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Application of Mycobacterium tuberculosis Rv1909c gene to preparation of medicaments for treating latent infection of Mycobacterium tuberculosis

A technology of Mycobacterium tuberculosis and rv1909c, which is applied in gene therapy, antibacterial drugs, and microbial-based methods, can solve the problems of low resolution and inaccurate quantification, achieve high sensitivity, reduce false positive interference, and specificity sex-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 overcomes the disadvantages of inaccurate quantification and low resolution of traditional PCR

Method used

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  • Application of Mycobacterium tuberculosis Rv1909c gene to preparation of medicaments for treating latent infection of Mycobacterium tuberculosis
  • Application of Mycobacterium tuberculosis Rv1909c gene to preparation of medicaments for treating latent infection of Mycobacterium tuberculosis
  • Application of Mycobacterium tuberculosis Rv1909c gene to preparation of medicaments for treating latent infection of Mycobacterium tuberculosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Under anaerobic and normal conditions, high expression of the Mycobacterium tuberculosis Rv1909c gene can inhibit the growth of Mycobacterium tuberculosis

[0041] 1. Construction of Mycobacterium tuberculosis Rv1909c Gene High Expression Plasmid

[0042] Using the Mycobacterium tuberculosis H37Rv genome as a template, the Rv1909c 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 The colonies were inoculated into 3ml of liquid LB medium containing kanamycin, shaken overnight at 37°C, and the plasmid was extracted from the bacterial solution for sequencing. The plasmid with correct sequencing was the constructed high-expression plas...

Embodiment 2

[0045] Example 2: Recombinant BCG constructed with the Mycobacterium tuberculosis Rv1909c gene

[0046] 1. Construction of recombinant BCG

[0047] Using the genomic DNA of Mycobacterium tuberculosis H37Rv strain as a template, the Rv1909c 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 ...

Embodiment 3

[0054] Embodiment 3: the recombinant subunit vaccine constructed with the Mycobacterium tuberculosis Rv1909c gene

[0055] 1. Construction of recombinant plasmids

[0056] Using the genomic DNA of Mycobacterium tuberculosis H37Rv strain as a template, the Rv1909c gene was amplified by PCR and cloned into the eukaryotic expression plasmid vector pcDNA3.1+ (such as Figure 4 shown), sequenced and identified. Prepare E.coliDH5α competent cells, identify the correct pcDNA-Rv1909c gene recombinant plasmid, transform into competent cells, and culture overnight at 37°C in LB medium. A large amount of extraction of recombinant plasmids adopts the endotoxin-removing plasmid mass extraction kit of Qiagan Company;

[0057] 2. Analysis of immune protection of recombinant subunit vaccine

[0058] The above subunit vaccine was immunized to BalB / c mice once every two weeks for a total of 3 times, and a PBS group was set as a control. Four weeks after the last immunization, the M.tb H37Rv...

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Abstract

The invention belongs to the field of biotechnology, and more specifically relates to a new application of a Mycobacterium tuberculosis Rv1909c gene to preparation of medicaments for treating latent infection of Mycobacterium tuberculosis. Researches show that Rv1909c has an important biological function in the process when Mycobacterium tuberculosis enters the latency; compared with Mycobacterium tuberculosis which grows normally, high expression of the Rv1909c gene inhibits growth of Mycobacterium tuberculosis. The invention also discloses a kit for detecting tuberculosis in latent period using the gene as a target, and a vaccine which is constructed based on the gene for preventing and treating latent infection of Mycobacterium tuberculosis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a new application of the Rv1909c gene of mycobacterium tuberculosis in the medicine 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] Mycobacterium tuberculosis is the pathogen 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. One of the main reasons for the success of Mycobacterium tuberculosis is that it can latent for a long time in the host cell, and it has a good adaptability to hypoxic conditions. After infecting the host, Mycobacterium tuberculosis will qu...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04A61K48/00A61K39/04A61P31/06C12R1/32
Inventor 孙娴李瑶姜君张鹭吴海刘军
Owner FUDAN UNIV
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