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Mycobacterium bovis BCG vaccine low-adhesion low-invasiveness mutant strain B2909

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 realize immune protection function, and no vaccine, and achieve the effect of reducing the growth rate

Active Publication Date: 2019-10-08
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Not only that, but for many special groups, especially immunocompromised groups (such as AIDS patients), BCG vaccine not only fails to realize its immune protection function, but also accelerates the occurrence of tuberculosis, which has great potential safety hazards
Even so, in the continuous development of tuberculosis vaccines, there is still no vaccine superior to BCG

Method used

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  • Mycobacterium bovis BCG vaccine low-adhesion low-invasiveness mutant strain B2909
  • Mycobacterium bovis BCG vaccine low-adhesion low-invasiveness mutant strain B2909
  • Mycobacterium bovis BCG vaccine low-adhesion low-invasiveness mutant strain B2909

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Screening and Identification of Mycobacterium bovis BCG Low Invasion Mutants

[0028] 1.1 High-throughput screening of low-invasive mutants of Mycobacterium bovis

[0029] 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

[0035] Example 2: Detection of the adhesion ability of Mycobacterium bovis BCG low-invasive mutants

[0036] In order to verify the adhesion ability of mutants, A549 cells were divided into 2 × 10 5 One per well was spread to a 12-well cell culture plate, and the mutant was inoculated into A549 cells with an infection ratio of 10:1, and the wild strain of BCG was set as a control. The mutants were incubated with A549 cells for 30 min at 4°C. After incubation, wash 3 times with phosphate buffered saline (ie, PBS, 0.01M, pH 7.4 PBS, Hyclone Company), then add 1 mL of 0.025% Triton X-100 to each well, place it at room temperature for 15 min, and use a pipette After the liquid container was blown repeatedly, 100 μL was taken for serial 10-fold dilution, and the lysate with an appropriate dilution factor was evenly spread on a 7H11 plate, and colony counting was performed after culturing in a 37°C incubator for about 21 days. The results showed that the adhesion ability of the B2...

Embodiment 3

[0037] Embodiment 3: Detection of the growth curve of Mycobacterium bovis BCG

[0038] Take the Mycobacterium bovis BCG wild strain and the mutant strain B2909 to inoculate the 7H9 liquid medium at a ratio of 1:100 (volume ratio), and place it 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 was significantly higher than that of the wild strain on the 21st day and the 27th day ( Image 6 ), both increased by 1.2 times. Statistically significant difference. It is suggested that the gene inactivated by the mutant strain B2909 has the function of reducing the growth rate of bacteria.

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Abstract

The invention belongs to the technical field of animal infectious disease prevention, and relates to a mycobacterium bovis BCG vaccine low-adhesion low-invasiveness mutant strain B2909. The mutant strain contains a mutant gene of mycobacterium bovis BCG vaccine FadD18, and a nucleotide sequence thereof is shown as SEQ ID NO: 1. The mutation site is located at the 3925305 site of the genome, and islocated at the 581 site of the FadD18 gene sequence, and the gene is a novel structural gene and a functional gene. The mutant strain of the invention has low adhesion, low invasiveness, high growthrate, and no morphological difference from the wild strain. The mutant strain B2909 is preserved at the China Center for Type Culture Collection with a preservation number of CCTCC NO: M 2018866. Themutant gene and the mutant strain thereof of the present invention are expected to be applied in a mycobacterium bovis pathogenic mechanism research, an immune mechanism research and preparation of avaccine or a drug for preventing 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 method for isolation, identification and preparation of Mycobacterium bovis BCG low-adhesion and low-invasiveness mutant strain B2909. The mutant strain contains the mutant gene of Mycobacterium bovis BCG FadD18. Compared with the wild strain, the mutant strain of the present invention has low adhesion and low invasion ability, and its later growth rate is significantly higher than that of the wild strain. Background technique [0002] Tuberculosis is an important chronic wasting zoonotic disease and the number one killer of human deaths caused by a single pathogen. In 2017, the disease has caused 1.3 million deaths worldwide [1]. In the development history of thousands of years, the pathogenic Mycobacterium tuberculosis has escaped the immune response of the host in various forms and co-evolved with humans, posing a huge thre...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N1/20C12R1/32
CPCC07K14/35
Inventor 陈颖钰李倩倩郭爱珍郭佳成师红铃胡长敏陈焕春
Owner HUAZHONG AGRI UNIV
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