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Mycoplasma bovis Mbov_0475 gene mutant strain and application thereof

A technology of mycoplasma bovis and gene, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems that hinder the development of new drugs and vaccines, unclear pathogenic mechanism, and hinder the effective prevention and treatment of bovine mycoplasma disease

Active Publication Date: 2020-10-09
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the unclear pathogenic mechanism of M. bovis seriously hinders the development of new drugs and vaccines, and thus hinders the effective prevention and treatment of M. bovis disease.

Method used

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  • Mycoplasma bovis Mbov_0475 gene mutant strain and application thereof
  • Mycoplasma bovis Mbov_0475 gene mutant strain and application thereof
  • Mycoplasma bovis Mbov_0475 gene mutant strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: Construction of Mycoplasma bovis insertion mutant library

[0023] The applicant isolated a strain of Mycoplasma bovis HB0801 from the diseased lung tissue of sick cattle in June 2008, and named it Mycoplasma bovis HB0801, Mycoplasma bovis HB0801. This strain has been disclosed in the patent document of CN 102220263A.

[0024] The pMT85 plasmid was donated by Dr. Eric Baranowski of the French Academy of Agricultural Sciences in France. It contains a mini-type Tn4001 transposon (mini-Tn4001), and the gentamicin resistance marker encoded by the aacA-aphD gene has been introduced into the transposon. Located between two inverted repeats (IRs) at both ends of the transposable fragment, the transposase gene (tnpA) is located outside the repeats and prevents transposition from occurring again (Baranowski et al 2010).

[0025] Use M.bovis HB0801 as the parent strain to construct a mutant library, collect the M.bovis cultured to the late logarithmic phase, wash tw...

Embodiment 2

[0026] Example 2. Genome sequencing and identification of mutant strains in the Mycoplasma bovis mutant library

[0027] 2.1 Genome sequencing to obtain the MbovP0475 mutant strain of Mycoplasma bovis

[0028] Using the Bacterial Genome Extraction Kit (purchased from Bao Biological Engineering Dalian Co., Ltd.), extract the total DNA of Mycoplasma bovis in the Mycoplasma bovis T9.55 mutant library, sequence the junction of the Tn4001 transposon and the Mycoplasma bovis genome, and sequence the results Compared with the whole genome sequence of Mycoplasma bovis HB0801, the results show that the gene Mbov_0475 involved in the mutant strain T9.55 contains the transposon insertion sequence, the size is 3438bp, the T9.55 mutant gene is Mbov_0475, the transposon insertion site After the 555965 site in the genome, and after the 555 site of the Mbov_0475 gene ( figure 1 ).

[0029] 2.2 Western blot to verify the expression defect of the mutant strain MbovP0475

[0030] The construc...

Embodiment 3

[0031] Example 3: Effects of Mutants on BoMac Cell Proliferation and Viability

[0032]3.1 Detection of cell viability after BoMac infection with Mycoplasma bovis

[0033] The cell viability of Mbov_0475 gene deletion / completion strain and wild strain HB0801 infected cells was detected by CCK-8 method for 12h, 24h, and 36h. 5000 BoMac cells per well were inoculated in a 96-well plate, cultured at 37°C, 5% CO2 until adherent, and blank wells (no cells) and control wells (only cells) were set, and 4 replicate wells were set for each group; Incubate overnight at 37°C, 5% CO2, and observe under an inverted microscope. Take out the cell plate and add 2.5×10 4 CFU Mycoplasma bovis wild strain HB0801 or Mbov_0475 gene deletion strain or Mbov_0475 gene complementation strain, 37°C, 5% CO2 static co-culture for 12h, 24h, 36h, add 10μL CCK-8 (purchased from Japanese colleagues) to each well, 37°C , 5% CO2Incubate for 1h, measure the absorbance value of each well at 450nm, calculate t...

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Abstract

The invention discloses a mutant strain of a mycoplasma bovis Mbov_0475 gene, which belongs to the technical field of animal infectious disease prevention. The mutant strain is named as Mycoplasma bovis T9.55, and is preserved in China Center for Type Culture Collection, a preservation number is CCTCC NO: M 2020082, and the nucleotide sequence of the Mbov_0475 gene is shown as SEQ ID NO: 1. The strain has the effects of inducing macrophage proliferation and improving macrophage activity, and because bovine macrophages are important immune cells of a host for resisting pathogenic infection, themutant strain is expected to play an important role in the field of mycoplasma bovis immune prevention and treatment.

Description

technical field [0001] The invention belongs to the technical field of prevention and treatment of animal infectious diseases, and in particular relates to a mutant strain of the mycoplasma bovis Mbov_0475 gene. The mutant strain can induce the proliferation of macrophages and improve the activity of macrophages. Background technique [0002] Mycoplasma bovis (M.bovis) belongs to the class Mollicutes, order Mycoplasmatles, family Mycoplasma, and genus Mycoplasma. It is an important pathogen that endangers the development of the cattle industry and exists widely in the world. , can infect cattle of any age, and its pathogenesis is mostly related to transportation stress, and in most cases it manifests as chronic pneumonia and arthritis. Lactating dairy cows mainly manifest as Mycoplasma bovis mastitis, and calves suffer from Mycoplasma bovis pneumonia and arthritis through milk infection. With the increase of clinical antibiotic resistance, the prevention and treatment of b...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12N15/31A61K39/02A61P31/04C12R1/35
CPCA61K39/0241A61K2039/52A61K2039/552A61P31/04C07K14/30C12N15/74C12N2800/90
Inventor 郭爱珍赵刚朱习芳陈颖钰胡长敏陈曦陈建国
Owner HUAZHONG AGRI UNIV
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