Mycoplasma bovis Mbov_0570 gene mutant strain and application thereof

A technology of Mycoplasma bovis and gene is applied in the field of Mycoplasma bovis Mbov_0570 gene mutant strain to prepare Mycoplasma bovis vaccines, which can solve problems such as unclear pathogenic mechanism, hindering effective prevention, control and treatment of Mycoplasma bovis, and hindering the development of new drugs and new vaccines.

Active Publication Date: 2020-09-25
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the unclear pathogenic mechanism of M. bovis seriously hinders the development of n...

Method used

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  • Mycoplasma bovis Mbov_0570 gene mutant strain and application thereof
  • Mycoplasma bovis Mbov_0570 gene mutant strain and application thereof
  • Mycoplasma bovis Mbov_0570 gene mutant strain and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0010] Embodiment 1: Construction of Mycoplasma bovis insertion mutant library

[0011] 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.

[0012] 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).

[0013] Using M.bovis HB0801 as the parent strain to construct a mutant library, the basic procedure is as follows: collect M.bovis cultured to t...

Embodiment 2

[0014] Embodiment 2: Screening and identification of Mycoplasma bovis T9.171 mutant

[0015] Using the bacterial genome extraction kit (purchased from Bao Bioengineering Dalian Co., Ltd.), the whole genome of the Mycoplasma bovis T9.171 mutant was extracted, and the junction of the Tn4001 transposon and the Mycoplasma bovis genome was sequenced. The genome sequence was compared, and the results showed that the T9.171 mutant gene was Mbov_0570, and the transposon insertion site was located after the 672416 site of the genome and after the 409 site of the Mbov_0570 gene, with a size of 3438bp ( figure 1 ).

Embodiment 3

[0016] Example 3: T9.171 mutant stimulates BoMac cells to reduce the ability to express cytokines

[0017] Culture and counting of Mycoplasma bovis: Take Mycoplasma bovis HB0801, Mycoplasma bovis MbovHB0801-150.2, and T9.171 to inoculate PPLO liquid medium at a ratio of 1:1000, and place it at 37°C, 5% CO 2 After culturing in the incubator for 36 hours to reach the logarithmic phase, count the CFUs. The method is as follows: Dilute the cultured bacterial solution by 10 times, take 10 μL of the appropriate dilution of the bacterial solution and spread it on the PPLO solid medium, at 37 ° C, Inverted culture, 5% CO 2 After culturing in the incubator for 3-7 days, count the colonies under a stereo microscope. The formula for calculating the number of colonies is: CFU / mL=number of colonies×dilution×100.

[0018] Place the BoMac in a 2×10 6 The cell density of each well was inoculated into a 6-well cell plate, cultured at 37°C, 5% CO2 until adherent, and added Mycoplasma bovis HB...

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Abstract

The invention discloses a Mycoplasma bovis Mbov_0570 gene mutant strain, which belongs to the technical fields of prevention and treatment of animal infectious diseases, is named as Mycoplasma bovis T9.171, and is preserved in China Center for Type Culture Collection (CCTCC). The preservation number of the Mbov_0570 gene is CCTCC NO: M 2020083, and the nucleotide sequence of the Mbov_0570 gene isshown as SEQ ID NO: 1. According to the invention, qRT-PCR is used for detecting the capacity of T9.171 strain for stimulating BoMac cells to express IL-8, which shows that the capacity of the mutantstrain for inducing the BoMac cells to express cytokines is reduced, so that the toxicity of the mutant strain is reduced, and the mutant strain is expected to play an important role in the fields ofmycoplasma bovis immune prevention and treatment as a vaccine strain.

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_0570 gene, which reduces the ability of inducing macrophages to express IL-8 cytokines, and has good application in the preparation of Mycoplasma bovis vaccines potential. 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 clinica...

Claims

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Application Information

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IPC IPC(8): C12N1/20A61K39/02A61P31/04C12R1/35
CPCA61K39/0241A61K2039/552A61P31/04C07K14/30
Inventor 郭爱珍赵刚伊赫萨努拉·希拉尼张慧陈颖钰胡长敏陈曦陈建国
Owner HUAZHONG AGRI UNIV
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