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Mycoplasma bovis VspX gene mutant strain, and construction method and application thereof

A technology of Mycoplasma bovis and a construction method, applied in the prevention and treatment of animal infectious diseases, the construction method of the strain and its application in the fields of Mycoplasma bovis pathogenic mechanism and immune prevention and control

Active Publication Date: 2020-09-08
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no domestic report on the use of min-Tn4001 transposon to construct a random insertion mutant library of Mycoplasma bovis

Method used

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  • Mycoplasma bovis VspX gene mutant strain, and construction method and application thereof
  • Mycoplasma bovis VspX gene mutant strain, and construction method and application thereof
  • Mycoplasma bovis VspX gene mutant strain, and construction method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0016] Embodiment 1: Mycoplasma bovis insertion mutant library construction

[0017] The pMT85 plasmid was donated by Dr. Eric Baranowski of the French Academy of Agricultural Sciences (Baranowski, Guiral et al. 2010), which contains a mini-Tn4001 transposon (mini-Tn Tn4001), which has been introduced into the transposon encoded by the aacA-aphD gene The gentamicin resistance marker, which is located between the two inverted repeats (IR) at both ends of the transposition fragment, and the transposase gene (tnpA) is located outside the repeat sequence, which can prevent transposition from occurring again.

[0018] Use M.bovis HB0801 as the parent strain to construct a mutant library, collect the M.bovis cultured to the late logarithmic phase, wash twice with cold DPBS buffer, and resuspend in 0.1M CaCl 2 The solution was incubated on ice for 30 min; the prepared Mycoplasma bovis competent cells were mixed with 3 μg pMT85 plasmid, 10 μg yeast tRNA and 1 mL 50% PEG8000. After 1 ...

Embodiment 2

[0019] Embodiment 2: Screening and Identification of Mycoplasma bovis VspX Gene Mutants

[0020] 1. Genome sequencing to screen mutant strains

[0021] The genomic DNA (gDNA) of the mutant strain in the mutant library was extracted by CTAB method, and sent to Wuhan Ruizhi Mofang Biotechnology Co., Ltd. to sequence the junction of the transposon sequence and the genome of the mutant strain, and the sequencing results were compared with the whole genome sequence of M.bovis HB0801 The comparison results show that there is a transposon sequence in the VspX gene (sequence shown in SEQ ID NO: 1), and the min-Tn4001 transposon insertion site is located after the 548342 site of the genome and at the 384 site of the VspX gene rear( figure 1 ), the mutant strain was named M.bovis T8.101.

[0022] 2. Western blot to verify protein expression of mutant strains

[0023] After culturing T8.101 and wild strains in PPLO medium for 48 hours, the cells were collected, and after lysing, the w...

Embodiment 3

[0024] Embodiment 3: growth characteristics test of Mycoplasma bovis mutant strain T8.101

[0025] The growth curve and colony morphology of the mutant strains were detected by routine methods.

[0026] 1. Growth curve determination

[0027] After recovering T8.101 and wild strains, they were inoculated on PPLO solid medium, 37°C, 5% CO 2 Cultured in the incubator for 72h. After picking 3 single clones of each strain, they were inoculated in 1mL PPLO liquid medium, cultured at 37°C for 96h, and the bacterial solution was spread on the PPLO plate every 12h. After 72h of culture, the number of colonies was counted under a microscope and Draw the growth curve of each bacteria. The results showed that the growth rate and growth state of T8.101 and the wild strain were similar without significant difference. They were in the logarithmic phase at 12-24h, at the plateau phase at 24-60h, and then entered the decline phase ( image 3 )

[0028] 2. Colony morphology observation

...

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Abstract

The invention discloses a mycoplasma bovis VspX gene mutant strain. The mycoplasma bovis VspX gene mutant strain is named as Mycoplasma bovis T8.101, is preserved in the China Center for Type CultureCollection (CCTCC), and has a preservation number of CCTCC NO: M 20191038, and the VspX gene has a nucleotide sequence shown as SEQ ID NO:1. The invention also discloses a construction method of the mutant strain and an application of the mutant strain in the fields of mycoplasma bovis pathogenic mechanism and immune prevention and control. The mutant strain does not express protein coded by the mutant gene, and the growth curve, colony morphology and size of the mutant strain are not obviously different from those of a wild strain. However, the binding capacity of the mutant strain to bovinepulmonary epithelial cell lines and fibronectin is remarkably reduced, so that the mutant strain has the potential of being applied to the fields of bovine mycoplasma pathogenic mechanism research andprevention and treatment drug research and development as a virulence attenuating strain.

Description

technical field [0001] The invention belongs to the technical field of prevention and control of animal infectious diseases, and specifically relates to a VspX gene mutant strain of Mycoplasma bovis, and also relates to a construction method of the strain and its application in the fields of pathogenic mechanism and immune prevention and control of Mycoplasma bovis. Background technique [0002] Mycoplasma bovis (M.bovis) is one of the important pathogens causing bovine respiratory diseases, leading to a variety of clinical symptoms, mainly including bronchopneumonia, mastitis, arthritis, genital tract inflammation and tenosynovitis. Due to its unclear pathogenic mechanism, there is still a lack of effective vaccines in clinical practice. Antibiotic treatment is ineffective, and the course of treatment is long, and drug resistance is easy to develop. This makes the prevention and control of this disease difficult, and causes huge economic losses to the beef and dairy industri...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12N15/90A61K39/02A61P31/04C12R1/35
CPCA61K39/0241A61K2039/523A61K2039/552A61P31/04C07K14/30C12N15/74C12N15/90C12N2800/90
Inventor 郭爱珍陈曦刘东明朱习芳张慧路豆昆陈颖钰胡长敏陈建国陈焕春
Owner HUAZHONG AGRI UNIV
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