Mycoplasma bovis Mbov_0280 gene mutant and application thereof

A Mycoplasma bovis and gene technology, applied in veterinary vaccines, bacterial antigen components, antibody medical components, etc., can solve the problems that hinder the development of new drugs and vaccines, the pathogenic mechanism is unclear, and the effective prevention and treatment of Mycoplasma bovis is hindered. question

Active Publication Date: 2020-09-25
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_0280 gene mutant and application thereof
  • Mycoplasma bovis Mbov_0280 gene mutant and application thereof
  • Mycoplasma bovis Mbov_0280 gene mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Embodiment 1: Construction of Mycoplasma bovis insertion mutant library

[0013] 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. The pMT85 plasmid, donated by Dr. Eric Baranowski of the French Academy of Agricultural Sciences, contains a mini-type Tn4001 transposon (mini-Tn4001), which has introduced a gentamicin resistance marker encoded by the aacA-aphD gene, which is located in Between the two inverted repeats (IRs) at both ends of the transposable fragment, the transposase gene (tnpA) located outside the repeats prevents further transposition (Baranowski et al 2010).

[0014] Use M.bovis HB0801 as the parent strain to construct a mutant library. The basic procedure is: collect M.bovis cultured to late logarithmic phase, wash twice with cold DPBS buffer, and resusp...

Embodiment 2

[0015] Example 2. Genome sequencing and identification of mutant strains in the Mycoplasma bovis mutant library

[0016] 2.1 Genome sequencing to obtain the Mbov_0280 mutant strain of Mycoplasma bovis

[0017] Using the Bacterial Genome Extraction Kit (purchased from Treasure Bioengineering Dalian Co., Ltd.), the total DNA of Mycoplasma bovis in the Mycoplasma bovis T9.297 mutant library was extracted, and the junction of the Tn4001 transposon and the Mycoplasma bovis genome was sequenced, and the sequencing results were Compared with the whole genome sequence of Mycoplasma bovis HB0801, the results showed that the gene Mbov_0280 involved in the mutant strain T9.297 contained a transposon insertion sequence with a size of 3438bp, and the T9.297 mutant gene was Mbov_0280, the transposon insertion site After the 323270 site in the genome, and after the 888 site of the Mbov_0280 gene ( figure 1 ).

[0018] 2.2 Western blot to verify the expression defect of the mutant strain Mb...

Embodiment 3

[0022] Example 3: Ability of Mycoplasma bovis Mutants to Induce Macrophage Apoptosis is Reduced

[0023] The T9.297 mutant strain and the HB0801 strain were expanded in vitro.

[0024] BoMac cells were divided into 5×10 per well 5 The cells were seeded in 6-well plates and incubated overnight at 37°C, 5% CO2. Remove the cell plate and add 5 × 10 8 CFU HB0801 or T9.297, and incubated at 37°C for 24h. Collect the supernatant and trypsinized adherent cells, centrifuge at 300g for 5min; remove the supernatant, wash the cells with pre-cooled PBS, centrifuge at 300g for 5min, repeat the above washing step once, and then use 100μl banding buffer (purchased from Nanjing Nuowei Zan Biotechnology Co., Ltd.) to resuspend the cell pellet, add Annexin-FITC / PI dye (purchased from Nanjing Nuoweizan Biotechnology Co., Ltd.) to stain the cells, and keep away from light for 10 minutes, and then add 400 μl banding buffer (purchased from Nanjing Biotechnology Co., Ltd.) to each well. Novozyme...

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Abstract

The invention discloses a mycoplasma bovis Mbov_0280 gene mutant and belongs to the technical field of animal infectious disease prevention and treatment. The mutant is named Mycoplasma bovis T9.297 is preserved in the China Center for Type Culture Collection and has a preservation number of CCTCC NO:M 2020085. The Mbov_0280 gene has a nucleotide sequence as shown in SEQ ID NO:1. The strain has the performance for inducing host macrophages apoptosis is weakened in comparison with a wild strain HB0801; the mutant has low toxicity since the bovine macrophages serve as a critical immune cell forresisting adventitious infection of a host; and the mutant favors a body to resisting mycoplasma bovis infection, and is expected to achieve a vital function in the field of mycoplasma bovis immunological control.

Description

technical field [0001] The invention belongs to the technical field of animal infectious disease prevention and control, and in particular relates to a mycoplasma bovis Mbov_0280 gene mutant strain, the ability of the mutant strain to induce macrophage apoptosis is reduced. 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 bovine mycoplasma disease has become an ur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21A61K39/02A61P31/04C12R1/35
CPCA61K39/0241A61K2039/552A61P31/04C07K14/30
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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