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Specific antigen protein for diagnosing tuberculosis and application thereof in preparing diagnosis kit

An antigenic protein and specific technology, applied in the field of tuberculosis diagnostic reagents, can solve the problems of long culture period, not being able to be used as a basis for diagnosis, restrictions, etc., and achieve the effects of reducing diagnostic costs, good diagnostic coincidence rate, and good social benefits

Inactive Publication Date: 2015-04-22
孙丽华
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it is the "golden indicator" for diagnosing pulmonary tuberculosis, its diagnostic rate is low and the culture period is long
2. Sputum tuberculosis culture, the results are highly reliable, and tuberculosis drug susceptibility test can be done, but it takes 6-8 weeks, and the application is limited
High false positive rate, easy to misdiagnose
2. Positive tuberculosis antibody detection in blood and sputum is also helpful for diagnosis, such as 38kDa and many fusion protein antigens including 38kDa antigen, which are used for the diagnosis of humoral immunity, but the false positive rate is also inevitable
[0011] (4) Other examinations can only be used as an auxiliary diagnosis and cannot be used as a basis for diagnosis
[0012] In the commonly used diagnostic techniques for pulmonary tuberculosis, there are problems such as low positive rate, long cycle, complicated operation, and high false positive rate. Therefore, there is an urgent need for a fast, simple, sensitive, and specific diagnostic method to quickly and accurately diagnose tuberculosis.

Method used

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  • Specific antigen protein for diagnosing tuberculosis and application thereof in preparing diagnosis kit
  • Specific antigen protein for diagnosing tuberculosis and application thereof in preparing diagnosis kit
  • Specific antigen protein for diagnosing tuberculosis and application thereof in preparing diagnosis kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1 Expression and purification of recombinant antigenic protein

[0025] The DNA sequence (SEQ ID NO.2) of the artificially synthesized antigen protein was stored in DH5α, and then the expression vectors of DH5α and pET28b were digested with NcoI and XhoI endonucleases respectively, and the target fragment and expression vector were recovered. 4 Under the action of DNA ligase, ligate at 4°C for 16 hours (see vector construction diagram figure 1), transformed into Escherichia coli BL21(DE3) by heat shock method, and cultivated overnight at 37°C. Pick colonies that grow well on the plate and have typical Escherichia coli characteristics and cultivate them in liquid LB medium. At the same time, add kanamycin with a final concentration of 30 μg / ml to the medium, and cultivate for 3 hours at 37°C and 220rpm , Extract the plasmid for enzyme digestion and sequencing identification, the enzyme map and sequencing map are in line with expectations. Pick colonies that ...

Embodiment 2

[0026] Example 2 Preparation of Human IFN-γ Detection Kit

[0027] Using the recombinant antigenic protein prepared in Example 1, the monoclonal antibody against IFN-γ was screened according to the standard monoclonal antibody screening method, and two monoclonal antibodies against different antigenic clusters of IFN-γ were obtained. Concentration-coated ELISA plate, overnight at 4°C, and then blocked at 37°C for 1-2 hours, then freeze-dried the ELISA plate, which is a commercial ELISA plate, and the other antibody is labeled with biotin according to standard methods . Dilute the interferon gamma standard (1 μg / ml) as required to form a standard curve control. According to the requirements of the experiment, take a suitable microplate strip, and put the unused microplate into the microplate bag, keep it at 4°C-8°C, and add the standard and positive control (interferon gamma standard: 1ng / ml), blank control and sample to be tested (supernatant after cultured fresh blood), 50...

Embodiment 3

[0028] Sensitivity and specificity detection of embodiment 3 tuberculosis diagnostic kits

[0029] Collect 3ml of fresh heparin anticoagulated venous blood from 50 cases of confirmed tuberculosis patients and 20 healthy cases of non-tuberculosis infection, each take 1ml of whole blood and culture it in a sterile culture plate, add recombinant antigen protein (concentration: 20μg / ml ) for IFN-γ stimulation, and the other well as a control, incubated at 37°C for 20 hours, centrifuged to collect the supernatant, 100 μl of the supernatant was added to the whole blood IFN-γ detection kit prepared in Example 2 for detection, and the data showed the diagnosis of tuberculosis The sensitivity of infected patients is 100%, and the specificity is as high as 98.2%. Compared with the clinical diagnosis results, this kit shows good diagnostic consistency.

[0030]

[0031]

[0032]

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PUM

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Abstract

The invention discloses a specific antigen protein for diagnosing tuberculosis and application thereof in preparing a diagnosis kit. The amino acid sequence of the antigen protein is disclosed as SEQ ID NO.1, or is a conservative mutant obtained by carrying out conservative mutation of addition, deletion, substitution and modification of one or more amino acids on the sequence. When the antigen protein is used for stimulation, the sensitivity of the tuberculosis antigen specific whole blood IFN-gamma diagnosis is 100%, the specificity is up to 98.2%, and therefore, the protein can be used for preparing reagents, kits, vaccines or drugs for diagnosing, preventing and / or treating diseases caused by Mycobacterium tuberculosis infection.

Description

technical field [0001] The invention belongs to the technical field of tuberculosis diagnostic reagents, and in particular relates to a specific antigenic protein for diagnosing tuberculosis and its use in preparing a diagnostic kit. Background technique [0002] Mycobacterium tuberculosis is one of the most threatening pathogens to human health. Tuberculosis is the main infectious disease among adults in the world today and has posed a serious challenge to international public health ([1] Khasnobis S., Escuyer V.E., Chatterjee D. Emerging therapeutic targets in tuberculosis: post-genomic era. Expert Opin. Ther. Targets, 2002, 6:21~40). According to the WHO report: about 2 billion people have been infected with tuberculosis in the world, there are about 20 million tuberculosis patients, 8 to 10 million new cases are reported every year, and about 3 million people die from tuberculosis every year, which is equivalent to one person every 10 seconds Died of tuberculosis. Afte...

Claims

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Application Information

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IPC IPC(8): C07K14/35C12N15/31C07K16/12G01N33/68G01N33/569A61K39/04A61P31/06C07K16/24
CPCC07K14/35A61K39/00C07K16/1289G01N33/5695
Inventor 陈刚
Owner 孙丽华
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