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Mycoplasma bovis protein gene MbovGdpP and application thereof

A technology of mycoplasma bovis and gene, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of economic loss in cattle breeding, undiscovered bacterial toxins and virulence factors, lack of specific and efficient control measures, etc.

Active Publication Date: 2019-05-14
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lack of specific and efficient control measures, the disease has caused huge economic losses to the cattle industry in my country
However, the virulence mechanism of M. bovis is still poorly understood, and classic bacterial toxins and virulence factors have not been discovered (Qi et al., 2012)

Method used

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  • Mycoplasma bovis protein gene MbovGdpP and application thereof
  • Mycoplasma bovis protein gene MbovGdpP and application thereof
  • Mycoplasma bovis protein gene MbovGdpP and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Identification of Screening of M. bovis Growth Deficiency Mutants

[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 cell infection model and 96-pin replicator constructed by the ruminant pathogen branch of the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University where the inventor works were used. High-throughput screening of libraries of M. bovis mutants. 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 the ...

Embodiment 2

[0042] Embodiment 2: Expression of Mycoplasma bovis MbovGdpP protein

[0043] 1. Synthesis of Mycoplasma bovis Mbov_0276 gene

[0044] Because E. coli is to the preference of codon, the codon UGA of tryptophan of encoding tryptophan in E. coli is used as terminator in E. coli in the present invention, therefore, when expressing M. bovis gene with E. coli, need carry out mycoplasma gene Mutation, the codon UGA is mutated to the codon UGG that can express tryptophan in E. coli. The applicant obtained a local isolate of Mycoplasma bovis from the lung tissue of a cattle farm in Yingcheng City, Hubei Province in June 2008, named it Mycoplasma bovis HB0801, Mycoplasma bovis HB0801, and sent it on February 1, 2010 It was deposited in the Chinese Type Culture Collection Center of Wuhan University, China. The deposit number is CCTCC NO: M2010040. The present invention utilizes the method of gene synthesis to synthesize the truncated expression sequence of the Mbov_0276 gene, and the ...

Embodiment 3

[0060] Embodiment 3: detection of enzyme activity of Mycoplasma bovis recombinant protein rMbovGdpP

[0061] 1. Detection of phosphodiesterase activity of Mycoplasma bovis rMbovGdpP recombinant protein

[0062] (1) Reaction system preparation: Add the following reaction solution to a 1.5mL EP tube: Tris-HCl (pH 7.0) 100mM, Mncl 2 10 mM, substrate cyclic dinucleotides (cyclic diadenylic acid c-di-AMP and cyclic diguanylic acid c-di-GMP) 50 μM, rMbovGdpP recombinant protein 5 μM. The total reaction system was 100 μL, and the prepared reaction system was mixed and reacted at 37°C.

[0063] (2) Determination of phosphodiesterase activity: the sample was detected by HPLC separation and detection method, and the specific settings were as follows: 10% methanol and 0.2% ammonium acetate were used as the mobile phase, the flow rate was 1mL / min, and the column oven was controlled at 25°C , the injection volume of the autosampler was 10 μL, the reaction products were separated, and th...

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Abstract

The invention belongs to the technical field of zoonosis prevention, and particularly relates to a mycoplasma bovis protein gene MbovGdpP and application thereof. Mbov_0276 is artificially synthesizedaccording to the mycoplasma bovis HB0801 genome sequence. An Mbov_0276 gene is modified for the preference of escherichia coli for codons; mycoplasma bovis tryptophan codons UGA are mutated into codons UGG for encoding tryptophan in escherichia coli to obtain escherichia coli recombinant proteins rMbovGdpP. The Mbov_0276 gene has the nucleotide sequence shown in SEQ ID NO:1, and the ended proteinhas the sequence shown in SEQ ID NO:2. A mutant strain T6.290 has the growth defect phenotype, has the small colony morphology on a PPLO culture medium, reduces the adhesion of EBL cells and improvesthe sensitivity to saline ions. The mutant strain can be expected to be applied in the mycoplasma bovis pathogenesis and the preparation of immunosuppressive medicines.

Description

technical field [0001] The invention belongs to the technical field of prevention and treatment of animal infectious diseases, and specifically relates to the Mycoplasma bovis protein gene MbovGdpP and its application. The protein can degrade cyclic dinucleotides, ultra-small nucleic acid fragments, ATP and ADP. The gene encoding the protein is Mbov_0276, which Mutant strain T6.290. Compared with the wild strain, the mutant strain showed significant growth defects under co-increase and decrease of cells, small colony morphological phenotype, reduced adhesion ability, and significantly reduced tolerance to salt ions including potassium ions and sodium ions. Background technique [0002] Mycoplasma bovis (M.bovis) is an important pathogen of cattle, causing bovine pneumonia, mastitis, arthritis and other symptoms. It was first discovered in the milk of cows with mastitis in the United States in 1961, and it was first reported that the pathogen caused bovine pneumonia in 1976....

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/16C12N1/21A61K38/46A61P31/04C12R1/19
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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