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Mycoplasma bovis mutant strain with growth defect under cell coculture and application

A technology of Mycoplasma bovis and mutant strains, applied in the direction of microorganism-based methods, bacteria, microorganisms, etc., can solve the problems that toxins, virulence factors and virulence islands have not been discovered, and are poorly understood

Active Publication Date: 2019-04-19
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the virulence mechanism of M. bovis is still poorly understood, and it has not been found that it has classic toxins, virulence factors and virulence islands.

Method used

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  • Mycoplasma bovis mutant strain with growth defect under cell coculture and application
  • Mycoplasma bovis mutant strain with growth defect under cell coculture and application
  • Mycoplasma bovis mutant strain with growth defect under cell coculture and application

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1: Screening and identification of growth-deficient mutants of Mycoplasma bovis

[0036] 1. High-throughput screening of growth-deficient mutants of Mycoplasma bovis

[0037] The Mycoplasma bovis mutant library was transferred to 24 96-well plates, and the cell co-culture growth defect experimental model and 96-pin replicator constructed by the Ruminant Pathogen Branch of the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University were used to test the Mycoplasma bovis mutant library. Perform high-throughput screening. Divide the EBL cells into 4 × 10 4 cell / cm 2 Spread to a 96-well cell culture plate, use a 96-pin replicator to inoculate the mutant library into the cells, at 37°C, 5% CO 2 Co-cultivate in the incubator for 72 hours, lyse the cells after a cycle of freeze-thaw (-80°C / +37°C), and use a 96-pin replicator to coat each strain of mutants on a PPLO solid plate, and store them at 37°C, 5% CO 2 Cultivate in an incubator fo...

Embodiment 2

[0042] Embodiment 2: Detection of the growth curve of the growth defect mutant of Mycoplasma bovis

[0043] 1. Detection of the growth curve of Mycoplasma bovis in EBL cells

[0044] (1) Mycoplasma bovis cultivation and counting: Take Mycoplasma bovis HB0801 and T9.386 and inoculate them in PPLO liquid medium at a ratio of 1:1000 respectively, at 37°C, 5% CO 2 After standing in the incubator for 36 hours and reaching the end of the logarithm, count the CFU. The cultured bacterial solution is about to be diluted 10-fold, and 10 μL of the appropriate dilution of the bacterial solution is applied to the PPLO solid medium, and placed upside down at 37°C, 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.

[0045] (2) EBL cell culture and counting: EBL cells were cultured in MEM complete medium (that is, MEM medium containing 10% heat-in...

Embodiment 3

[0051] Embodiment 3: Morphological observation of growth defect mutants co-cultured with Mycoplasma bovis

[0052] Dilute the Mycoplasma bovis HB0801 and T9.386 strains cultivated to the end of the logarithm, respectively, and spread them on PPLO solid medium, at 37°C, 5% CO 2 After culturing in the incubator for 3-7 days, the colony morphology of mycoplasma was observed under a stereo microscope, and the results showed that the colony of the T9.386 mutant strain was smaller than that of the HB0801 strain ( Image 6 ).

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Abstract

The invention belongs to the technical field of zoonosis prevention and treatment, and particularly relates to a mycoplasma bovis mutant strain with a growth defect under cell coculture and application. The mycoplasma bovis Mbov-0328 gene deletion mutant strain T9.386 is sifted out from a mycoplasma bovis gene mutant library, and the gene is coded to form cyclic dinucleotide phosphodiesterase. When the mutant strain and a bovine embryo pneumonocyte are co-cultured, the remarkable growth defect phenotype is shown; the mutant strain shows small colonial morphology on a PPLO solid culture medium.Proteomics between the mutant strain and a wild strain shows a remarkable difference expression spectrum. The mutant strain has 38 differential expression proteins, wherein 30 proteins are in up-regulated expression, and 8 proteins are in down-regulated expression. The mutant strain can be applied to the field of mycoplasma bovis metabolic physiology, pathopoiesia and immune prevention.

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 mutant strain of mycoplasma bovis with growth defects under cell co-cultivation and its application. A Mycoplasma bovis Mbov_0328 gene deletion mutant strain T9.386 was screened from the M. bovis gene mutant library. The gene encodes cyclic dinucleotide phosphodiesterase (CDNPase). The mutant strain exhibited a marked growth defect phenotype when co-cultured with bovine embryonic lung cells (EBL), showing small colony morphology on PPLO solid medium. The proteomics between the mutant strain and the wild strain showed a significant differential expression profile. The mutant strain is expected to be applied in pathogenicity and immune control of Mycoplasma bovis. Background technique [0002] Mycoplasma bovis (M.bovis) is an important pathogen of cattle, which can cause symptoms such as pneumonia, arthritis, mastitis and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12R1/35
CPCC12N9/16C12Y301/04017
Inventor 郭爱珍朱习芳董亚旗李茜茜陈颖钰胡长敏陈焕春
Owner HUAZHONG AGRI UNIV
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