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Coating antigen for detecting mycoplasma synoviae antibody, kit and detection method thereof

A technology of mycoplasma synovialum and coated antigen, which is applied in the field of kits and its detection, detection of coated antigen of mycoplasma synovialum antibody, which can solve the problems of poor antigen specificity and sensitivity, and inability to effectively evaluate vaccine antibodies , achieve good reactogenicity, prevent and control the effect of mycoplasma synovial bursa infection

Pending Publication Date: 2022-04-12
WENS FOODSTUFF GRP CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The immune effect of the vaccine requires effective antibody detection to achieve the purpose of preventing and controlling Mycoplasma synovialum infection, but the specificity and sensitivity of the antigen in the existing detection methods are poor, and the antibody evaluation of the vaccine cannot be effectively carried out

Method used

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  • Coating antigen for detecting mycoplasma synoviae antibody, kit and detection method thereof
  • Coating antigen for detecting mycoplasma synoviae antibody, kit and detection method thereof
  • Coating antigen for detecting mycoplasma synoviae antibody, kit and detection method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0042] A coating antigen for detecting mycoplasma synovialum antibody, the coating antigen is a recombinant EFG protein; the amino acid sequence of the recombinant EFG protein is as shown in SEQ ID NO: 1; the nucleotide of the recombinant EFG protein The sequence is shown in SEQ ID NO: 2, and the GC content of the sequence has been optimized to facilitate expression.

[0043] The preparation method of the coated antigen is:

[0044] 1. EFG gene primer design:

[0045] Referring to the full-length sequence of the EFG protein gene of Mycoplasma synoviae (NCBI gene sequence number: Sequence ID: WP_011283211.1), after codon optimization, the EFG target gene sequence was obtained. Design a pair of primers to amplify the EFG target gene sequence, the primer sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4:

[0046] SEQ ID NO: 3: 5'-ATGGCACGTGATTATGATCTGAAAG-3';

[0047] SEQ ID NO: 4: 5'-TTCTTCATCTTTTATTCTGAATATTAC-3';

[0048] The target gene sequence size of EFG protein is ...

Embodiment 2

[0057] A test kit for detecting mycoplasma synovial bursae antibody, said test kit includes the recombinant EFG protein purified in Example 1 as the coated antigen of the enzyme label plate, the enzyme label plate, the coating solution, the blocking solution, the enzyme label two Anti-, TMB chromogenic solution, stop solution, washing solution and serum diluent; the coating solution is carbonate buffer; the washing solution is PBST; the enzyme-labeled secondary antibody is enzyme-labeled goat anti-rabbit antibody; The blocking solution is 5% skimmed milk powder; the serum diluent is 5% skimmed milk powder.

Embodiment 3

[0059] The method that adopts the kit described in embodiment 2 to detect mycoplasma synovialum antibody may further comprise the steps:

[0060] S1; Dilute the coated antigen with carbonate buffer to a concentration of 0.8 μg / mL, add 100 μL / well into the microtiter plate, and coat overnight at 4°C;

[0061] S2: Remove the coating solution in S1, wash with PBST 4 times, 2 min each time, pat dry, add 100 μL of 5% skimmed milk powder blocking solution to each well, and block at 4°C for 16 h.

[0062] S3: Remove the blocking solution in S2, wash with PBST 4 times, each time for 2 minutes, pat dry, add 100 μL of the serum sample to be tested diluted 1:800 times with 5% skimmed milk powder to each well, and incubate at 37°C for 90 minutes;

[0063] S4: remove the serum sample in S3, wash with PBST 4 times, 2 min each time, pat dry, and incubate with goat anti-rabbit enzyme-labeled secondary antibody diluted 1:2000 times with PBS, and incubate at 37°C for 120 min;

[0064] S5: Remo...

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Abstract

The invention relates to the technical field of biology, in particular to a coating antigen for detecting a mycoplasma synoviae antibody, a kit and a detection method thereof. Detecting that a coating antigen of the mycoplasma synoviae antibody is a recombinant EFG protein; the amino acid sequence of the recombinant EFG protein comprises a sequence as shown in SEQ ID NO: 1; the nucleotide sequence of the recombinant EFG protein comprises a sequence as shown in SEQ ID NO: 2. The recombinant EFG protein is used as the coating antigen of the ELISA method for detecting the mycoplasma synoviae antibody, can be used for clinical detection of the mycoplasma synoviae serum antibody, and efficiently detects the mycoplasma synoviae antibody in a farm; according to the present invention, the recombinant EFG protein is adopted as the coating antigen of the ELISA method for detecting the mycoplasma synoviae antibody, such that the detection method has good reactogenicity, specificity, sensitivity and repeatability, can be used for clinical mycoplasma synoviae serum antibody detection, provides certain guidance for the formulation of the mycoplasma synoviae vaccine immune program, and provides the important significance for the clinical detection of the mycoplasma synoviae serum antibody. Therefore, the aim of preventing and controlling mycoplasma synoviae infection is achieved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a coating antigen for detecting mycoplasma synovialum antibody, a kit and a detection method thereof. Background technique [0002] Mycoplasma synoviae (Mycoplasma synoviae, MS) is one of the main pathogenic mycoplasma in poultry, and its main infection group is chickens or turkeys. After poultry infection with MS, the main manifestations are exudative synovial membrane and tendon sheath. Meningitis can also cause subclinical respiratory symptoms and air sacculitis. MS can infect chickens and turkeys of all ages, but young chickens are the most infective, and once infected, they will carry the bacteria for life. MS infection is mostly chronic or recessive infection, and acute cases are rare. Clinically, it often manifests as mixed infection with NDV, IBV, IBDV, Escherichia coli and other pathogens. MS is mainly transmitted horizontally through contact between healthy chickens and d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/543
Inventor 周庆丰魏晓娜孙乾晋陈苇严专强周祺尹丽娟陈峰
Owner WENS FOODSTUFF GRP CO LTD
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