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Mycoplasma bovis MbovP579 protein and application thereof

A technology of mycoplasma bovis and antigenic protein, applied in the direction of material inspection products, instruments, peptides, etc.

Pending Publication Date: 2017-09-01
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current diagnostic methods can no longer meet the needs of the prevention and control of Mycoplasma bovis, so it is urgent to find specific targets for the diagnosis of Mycoplasma bovis and develop a detection kit with high specificity and sensitivity

Method used

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  • Mycoplasma bovis MbovP579 protein and application thereof
  • Mycoplasma bovis MbovP579 protein and application thereof
  • Mycoplasma bovis MbovP579 protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: Expression of Mycoplasma bovis Mbov579 protein

[0039] 1.1 Cloning and expression of Mycoplasma bovis Mbov_579 gene

[0040]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.

[0041] 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: taking the Mbo...

Embodiment 2

[0078] Example 2: Application of Mycoplasma bovis MbovP579 Protein in Diagnosis of Natural Infection of Mycoplasma bovis Wild Strain

[0079] 2.1 Using the rMbovP579 recombinant protein of Mycoplasma bovis as an antigen, the natural infection of Mycoplasma bovis field strains was diagnosed by indirect ELISA method

[0080] The specific operation procedure of the ELISA method:

[0081] (1) Antigen coating: use coating solution (Na 2 CO 3 1.59g, NaHCO 3 2.93g, add ddH 2 (2 to 1000ml) dilute and concentrate the rMbovP579 protein, add 100 μl of coating solution after diluting the rMbovP579 protein in the ratio of 100 ng per hole and add to a 96-well ELISA plate, and coat overnight at 4 ° C. Discard the coating solution, add 300μl PBST (PBS:NaCl 8.0g, KCl 0.2g, NaCl 2 HPO 4 12H 2 O 2.9g, NaH 2 PO 4 0.2g, add ddH 2 0 to 1000ml. PBST: add 0.05% Tween-20 to PBS) and wash 3 times, 2 minutes each time.

[0082] (2) Blocking: Add 100 μl of 5% skimmed milk (5g skimmed milk ...

Embodiment 3

[0091] Embodiment 3: the assembly of Mycoplasma bovis rMbovP579 protein diagnosis kit for natural infection of Mycoplasma bovis

[0092] The kit of the present invention comprises 2 Mycoplasma bovis rMbovisP579 antigen-coated plates (96 wells / block, 100ng / well), 1 tube (0.5ml / tube) of Mycoplasma bovis antibody ELISA negative and positive control, 1 bottle of sample diluent ( 40ml / bottle), goat anti-bovine enzyme-labeled secondary antibody (IgG Fc-HRP) (purchased from southernbiotech company) 1 tube (200μl / tube), 20 times concentrated washing solution 1 bottle (30ml / bottle), TMB substrate for color development Solution A, 1 bottle of TMB substrate chromogenic solution B (10ml / bottle; purchased from Wuhan Keqian Animal Biological Products Co., Ltd.), 1 bottle of stop solution (10ml / bottle, purchased from Wuhan Keqian Animal Biological Products Co., Ltd.) Products, LLC).

[0093] (1) Preparation of 20 times concentrated washing solution: NaCl 160g, KCl 4g, NaCl 2 HPO4·12H2O 58g...

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Abstract

The invention specifically relates to a mycoplasma bovis MbovP579 protein and application thereof. The protein is obtained by cloning and expression from mycoplasma bovis HB0801 genome, and is a specific immunogenic protein with unknown function. The mycoplasma bovis HB0801 genome is adopted as the template to clone Mbov_579 gene, the Mbov_579 gene is modified according to the codon preference of escherichia coli, the mycoplasma bovis tryptophan codon UGA is mutated into codon UGG encoding tryptophan in escherichia coli, and the recombinant rMbovP579 expressed in escherichia coli can be obtained. The nucleotide sequence of the protein's gene is shown as the 1-2187th basic groups in SEQ ID NO:13, and the amino acid sequence of the protein is shown as SEQ ID NO:14. The recombinant protein can be used as an antigen protein in a mycoplasma bovis infection and vaccine immunodiagnostic kit.

Description

technical field [0001] The invention belongs to the technical field of animal infectious disease prevention and control, and specifically relates to a Mycoplasma bovis MbovP579 protein and application thereof. Said protein can be used as an antigenic protein in the preparation of a diagnostic kit for mycoplasma bovis infection. Background technique [0002] Mycoplasma bovis (M.bovis) is an important pathogen that mainly causes respiratory diseases and arthritis in beef cattle and dairy cattle of any age. The main clinical symptom is pneumonia, and it can also cause mastitis, arthritis, reproductive tract inflammation, Conjunctivitis, otitis media and other symptoms, the incidence rate is 40%, and the mortality rate is 10%. 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. In 2008, respiratory diseases were prevalent in beef cattle import...

Claims

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Application Information

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IPC IPC(8): C07K14/30C12N1/21G01N33/68G01N33/569
CPCC07K14/30G01N33/569G01N33/68G01N2333/30
Inventor 郭爱珍安瓦尔赵刚费萨尔陈曦陈颖钰胡长敏陈焕春
Owner HUAZHONG AGRI UNIV
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