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sRNA gene for regulating and controlling stress adaptability of Mycobacterium bovis and application of sRNA gene

A technology of Mycobacterium bovis, adaptability, applied in the field of molecular biology, can solve the problems of lack of vaccine, lack of diagnostic gold standard, etc.

Pending Publication Date: 2021-12-31
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the current severe situation of animal tuberculosis lack of vaccines, lack of diagnostic gold standard, and severe drug-resistant bacteria, the research of sRNAs undoubtedly provides a new idea for the development of new prevention and control methods for zoonotic tuberculosis

Method used

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  • sRNA gene for regulating and controlling stress adaptability of Mycobacterium bovis and application of sRNA gene
  • sRNA gene for regulating and controlling stress adaptability of Mycobacterium bovis and application of sRNA gene
  • sRNA gene for regulating and controlling stress adaptability of Mycobacterium bovis and application of sRNA gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Screening of sRNA ncBCG427 and detection of stress adaptation

[0025] 1. RNA-Seq sequencing, data processing and sRNA annotation of Mycobacterium bovis

[0026] Mycobacterium bovis BCG Tokyo strain (gifted by Professor Liu Junyan of Wuhan University) was revived and cultured to infect macrophages for 24 hours, and intracellular bacteria (BCG in the cells after infection of macrophages) and extracellular bacteria (infected macrophages) were collected. After phagocytosis, the extracellular BCG collected from the culture medium) was subjected to RNA-seq sequencing.

[0027]First, the reads containing more than 2-N bases in the original reads of the sequencing data are removed, and then the resulting reads are removed from the low-quality bases, too short reads (less than 13nt) and the adapter is excised. Cleanreads were obtained using the FASTX toolkit. Use Bowtie2 to align the obtained clean reads mapping to the M.tb 1458 (NZ_CP013475) and BCG (NC_012207) ge...

Embodiment 2

[0057] Example 2: Construction and phenotypic determination of ncBCG427 overexpression strain

[0058] 1. Construction of ncBCG427 overexpression strain

[0059] Using the Mycobacterium smegmatis genome as a template, the ribosomal promoter rrnB (-200~-8) fragment was amplified, and pMV261 was transformed into pMV261-P rrnB , to achieve high expression of ncRNA.

[0060] Using the genome as a template to amplify ncBCG427, PCR cycle conditions: pre-denaturation at 98°C for 5 minutes; denaturation at 98°C for 30 s, annealing for 30 s, extension at 72°C for 2 min, and 30 cycle reactions; complete extension at 72°C for 10 min. The reaction system is: template (20ng) 2μL, upstream and downstream primers (10μM) 0.5μL each, 2×Phanta Max Master Mix (Vazyme) 25μL, ddH 2 O 22 μL. The total volume is 50 μL.

[0061] Upstream primers:

[0062] 5'-agcttATGCACGACGACCCTCGCCTCCTCCCCCCCCTGGCGGGGGTCGTCCCTTTTGGCAGa-3' (SEQ ID NO: 2)

[0063] Downstream primers:

[0064] 5'-agcttCTGCCAAAAG...

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Abstract

The invention discloses an sRNA (Ribonucleic Acid) gene for regulating and controlling the stress adaptability of Mycobacterium bovis, and belongs to the field of molecular biology, and the nucleotide sequence of the sRNA gene is shown as SEQ ID NO: 1. The sRNA gene has the remarkable characteristic of regulating the adaptive capacity of the Mycobacterium bovis to carbon starvation, acidification pressure and membrane pressure conditions, and can regulate the bacterial growth, biofilm formation, cell wall permeability, invasion ability to host cells and the like of the Mycobacterium bovis, so that the stress adaptability of the Mycobacterium bovis is regulated and controlled. The Mycobacterium bovis can be used for preparing vaccines or medicines for preventing and treating tuberculosis, and has potential application prospects in the aspects of reducing the bacterial toxicity of the Mycobacterium bovis, improving the sensitivity of the Mycobacterium bovis to anti-tuberculosis medicines and the like; and besides, the gene also has an extremely important reference value in the aspects of clarification of related pathogenic mechanisms of the Mycobacterium bovis and even Mycobacterium tuberculosis complex groups, development of novel diagnostic reagents and the like.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to an sRNA gene regulating the adversity adaptability of Mycobacterium bovis and its application. Background technique [0002] Zoonotic tuberculosis is a chronic wasting disease caused by Mycobacterium tuberculosis Complex (MTBC) infection. Almost all vertebrates, including humans, are susceptible. Among them, Mycobacterium bovis (M.bovis) is the main pathogenic bacterium that causes tuberculosis in animals. It is highly homologous to Mycobacterium tuberculosis (M.tb), which mainly causes tuberculosis in humans, with 99.95% gene level similarity, so the infection spectrum crosses each other. [0003] The research results show that the overall individual prevalence rate of bovine tuberculosis in my country from 2010 to 2019 is about 2.4%, and about 259,000 cattle are infected (Gong QL, et al., 2021); Among tuberculosis cases, about 140,000 people, or 1.8% of all cases, were infected...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/74C12N1/21C12R1/32
CPCC12N15/113C12N15/74C12N2310/14
Inventor 陈颖钰郭爱珍翟文俊章恺伦胡长敏陈建国陈曦陈焕春
Owner HUAZHONG AGRI UNIV