million people in my country have been infected with tuberculosis, 130,000 people die of tuberculosis every year, and there are about 120,000 new patients every month. In 2006, tuberculosis has become the first infectious disease in my country, and it is very urg...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/62A61K48/00A61P31/06
Inventor 熊思东高海峰徐薇
Owner FUDAN UNIV
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