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Mycoplasma bovis multifunctional protein CDNPase

A technology of mycoplasma bovis and protein, applied in the direction of biochemical equipment and methods, applications, botanical equipment and methods, etc., can solve problems such as little known, undiscovered toxins, virulence factors and virulence islands

Inactive Publication Date: 2018-10-16
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 multifunctional protein CDNPase
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  • Mycoplasma bovis multifunctional protein CDNPase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Screening and identification of growth-deficient mutants of Mycoplasma bovis

[0039] (1) High-throughput screening of growth-deficient mutants of Mycoplasma bovis

[0040] Using the PEG8000-mediated transformation method, the Tn4001 transposon mutant library of Mycoplasma bovis HB0801 was successfully constructed (Mahairas and Minion, 1989). The mutant library contained a total of 2285 mutants, which had been stored separately for individual strains. High-throughput screening of the Mycoplasma bovis mutant library was performed using the growth-deficient experimental cell infection model and the 96-pin replicator constructed in our laboratory. The specific steps are as follows: spread bovine embryonic lung cells (Embryonic bovine lung, EBL) cells to 96-well cell culture plate according to 4 × 104 cells / cm2, and use 96-pin replicator to inoculate the mutant library into the wells of the cell culture plate respectively. Co-cultivate for 72 hours in a carbon d...

Embodiment 2

[0045] Embodiment 2: Expression of Mycoplasma bovis CDNPase protein

[0046] 1. Cloning and expression of Mycoplasma bovis Mbov_0328 gene

[0047] 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.

[0048] 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 self-designed PCR primers to mutate the gene, and the specific steps are: take the Mbov...

Embodiment 3

[0084] Example 3: Multifunctional Verification of Mycoplasma bovis rCDNPase Recombinant Protein

[0085] 1. Phosphodiesterase Activity Test of Mycoplasma bovis rCDNPase Recombinant Protein

[0086] (1) Reaction system preparation: Add the following reaction solution to a 1.5ml EP tube, Tris-HCL (pH 7.0) 100mM, Mncl 2 10mM, substrate cyclic dinucleotide (cyclic diadenylic acid c-di-AMP and cyclic diguanylic acid c-di-GMP) 50μM, rCDNPase recombinant protein 5μM, the total reaction system is 100mL. Mix the prepared reaction system Evenly, react at 37°C.

[0087] (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 the wavelength of the ultraviol...

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Abstract

The invention belongs to the technical field of prevention and control of animal infectious diseases, and in particular relates to a mycoplasma bovis multifunctional protein gene CDNPase. The proteingene Mbov-0328 is obtained by conducting cloning from mycoplasma bovis HB0801 genome. In accordance with preference of escherichia coli to codon, the Mbov-0328 gene is modified by the inventor, and mycoplasma bovis tryptophan codon UGA is mutated into codon UGG for coding tryptophan in escherichia coli, so that recombinant protein rCDNPase, which is expressed by the escherichia coli, is finally obtained. A nucleotide sequence of the protein coding gene cloned by the invention sis shown as the 1-969th base groups in SEQ ID NO:11, and a sequence of the protein is shown as SEQ ID NO:12. The recombinant protein has activities of phosphodiesterase and nuclease of an ultra-small fragment.

Description

technical field [0001] The invention belongs to the technical field of animal infectious disease prevention and control, and specifically relates to a multifunctional protein CDNPase of mycoplasma bovis, which can degrade cyclic dinucleotides and ultra-small fragment nucleic acids. Background technique [0002] Mycoplasma bovis (M.bovis) is an important pathogenic pathogen of cattle, which can cause symptoms such as pneumonia, arthritis, mastitis and keratoconjunctivitis in cattle. Mycoplasma bovis was first isolated from the milk of cows suffering from mastitis in the United States in 1961, and it was confirmed to be an important pathogen causing pneumonia in 1976. The disease is susceptible to the breathing of beef cattle and dairy cattle of any age, and the incidence rate of infected cattle is 40%, and the mortality rate is 10%. At present, mycoplasma bovis is widely prevalent in the world, and has become a major infectious disease and a common disease that endangers the...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/16C12N9/22C12N1/21C12R1/19
CPCC12N9/16C12N9/22
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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