Antigen composition for detecting mycoplasma bovis antibody, kit and application thereof

A technology of Mycoplasma bovis and a composition is applied in the field of animal infectious disease prevention and treatment, which can solve the problems of cross-agglutination, insufficient sensitivity, misjudgment of clinical serum antibody diagnosis, etc., and achieves the effect of high detection rate, balancing reactogenicity and sensitivity

Active Publication Date: 2021-06-18
SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The shared antigenic protein causes certain cross-agglutination phenomenon between Mycoplasma bovis and the above-mentioned Mycoplasma antiserum, which leads to misjudgment of clinical serum antibody diagnosis
Existing imported or domestic detection kits are not sensitive enough when detecting serum antibodies of domestic epidemic strains

Method used

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  • Antigen composition for detecting mycoplasma bovis antibody, kit and application thereof
  • Antigen composition for detecting mycoplasma bovis antibody, kit and application thereof
  • Antigen composition for detecting mycoplasma bovis antibody, kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the acquisition of mycoplasma bovis specific antigenic protein

[0035] 1.1 Acquisition of complete gene sequences of different strains of Mycoplasma bovis

[0036] The full gene sequences of 33 representative strains of Mycoplasma bovis (Mycoplasma bovis) were found from the National Center for Biotechnology Information (NCBI) in the United States. , Journal of Livestock Ecology, 2017, 38(2):64-66" 1.4, hereinafter referred to as "Mycoplasma bovis Shandong strain"), and its full gene sequence was obtained through company sequencing.

[0037] 1.2 Computer prediction analysis of antigenic protein

[0038] Using online biological software PSORTb, Vaxign 2Beta, BLASTp, etc. to analyze the protein localization, transmembrane region, antigen parameters and homology comparison of different M. bovis strains, the following candidate M. bovis specific antigen proteins 4979.1, 0148.1, 2700.1 were obtained.

[0039] 1.3 Codon optimization of candidate Mycoplasma bo...

Embodiment 2

[0056] Embodiment 2, the reactogenicity analysis of different Mycoplasma bovis specific antigenic protein

[0057] The purified antigenic protein obtained in step 1.4 of Example 1 was respectively coated on the reaction wells of the ELISA plate according to the coating concentration gradient of 32.0, 16.0, 8.0, 4.0, 2.0, 1.0, 0.5, 0.25 μg / mL.

[0058] The inactivated culture of Mycoplasma bovis Shandong strain was used to immunize New Zealand rabbits to prepare positive serum for the study of its reactogenicity, and the serum of non-immunized New Zealand white rabbits was used as negative control.

[0059] Preparation of buffer:

[0060] Coating buffer (pH9.6 carbonate buffer): 1.59g of sodium carbonate, 2.93g of sodium bicarbonate, dilute to 1000ml with ultrapure water, confirm that the pH value is 9.6 with a pH meter, filter with a 0.44μm membrane Backup.

[0061]Washing buffer: weigh 8.0g sodium chloride, 0.2g potassium dihydrogen phosphate, 6.29g disodium hydrogen phosph...

Embodiment 3

[0087] Embodiment 3, the reaction specificity analysis of Mycoplasma bovis specific antigenic protein

[0088] Carry out according to the method of embodiment 2, difference is: 4979.1, 0148.1 and 2700.1 proteins use 2μg / ml, 8μg / ml and 20μg / ml respectively as coating antigen to carry out the coating of ELISA plate; The source positive serum was replaced with positive serum of Mycoplasma agalactiae, Mycoplasma alcaligenes, Mycoplasma mycoplasma subsp. goat, or Mycoplasma subsp. filamentous for specificity determination.

[0089] The preparation method of the positive serum of above-mentioned Mycoplasma agalactiae, Mycoplasma alcaligenes, Mycoplasma filamentous subspecies goat subspecies, or Mycoplasma filamentous subspecies is as follows:

[0090] Mycoplasma agalactiae (CVCC344), Mycoplasma alcaligenes (YX), Mycoplasma filiformis subspecies goat subspecies (CVCC3009), or Mycoplasma filamentous subspecies filamentous subspecies (CVCC378) were all from Binzhou animal husbandry Th...

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Abstract

The invention discloses an antigen composition for detecting a mycoplasma bovis antibody, a kit and application of the antigen composition. The antigen composition comprises a first antigen, and the amino acid sequence of the first antigen comprises a sequence as shown in SEQ ID No.1. The antigen composition further comprises a second antigen, and the amino acid sequence of the second antigen comprises a sequence as shown in SEQ ID No.2. When the antigen composition provided by the invention is used as a coating antigen of an ELISA method, the detection rate of domestic bovine mycoplasma epidemic serotypes is obviously higher than that of existing imported kits.

Description

technical field [0001] The invention belongs to the technical field of prevention and treatment of animal infectious diseases, and relates to an antigen composition, a kit for detecting antibodies to mycoplasma bovis and applications thereof. Background technique [0002] From 2015 to 2019, epidemiological surveys showed that the incidence of bovine mycoplasma disease is increasing in China, which can cause disease in dairy and beef cattle, and cause serious economic losses to some cattle farms. Mycoplasma bovis can cause various diseases such as arthritis, pneumonia and miscarriage in cattle. At present, it mostly appears in the form of pneumonia in China. [0003] Mycoplasma bovis and Mycoplasma agalactiae, Mycoplasma agalactiae, Mycoplasma filamentous subspecies goat subspecies, Mycoplasma filamentous subspecies, etc. have some common antigenic proteins, especially with Mycoplasma agalactiae in the variable surface lipoprotein (Variablesurface lipoproteins (Vsps) are a g...

Claims

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Application Information

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IPC IPC(8): C07K14/30C12N15/31G01N33/569G01N33/543
CPCC07K14/30G01N33/56933G01N33/543G01N2333/30
Inventor 李书光刘吉山沈志强肖跃强曲光刚杨立芳赵家磊
Owner SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY
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