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Application of mycobacterium tuberculosis Rv0324 gene in preparation of preparation for treating mycobacterium tuberculosis latent infection

A technology of Mycobacterium tuberculosis, rv0324, applied in gene therapy, microorganism-based methods, antibacterial drugs, etc., can solve the problems of inaccurate quantification, low resolution, etc., to reduce false positive rate, reduce sensitivity, detect Sensitivity-enhancing effect

Inactive Publication Date: 2016-08-17
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since only the product of the exponential phase reaction is detected and the signal amplification effect of the fluorophore, it can overcome the shortcomings of traditional PCR, such as inaccurate quantification and low resolution.

Method used

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  • Application of mycobacterium tuberculosis Rv0324 gene in preparation of preparation for treating mycobacterium tuberculosis latent infection
  • Application of mycobacterium tuberculosis Rv0324 gene in preparation of preparation for treating mycobacterium tuberculosis latent infection
  • Application of mycobacterium tuberculosis Rv0324 gene in preparation of preparation for treating mycobacterium tuberculosis latent infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Under anaerobic and normal conditions, high expression of the Mycobacterium tuberculosis Rv0324 gene can inhibit the growth of Mycobacterium tuberculosis

[0037] 1. Construction of high expression plasmid of Mycobacterium tuberculosis Rv0324 gene

[0038] Using the Mycobacterium tuberculosis H37Rv genome as a template, the Rv0324 gene was amplified by PCR and cloned into the Escherichia coli-Mycobacterium tuberculosis shuttle plasmid pMV261 (such as figure 1 shown), transformed into Escherichia coli DH5a competent cells, the transformation solution was spread on a solid LB medium plate containing 0.05mg / ml kanamycin, cultured upside down at 37°C, positive clones were screened, and single clones were picked Inoculate the colonies into 3ml of liquid LB medium containing kanamycin, shake overnight at 37°C, extract the plasmids from the bacterial solution for sequencing, and the plasmids with correct sequencing are the constructed high-expression plasmid pMV261-...

Embodiment 2

[0041] Example 2: Recombinant BCG constructed with the Mycobacterium tuberculosis Rv0324 gene

[0042] 1. Construction of recombinant BCG

[0043] Using the genomic DNA of Mycobacterium tuberculosis H37Rv strain as a template, the Rv0324 gene was amplified by PCR and cloned into the Escherichia coli-Mycobacterium tuberculosis shuttle plasmid pMV361 (such as image 3 Shown), after the recovery of BCG, inoculate into 400ml 7H9 (adding 10% ADC and glycerol) culture medium for 10-14 days, in the logarithmic growth phase, add 100ml of 20% glycine to a final concentration of 4%, continue to cultivate for 24h, Glass beads were used to break the bacterial film, and washed with 10% glycerin to prepare BCG competent cells. The competent cells were transferred to the electroporation cup, and electroporation was carried out at 2.5kv / cm, 25μF, and 1000 ohms. The transformation solution was added with 15ml 7H9 (adding 10% ADC, glycerol, and Tween80) culture medium, cultivate overnight at 3...

Embodiment 3

[0050] Embodiment 3: the recombinant subunit vaccine constructed with the Mycobacterium tuberculosis Rv0324 gene

[0051] 1. Construction of recombinant plasmids

[0052] Using the Mycobacterium tuberculosis H37Rv strain genomic DNA as a template, the Rv0324 gene was amplified by PCR and cloned into the eukaryotic expression plasmid vector pcDNA3.1+ (such as Figure 4 Shown), sequencing identification, preparation of E.col iDH5α competent cells, identification of the correct pcDNA-Rv0324 gene recombinant plasmid transformed into competent cells, in LB medium, 37 ° C overnight culture, a large number of recombinant plasmids extracted by Qiagan company Endotoxin-free plasmid mass extraction kit;

[0053] 2. Analysis of immune protection of recombinant subunit vaccine

[0054] The above-mentioned subunit vaccine was immunized to BalB / c mice once every two weeks for a total of 3 times. At the same time, the PBS group was set as a control. Four weeks after the last immunization, ...

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Abstract

The invention belongs to the technical field of biology, and more specifically relates to an application of mycobacterium tuberculosis Rv0324 gene in preparation of a preparation for treating mycobacterium tuberculosis latent infection. Through research, Rv0324 has important biological function during a process that mycobacterium tuberculosis enters into a latent state, compared with the normally grown mycobacterium tuberculosis, high expression of the Rv0324 gene can inhibit the growth of mycobacterium tuberculosis. The invention also discloses a kit by taking the gene as a target for detecting tuberculosis in a latent stage and a vaccine constructed by the gene and used for preventing and treating mycobacterium tuberculosis latent infection.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to the use of the Rv0324 gene of mycobacterium tuberculosis in preparing preparations for treating latent infection of mycobacterium tuberculosis. The invention also relates to a vaccine for preventing and treating latent infection of mycobacterium tuberculosis constructed with the gene. Background technique [0002] The prior art discloses that Mycobacterium tuberculosis is the pathogenic bacterium that causes tuberculosis. According to the report of the World Health Organization, 1 / 3 of the world's population is infected with Mycobacterium tuberculosis, and at least 3 million people die from the disease every year. Tuberculosis is still a very important infectious disease. Studies have shown that Mycobacterium tuberculosis can remain latent in host cells for a long time, and it is well adapted to hypoxic conditions. After infecting the host, Mycobacterium tuberculosis will qu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04A61K48/00A61K39/04A61P31/06C12R1/32
Inventor 孙娴李瑶姜君吴海张鹭杨秀方
Owner FUDAN UNIV
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