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Streptodornase B antigen and application thereof

An antigen and nucleic acid sequence technology, applied in the field of genetic engineering, can solve problems such as deviation of detection results, complicated operation, and low yield, and achieve the effects of no safety risk, simple preparation method, and high sensitivity

Active Publication Date: 2020-06-02
东方海洋(北京)医学研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, natural DNaseB is mostly used in the detection of anti-DNase B antibodies. However, the purification process of natural DNaseB is complicated and the acquisition cost is expensive.
Fermenting GAS first to obtain a large amount of culture fluid containing secreted DNaseB not only requires a long growth period, but also poses serious safety risks
Secondly, the purification process after obtaining the culture medium is cumbersome and the yield is relatively low
Third, the natural crude extract contains known and unknown streptococcal products, which will interfere with the test results and amplify the test data, resulting in deviations in the test results, thus failing to meet the needs of clinical testing
On the other hand, most of the currently commercialized anti-DNase B antibody detection reagents use microtitration and immunoturbidimetric methods, which are not only complicated to operate, but also insensitive to changes in antibody concentration.

Method used

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  • Streptodornase B antigen and application thereof
  • Streptodornase B antigen and application thereof
  • Streptodornase B antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation of the DNaseB fusion dominant epitope antigen of the present application

[0033] 1. Amino acid sequence and coding sequence of DNase B fusion dominant epitope antigen

[0034] The original DNaseB (streptococcal DNase B) in Genebank has a full length of 271 amino acids, contains a 43aa leader peptide and 228 amino acids of a mature protein, and the sequence of the mature protein is shown in SEQ ID NO.3 in the sequence table, with a molecular weight of about It is 25.4kD. The DNaseB antigen of the present application (also known as DNase B fusion-dominant epitope antigen, which is different from the original DNaseB) has undergone gene optimization and epitope screening, and its amino acid sequence is shown in SEQ ID NO.1 in the sequence listing. The optimized gene sequence encoding DNaseB antigen is shown as SEQ ID NO.2 in the sequence listing.

[0035] 2. Expression and purification of DNaseB genetic engineering fusion antigen

[0036] According...

Embodiment 2

[0040] Example 2 Activity Identification of DNaseB Genetic Engineering Fusion Antigen

[0041] The DNase B fusion-dominant epitope antigen obtained after purification was routinely subjected to 10% SDS-PAGE electrophoresis, transferred to a membrane, and blocked with 5% skimmed milk powder at room temperature for 1 h. Specific anti-DNase B antibody and non-specific antibody (anti-actin antibody) diluted in blocking solution were incubated overnight at 4°C. After washing the membrane three times at room temperature with TBST, the membrane was incubated with horseradish peroxidase-labeled secondary antibody for 1 h. The membrane was washed three times with TBST at room temperature, and the positive bands were displayed by ECL method, scanned and photographed. The result is as Figure 4 As shown, the obtained DNase B fusion-dominant epitope antigen can be well recognized by specific anti-DNase B antibodies, but not by non-specific antibodies, indicating that the obtained DNase ...

Embodiment 3

[0042] Example 3 Establishment of Anti-DNaseB Antibody Double Antigen Sandwich Detection Kit

[0043] 1. Anti-DNaseB Antibody Double Antigen Sandwich Detection Kit

[0044] Anti-DNaseB Antibody Double Antigen Sandwich Detection Kit mainly includes:

[0045] ELISA plate coated with DNase B fusion-dominant epitope antigen, horseradish peroxidase (HRP)-labeled DNase B fusion-dominant epitope antigen, sample diluent, washing solution, TMB chromogenic substrate A solution, TMB chromogenic substrate Color substrate B solution and reaction termination solution.

[0046] Sample diluent: 1% BSA, 5mM EDTA, 5% goat serum, 0.1% Proclin-300, 0.1% Triton-X100, 1.78% NaCl, 0.04% Thimerosal.

[0047] Washing solution: 5.63% Na 2 HPO 4 12H 2 O, 0.672% NaH 2 PO 4 2H 2 O, 17% NaCl, 1% Tween-20.

[0048] TMB chromogenic substrate A solution: 0.32% citric acid (monohydrate), 0.06% hydrogen peroxide solution, 2.72% NaAc·3H 2 o

[0049] TMB chromogenic substrate solution B: 0.04% EDTA-Na ...

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Abstract

The invention discloses streptodornase B antigen and an application thereof, and belongs to the technical field of genetic engineering. The amino acid sequence of the antigen is shown as SEQID NO.1. The DNaseB antigen can be efficiently expressed, and has high activity. A dual-antigen sandwich method established with the antigen is used for detecting DNaseB resisting antibodies, is high in sensitivity and good in specificity, and is suitable for clinical examination.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a streptococcal DNase B antigen and an application thereof. Background technique [0002] Group A streptococci (group A streptococci), also known as group A hemolytic streptococci or streptococcus pyogenes, is one of the most important pathogens in human bacterial infections, and the infections caused mainly include acute pharyngitis and acute tonsillitis , can also cause lung infection, scarlet fever, skin and soft tissue infection, and systemic infection. The bacterium is also an indirect cause of allergic diseases rheumatic fever and acute glomerulonephritis. The incidence of serious infections caused by group A streptococcus has increased significantly in recent years, which has also attracted greater attention to this type of bacterial infection. [0003] Streptolysin "O" is an exotoxin produced by Group A Streptococcus, which can dissolve red blood...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N15/55C12N15/70C12N1/21G01N33/569G01N33/573C12R1/19
CPCC12N9/22C12N15/70G01N33/573G01N33/56944G01N2333/922
Inventor 冯晓燕张贺秋王超男张玲危利
Owner 东方海洋(北京)医学研究院有限公司
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