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Mycoplasma bovis Mbov0703 gene mutant strain and application thereof

A Mycoplasma bovis, gene technology, applied in the direction of bacterial antigen components, microbe-based methods, microbes, etc., can solve the problems of molecular regulation mechanism and typical virulence factors, and achieve the ability to adhere to cells and induce inflammatory reactions Effects of reduced capacity, weakened virulence, and reduced biofilm-forming ability

Active Publication Date: 2022-07-22
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, to date, the molecular regulatory mechanisms and canonical virulence factors of M. bovis remain unclear

Method used

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  • Mycoplasma bovis Mbov0703 gene mutant strain and application thereof
  • Mycoplasma bovis Mbov0703 gene mutant strain and application thereof
  • Mycoplasma bovis Mbov0703 gene mutant strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Construction and identification of Mycoplasma bovis Mbov_0703 gene deletion / apoptosis strain

[0025] 1.1 Construction of Mycoplasma bovis Mbov_0703 gene deletion / replenishment strain

[0026]The Mbov_0703 gene deletion strain T9.210 was screened out from the Mbov_0703 mutant library constructed earlier in the laboratory, and the Mbov_0703 gene deletion was verified at the protein level by Western blot; Amplify the Mbov_0703 gene, clone the target fragment into the pOH / P plasmid, and obtain a complementing plasmid; use the transformation method mediated by PEG8000 (purchased from SIGMA) to transform the constructed recombinant plasmid into the Mbov_0703 gene deletion strain, and in a solid culture Culture medium for 3-7 days, and select positive clones by resistance. After the positive clones were expanded and cultured, the total protein of the bacteria was extracted, and the expression of MbovP0703 protein in the recombinant bacteria was verified by Western...

Embodiment 2

[0029] Example 2: Environmental resistance and biofilm formation ability of mutants

[0030] 2.1 Determination of the viability of M. bovis mutant strain T9.210 under heat-stimulated conditions

[0031] Take 1 ml of Mycoplasma bovis HB0801 grown in logarithmic phase, the mutant strain T9.210 and the complementing strain CT9.210 for colony counting, and adjust to 10 according to the counting results. 6 CFU / ml, and then continue to culture at 42°C for 15min, 60min, 120min, and take 100μl of bacterial solution for colony counting within the specified time. The results showed that after culturing for 60 min, the number of colonies of the mutant strain was significantly lower than that of the wild strain and the complemented strain ( image 3 ).

[0032] 2.2 Biofilm formation assay of M. bovis mutant strain T9.210

[0033] Take Mycoplasma bovis HB0801, mutant strain T9.210 and replenishing strain CT9.210 grown in logarithmic phase, add 10 μl of bacterial liquid to a 48-well cell...

Embodiment 3

[0035] Example 3: Determination of the ability of mutant strains to adhere to EBL of bovine lung epithelial cells

[0036] Take an appropriate amount of counted Mycoplasma bovis HB0801, the mutant strain T9.210 and the replenishing strain CT9.210, washed three times with PBS, and resuspended in an equal volume with the cell culture medium DMEM, and infected EBL according to the ratio of infection ratio (MOI) to 1000 cells at 37 °C, 5% CO 2 Infected for 30min, 60min, and 120min respectively under the conditions, washed three times with sterile PBS, added a certain amount of DMEM medium, and repeatedly freeze-thawed at -80°C / 37°C to fully lyse the cells, and then counted CFU. The results showed that the adhesion ability of T9.210 was significantly lower than that of the wild strain and the complementing strain after 30min and 60min adhesion ( Figure 5 ).

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Abstract

The invention discloses a mycoplasma bovis gene mutant strain and belongs to the technical field of prevention and treatment of animal infectious diseases, the strain is named as mycoplasma bovis T9.210 and preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M 2022260, and a mutated gene is an Mbov0703 gene with a nucleotide sequence as shown in SEQ ID NO: 1. The capacity of adhering bovine lung epithelial cells and the capacity of inducing bovine lung macrophage inflammatory response of the mutant strain are reduced, and the biofilm forming capacity is reduced, so that the toxicity is weakened, the stress resistance is reduced, and the mutant strain has an application prospect in the fields of bovine mycoplasma pathopoiesis and immune control.

Description

technical field [0001] The invention belongs to the technical field of prevention and control of animal infectious diseases, and in particular relates to a gene mutant strain of Mycoplasma bovis, which has weakened virulence and reduced stress resistance due to the mutation of the Mbov_0703 gene. Background technique [0002] Mycoplasma bovis (M.bovis) belongs to the phylum Firmicutes, the class of Mildew, the order Mycoplasma, the family of Mycoplasma, and the genus of Mycoplasma. It is an important pathogen that infects beef and dairy cattle. Arthritis, synovitis, keratoconjunctivitis, meningitis, myocarditis, reproductive system disorders and other diseases (Shi Lei et al., 2008). At present, due to the increasing resistance to antibiotics and the lack of effective vaccines, the prevention and treatment of Mycoplasma bovis has become an important problem affecting the development of the cattle industry, and the solution to this problem depends on the development of new dr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21A61K39/02A61P31/04C12R1/35
CPCC07K14/30A61K39/0241A61P31/04A61K2039/522A61K2039/552Y02A50/30
Inventor 郭爱珍张慧张怡秋路豆昆赵刚陈颖钰陈曦陈建国胡长敏
Owner HUAZHONG AGRI UNIV
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