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Antigen composition for detecting chicken mycoplasma synoviae antibody, kit and application of kit

A technology of mycoplasma synovialum and its composition, which is applied in the field of prevention and control of animal infectious diseases, can solve the problems of low antibody positive rate, false negative, weak target protein antibody, etc., and achieve a balance of reactogenicity and sensitivity, and a high detection rate Effect

Active Publication Date: 2021-06-18
SHANDONG LVDU BIO SICIENCE & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of laboratory application, during the serological investigation of SPF chickens immunized with domestic popular strains and domestic diseased chicken flocks, the positive rate of antibodies using imported detection methods is low, and the antibody evaluation of vaccines cannot be effectively carried out
The main reason for this problem may be that the coating antigen component of the detection kit is single, and the expression level of some proteins of Mycoplasma synoviae decreases during the immune evasion process of infection, resulting in weak antibodies to the target protein, which leads to the loss of existing commercial reagents. Cases where the cartridges show false negatives in the test

Method used

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  • Antigen composition for detecting chicken mycoplasma synoviae antibody, kit and application of kit
  • Antigen composition for detecting chicken mycoplasma synoviae antibody, kit and application of kit
  • Antigen composition for detecting chicken mycoplasma synoviae antibody, kit and application of kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, the acquisition of mycoplasma chicken synovial bursa specific antigenic protein

[0037] 1.1 Acquisition of complete gene sequences of different strains of Mycoplasma gallinae bursa

[0038] The complete gene sequence of the representative No. 53 strain of Mycoplasma synoviae (Mycoplasma synoviae, MS) was obtained from the National Center for Biotechnology Information (NCBI) in the United States.

[0039] 1.2 Computer prediction analysis of antigenic protein

[0040] Use the online biological software PSORTb, Vaxign 2Beta, BLASTp, etc. to analyze the protein localization, transmembrane region, antigenic parameters and homology comparison of different strains of Mycoplasma gallisynovii, and obtain the following candidate Mycoplasma gallisovesia specific antigenic proteins 3540.1, 1919.1 and 2050.1.

[0041] 1.3 Codon optimization and construction of expression vectors for candidate Mycoplasma synovialis-specific antigenic proteins

[0042] Use DNAstar so...

Embodiment 2

[0058] Embodiment 2, the reactogenicity analysis of different mycoplasma synovial bursae specific antigenic protein

[0059] 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 gradients of 32.0, 16.0, 8.0, 4.0, 2.0, 1.0, 0.5, 0.25 μg / mL.

[0060] Use the fermented culture of Mycoplasma gallinarum CVCC385 (China Veterinary Microorganism Culture Collection and Management Center, http: / / cvccinfo.ivdc.org.cn / web / shopcart / ProductsSetting_Virus_Detail.aspx?id=CVCC385) to inactivate and immunize 30 Day-old SPF chickens were immunized once at the age of 45 days, and blood samples were collected at the age of 70 days as positive serum for future use. The research on its reactogenicity was carried out, and the serum of non-immunized SPF chickens was used as a negative control.

[0061] Preparation of buffer:

[0062] Coating buffer (pH9.6 carbonate buffer): 1.59g of sodium carbonate, 2.93g o...

Embodiment 3

[0091] Embodiment 3, the combination and optimization of different mycoplasma synovial bursae specific antigenic proteins

[0092] 1. Determination of the combination ratio of different Mycoplasma synovialum specific antigenic proteins

[0093] According to the reactogenicity comparison of the OD450 value of each antigen protein in Example 2, when the OD450 value of 3540.1, 1919.1 and 2050.1 is 1, the coating concentration is about 22, 4 and 2 μg / mL respectively, so the following coating antigen combination is set And the mass ratio of its antigen:

[0094] Combination 1: The mass ratio of 3540.1 and 1919.1 is 11:2;

[0095] Combination 2: The mass ratio of 1919.1 and 2050.1 is 2:1;

[0096] Combination 3: The mass ratio of 3540.1 and 2050.1 is 11:1;

[0097] Combination 4: The mass ratio of 3540.1, 1919.1 and 2050.1 is 11:2:1.

[0098] 2. Optimum coating concentration screening of the combination of Mycoplasma gallisovus-specific antigenic protein

[0099] In step 1, the...

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Abstract

The invention discloses an antigen composition for detecting a chicken mycoplasma synoviae antibody, a kit and application of the kit. 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.3. 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. The antigen composition further comprises a third antigen, and the amino acid sequence of the third antigen comprises a sequence as shown in SEQ ID No.1. When the antigen composition provided by the invention is used as a coating antigen of an ELISA method, the detection rate of clinical sample 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 and an application thereof for detecting antibodies to mycoplasma synovial bursae. Background technique [0002] Mycoplasma synovialum (alias: Mycoplasma synovialum) infection was first reported in 1954, and the affected chickens mainly showed synovitis; the onset age of Mycoplasma synovialum is generally about 30 days old, and the sick chickens have disheveled feathers and depression , decreased feed intake, unable to stand. Some flocks will also get sick at the age of 15 to 20 days, which should be related to maternal antibodies and vertical transmission. Membrane proteins of M. synovioides contain variable surface lipoproteins and hemagglutinin proteins, which have properties to evade host immune responses. The hemagglutinin protein is the main target of the body's immune response against Mycoplasma s...

Claims

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Application Information

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IPC IPC(8): C07K14/30C12N15/31G01N33/68G01N33/569G01N33/543G01N33/577
CPCC07K14/30G01N33/6854G01N33/56933G01N33/54393G01N33/577G01N2469/20
Inventor 李书光沈志强李峰程立坤苗立中杨立芳赵家磊
Owner SHANDONG LVDU BIO SICIENCE & TECH
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