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Polyepitope tuberculosis gene vaccine and its prepn process

A gene vaccine and multi-epitope technology, applied in the direction of bacterial antigen components, antibacterial drugs, etc., can solve the problem of low level of vaccine-induced cellular immune response, and achieve the effect of enhancing the level of immune response against Mycobacterium tuberculosis

Inactive Publication Date: 2007-12-19
上海欣安基因免疫与疫苗研究开发有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The object of the present invention is to provide a multi-epitope tuberculosis gene vaccine and its preparation method, which solves the technical problem of the low level of vaccine-induced cellular immune response in the prior art

Method used

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  • Polyepitope tuberculosis gene vaccine and its prepn process
  • Polyepitope tuberculosis gene vaccine and its prepn process
  • Polyepitope tuberculosis gene vaccine and its prepn process

Examples

Experimental program
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Embodiment 1

[0032] Immunological reagents and materials: goat anti-mouse CXCR3 polyclonal antibody (Sant Clous company); FITC-labeled donkey anti-goat IgG (ELISA titer 1:5000, Huamei Bioengineering Co., Ltd.); 2ml and 1ml sterile syringes (Mesha Wa Medical Industry Co., Ltd.). Embodiment 1: Design, construction, identification and expression of HEAT gene vaccine

[0033] Using molecular biology software and network database prediction methods, combined with the relevant reports of the existing tuberculosis protein epitope research, it was determined that the full-length gene of Mycobacterium tuberculosis HSP65, the 1-20 gene and the 61-81 position of ESAT-6 Gene, Ag85A 62-84 gene, Ag85B 121-155 gene, Ag85A 143-166 gene, Ag85B 234-256 gene and MPT64C terminal 177-228 gene are connected in series to construct the gene vaccine of the present invention.

[0034]The present invention utilizes the PCR method and the direct DNA sequence synthesis method to amplify the target gene, and finally a...

Embodiment 2

[0101] Example 2 Construction of multi-epitope tuberculosis gene vaccine

[0102] Using the extracted Mycobacterium tuberculosis H37Rv strain (provided by the Tuberculosis Department of Shanghai Center for Disease Control and Prevention) DNA as a template, use HSP65 upstream (SEQ ID NO: 19) and downstream (SEQ ID NO: 20) specific primers by PCR Methods The HSP65 fragment was amplified, and the PCR product was recovered and purified by low melting point gel.

[0103] The designed cohesive-end complementary DNA sequence 1-7, with T 4 DNA ligase was ligated, and the ligated products were ligated with HSP65 and MPT64 amplified by PCR respectively; using the ligated products as templates, specific primers for HSP65 upstream (SEQ ID NO: 19) and MPT64 downstream (SEQ ID NO: 29) were passed through The PCR method amplifies the full-length gene fragment of the multi-epitope gene vaccine. The results are shown in Figure 2, and a full-length coding gene of 2296 bp was obtained, which ...

Embodiment 3

[0110] Example 3 Construction of pcDNA3-HEAT eukaryotic expression vector and plasmid purification

[0111] The amplified HEAT coding gene fragment was double digested with EcoR I and Xba I, and the digested fragment was recovered, ligated with the corresponding digested vector pcDNA3, the ligated product was transformed into Escherichia coli DH5α, and the transformed colonies were screened with ampicillin. After identification by PCR, enzyme digestion and sequencing, the pcDNA3-HEAT eukaryotic expression plasmid was successfully constructed (Fig. 2A, 2B, 2C).

[0112] The recombinant plasmid pcDNA3-HEAT was transformed into Escherichia coli DH5α, positive clones were screened, cultured with shaking in LB (Amp100μg / ml) liquid medium for 15 hours, the bacteria were collected, and impurities and bacterial endotoxins were removed according to the QIAGEN Plasmid Mega Kit, and finally purified Plasmid DNA, our gene vaccine pcDNA3-HEAT.

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Abstract

The present invention discloses one kind of polyepitope tuberculosis gene vaccine comprising one kind of carrier and one inserted segment of target gene, which has in the 5' end one whole length HSP65 gene, and in the 3' end serially arranged selected epitope genes, including ESAT-6Á€1-20 positions genes and 61-81 positions genes of ESAT-6, 62-84 positions genes of Ag85A, 121-155 positions genes of Ag85B, 143-166 positions genes of Ag85A, 234-256 positions genes of Ag85B and 177-228 positions genes of MPT64C. The present invention discloses the application of the polyepitope tuberculosis gene vaccine. Mouse immunizing experiment shows that the gene vaccine can well induce humoral immunity response. The gene vaccine of the present invention can well induce Th1 type immunity response to mycobacterium tuberculosis and enhance the anti-mycobacterium tuberculosis immunity response level.

Description

technical field [0001] The invention relates to a recombinant gene vaccine, in particular to a vaccine for treating and preventing tuberculosis and a preparation method thereof, in particular to a multi-epitope tuberculosis gene vaccine and a preparation method thereof. Background technique [0002] Studies have shown that heat shock protein 65 (HSP65), one of the members of the heat shock protein 60 family, is highly conserved in prokaryotic and eukaryotic cells, and the HSP sequences from bacteria to human cells have high homology. The full length of Mtb is 1623bp, 540 amino acids, and the molecular weight is 65kD. Under normal circumstances, HSP participates in some important cellular physiological activities, such as protein translocation, folding and assembly, and plays an important role as a molecular chaperone. During Mtb infection, HSP65 is one of the most important immunoprotective antigens against its invasion. In Mtb-infected mice, 20% of effector T cells recogn...

Claims

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Application Information

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IPC IPC(8): A61K39/04A61P31/06
Inventor 熊思东高海峰王缨徐薇
Owner 上海欣安基因免疫与疫苗研究开发有限公司
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