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Mycobacterium bovis bacillus calmette guerin vaccine low-invasiveness mutant strain B2801

A technology of mycobacterium bovis and mutant strains, applied in the direction of bacteria, biochemical equipment and methods, applications, etc., can solve the problems of accelerating tuberculosis, inability to achieve immune protection function, and no superiority to BCG, to reduce the growth rate and significantly Rapid growth performance, enhanced intracellular survival effect

Active Publication Date: 2019-05-31
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although BCG has undoubtedly saved tens of thousands of lives, for many special groups, especially immunocompromised groups (such as AIDS patients), BCG not only fails to achieve its immune protection function, but accelerates the occurrence of tuberculosis
In order to solve this problem, more and more scholars are committed to developing a safer and more effective tuberculosis vaccine, but there is still no vaccine superior to BCG

Method used

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  • Mycobacterium bovis bacillus calmette guerin vaccine low-invasiveness mutant strain B2801
  • Mycobacterium bovis bacillus calmette guerin vaccine low-invasiveness mutant strain B2801
  • Mycobacterium bovis bacillus calmette guerin vaccine low-invasiveness mutant strain B2801

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Screening and Identification of Mycobacterium bovis BCG Low Invasion Mutants

[0031] 1.1 High-throughput screening of low-invasive mutants of Mycobacterium bovis

[0032] The clones in the Mycobacterium bovis BCG mutant library were transferred one by one to 7H9 liquid medium (7H9 liquid medium was purchased from BD Company) for culture, and the A549 cell (gifted by Professor Luiz Bermudez, Oregon State University, USA) infection model was used to infect the cattle. High-throughput screening of mycobacterial BCG mutant libraries. A549 cells were cultured in a 12-well cell culture plate until they grew into a monolayer and reached 2×10 5 After each well, add bacteria according to the infection ratio of 10:1, at 37°C, 5% CO 2 Add gentamicin (final concentration: 100 μg / ml) to the incubator for 1 hour to kill the extracellular bacteria, and after washing thoroughly, use Triton X-100 (Bio-Rad) to lyse the cells, collect the intracellular bacteria and coat the ...

Embodiment 2

[0038] Example 2: Detection of Intracellular Survival Ability of Mycobacterium bovis BCG Low Invasion Mutant

[0039] In order to verify the intracellular survival ability of the mutant, A549 cells were divided into 2×10 5 One per well was placed on a 12-well cell culture plate, and the B2801 mutant strain was inoculated into A549 cells with an infection ratio of 10:1, and the wild strain of Mycobacterium bovis BCG was set as a control. Mutant B2801 and A549 cells were incubated at 37°C, 5% CO 2 After incubating for 1 h, discard the supernatant, add phosphate buffer (0.01M, pH 7.4 PBS, Hyclone Company) to wash thoroughly for 3 times, then add gentamicin (final concentration 100 μg / ml) to kill extracellular bacteria, and then Placed at 37°C, 5% CO 2 Cultured in the incubator, this time was recorded as infection 0h. After 4h, 12h, and 24h of infection, the cells were fully washed with PBS, and the number of mutant colonies was quantitatively determined by colony counting afte...

Embodiment 3

[0040] Embodiment 3: Detection of the growth curve of Mycobacterium bovis BCG

[0041] Take the Mycobacterium bovis BCG wild strain and the mutant strain B2801 to inoculate 7H9 liquid medium at a ratio of 1:100 (volume ratio), and let it stand at 37°C, 5% CO 2 Continuous culture in the incubator for 27 days, take appropriate bacterial solution every 3 days to measure OD 630 The results showed that the growth rate of the mutant strain in the logarithmic phase was significantly higher than that of the wild strain ( Figure 6 ). Compared with the wild strain, the mutant strain B2801 increased by 1.2, 1.1, 1.2, 1.1 and 1.1 times on the 12th day, 15th day, 18th day, 21st day and 24th day of growth, respectively. Statistically significant difference. It is suggested that the mutant B2801 gene has the function of reducing the growth rate of bacteria.

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Abstract

The invention belongs to the technical field of infectious disease prevention and control of animals, and relates to a mycobacterium bovis bacillus calmette guerin vaccine low-invasiveness mutant strain B2801. The mutant strain contains a mutant gene of a mycobacterium bovis BCG_2658, the nucleotide sequence of the mutant gene is shown as SEQ ID NO.1, a mutation site is located behind a site 2922763 of a genome and behind a site 748 of the BCG_2658 gene sequence, and the mutant strain is a novel structural gene and a functional factor. The mutant strain has low invasiveness, low intracellularsurvival capacity, high growth speed, no cord-like structure and microcolony morphology. The mutant strain is preserved at the China Center for Type Culture Collection, with the preservation CCTCC NO:M 2018864. The seperated mutant gene and the mutant strain thereof can be expected to be applied to research on the pathogenic mechanism and immune mechanism of mycobacterium bovis and preparation ofdrugs for preventing and treating bovine tuberculosis.

Description

technical field [0001] The invention belongs to the technical field of animal infectious disease prevention and control, and specifically relates to a low-invasive mutant strain of Mycobacterium bovis BCG_2801, which contains a mutant gene of Mycobacterium bovis BCG_2658. Compared with the wild strain, the present invention The mutants showed low invasiveness, low intracellular viability, high growth rate, no cord-like structure and small colony morphology. Background technique [0002] Bovine tuberculosis is a chronic wasting zoonotic disease that occurs in many animals and humans, and has brought huge economic losses to the world's cattle industry. According to the OIE report, in 2014-2015, among the 180 member countries reporting through the World Animal Health Information System, only 7 member countries reported no bovine tuberculosis in their cattle. Mycobacterium bovis (M.bovis) is the main pathogenic bacterium that causes bovine tuberculosis, and it has 99.95% homolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N1/21C12R1/32
Inventor 陈颖钰郭佳成郭爱珍李倩倩师红玲胡长敏陈焕春
Owner HUAZHONG AGRI UNIV
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