Application of mycobacterium tuberculosis protein Rv1508c to preparation of antituberculotic drug sensitizer

A Mycobacterium tuberculosis, anti-tuberculosis technology, applied in the direction of antibacterial drugs, peptide/protein components, medical preparations containing active ingredients, etc., can solve the problems of drug resistance and aggravate the burden of patients, and achieve increased sensitivity, Effects of increasing drug sensitivity

Active Publication Date: 2018-08-03
WUHAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In order to avoid the emergence of drug resistance during drug treatment, the combination of anti-tube

Method used

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  • Application of mycobacterium tuberculosis protein Rv1508c to preparation of antituberculotic drug sensitizer
  • Application of mycobacterium tuberculosis protein Rv1508c to preparation of antituberculotic drug sensitizer
  • Application of mycobacterium tuberculosis protein Rv1508c to preparation of antituberculotic drug sensitizer

Examples

Experimental program
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Effect test

Example Embodiment

[0021] [Example 1] Construction and identification of the recombinant pMV261 prokaryotic shuttle plasmid of partial genes in the RD6 region, RD9 and RD15 regions

[0022] After extracting the H37Rv genome, use the RD6, RD9 and RD15 region gene primers (as shown in Table 1) to obtain the RD6, RD9 and RD15 region genes from the H37Rv genome by PCR, and recover the PCR products with the rubber tapping recovery kit. The recovered product and the pMV261 vector were double-digested with enzymes BamHI, EcoRI, and HindIII. After recovery, they were ligated with high-efficiency T4 ligase Ligation high and transformed into E.coli DH5, plated and cultured overnight. Single cell clones were picked, plasmids were extracted and sequenced. Successfully constructed 11 recombinant plasmids, which were successfully identified by double enzyme digestion. figure 1 The molecular weight of each gene has been marked. Sequencing results showed that the sequence was correct.

[0023] Table 1: RD6 r...

Example Embodiment

[0026] [Example 2] Shuttle recombinant plasmid pMV261-Rv series plasmid electroporation to Mycobacterium smegmatis

[0027] After preparing the competent Mycobacterium smegmatis, add 1 μL of the recombinant plasmid to 200 μL of the competent Mycobacterium smegmatis, bathe in ice for 15-20 minutes, perform aseptic operation, mix well, and then transfer to the electroporation cup. After 18-25ms of electroporation with an electroporator from Bio-Rad, USA, add 1ml of mycobacterial disease medium, incubate at 37°C and 100rpm for 1 hour, then spread on 7H11 solid medium containing Kan, pick positive clones after 3 days, and make colonies PCR identification. The results of PCR showed that when the rMS:RDs colonies were used as templates, RDs bands of the correct size could be amplified. The result is as figure 2 shown.

Example Embodiment

[0028] [Example 3] rifampicin, isoniazid and streptomycin are to the mensuration of the half-inhibitory concentration of recombinant Mycobacterium smegmatis

[0029] In order to screen the genes sensitive or resistant to rifampicin, isoniazid and streptomycin in the RD region, we will combine Mycobacterium smegmatis and recombinant Mycobacterium After 2 days of co-culture with hydrazine and streptomycin, use a microplate reader The A600 value of the bacteria was detected, and the recombinant Mycobacterium smegmatis was cultivated without adding drugs as a negative control, and the wells only added with medium were used as a blank control. Use Graphpad Prism5 to calculate IC50 value, and then use GraphpadPrism5 to draw a histogram, and do biostatistical analysis, the results are as follows image 3 shown. The results showed that rifampicin and isoniazid had no significant effect on the above genes; while in the streptomycin group, streptomycin had no significant effect on My...

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Abstract

The invention provides an application of mycobacterium tuberculosis protein Rv1508c to preparation of an antituberculotic drug sensitizer, and belongs to the technical field of biology. It is determined for the first time that the mycobacterium tuberculosis functional protein Rv1508c can improve sensitivity of mycobacterium tuberculosis to streptomycin, that is, the protein can promote a streptomycin drug to kill mycobacterium, and the protein can serve as a drug resistance reversal agent and a streptomycin sensitizer of an antituberculotic drug. New path and means can be provided for tuberculosis treatment by the mycobacterium tuberculosis functional protein Rv1508c as a new auxiliary component of the antituberculotic drug.

Description

technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to the application of mycobacterium tuberculosis functional protein Rv1508c in the preparation of anti-tuberculosis drug sensitizers. Background technique [0002] Tuberculosis is a chronic infectious disease that seriously endangers the health of humans and animals. Tuberculosis caused by Mycobacterium tuberculosis is a zoonotic tuberculosis. Nearly one-third of the world's population is currently infected with Mycobacterium tuberculosis. According to WHO's "Global Tuberculosis Report 2016", it is estimated that there will be 10.4 million new tuberculosis cases in 2015, and 1.4 million deaths due to tuberculosis, exceeding the death toll from other infectious diseases. sum. The number of tuberculosis patients in my country ranks third in the world. [0003] The number of multi-drug-resistant tuberculosis patients is growing very rapidly. According to data, in 2012, the num...

Claims

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Application Information

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IPC IPC(8): A61K38/16A61P31/06G01N33/68G01N33/94
CPCA61K38/164A61P31/06G01N33/68G01N33/9446
Inventor 章晓联左睿琪
Owner WUHAN UNIV
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