Screening and application of active tuberculosis diagnosis molecules

A technology for active tuberculosis and tuberculosis, applied in the biological field, can solve the problems of inability to distinguish active tuberculosis and latent tuberculosis, clinical application limitations, and sensitivity to be improved

Active Publication Date: 2017-04-26
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with TST, IGRAs have higher specificity in areas where BCG is widely vaccinated due to the use of antigens encoded by RD (region of difference) region deletion genes, such as CFP-10, ESAT-6 and TB7. Responsibility still needs to be improved, especially in immunosuppressed populations, there will be more indeterminate results
In addition, neither TST nor IGRAs can distinguish active TB from latent TB, which greatly limits their clinical application in areas with high TB ​​prevalence.

Method used

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  • Screening and application of active tuberculosis diagnosis molecules
  • Screening and application of active tuberculosis diagnosis molecules
  • Screening and application of active tuberculosis diagnosis molecules

Examples

Experimental program
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Effect test

preparation example Construction

[0111] The preparation method of the antigen of the present invention has the following steps:

[0112] (1). Transform or transduce a suitable host cell with a polynucleotide encoding the positive antigen of the present invention (or its variant), or with a recombinant expression vector containing the polynucleotide;

[0113] (2). Host cells cultured in a suitable medium;

[0114] (3). Isolate and purify the antigen of the present invention from culture medium or cells.

[0115] In the present invention, polynucleotide sequences can be inserted into recombinant expression vectors. Methods well known to those skilled in the art can be used to construct expression vectors containing the antigen-encoding DNA sequence and appropriate transcriptional / translational control signals. A vector comprising the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.

...

Embodiment 1

[0153] High-throughput screening of important antigens of Mycobacterium tuberculosis

[0154] 1. Prediction of Mycobacterium tuberculosis secretion and transmembrane proteins

[0155] Download the Mycobacterium tuberculosis genome database and the corresponding protein sequence from NCBI; import the protein sequence into the signal peptide online prediction software SignalP 3.0 (http: / / www.cbs.dtu.dk / services / SignalP / ) for prediction.

[0156] Import the protein sequence into the transmembrane structure online prediction software (http: / / www.cbs.dtu.dk / services / TMHMM / ) for prediction.

[0157] According to the prediction analysis, the inventors selected more than 600 proteins, including sequences of signal peptide proteins, and sequences with transmembrane structures.

[0158] 2. Gene cloning

[0159] Use conventional methods to design primers, amplify the target gene by PCR, recover the target fragment, digest it, clone the target fragment into the conventional commercially...

Embodiment 2

[0183] Serological Value Evaluation of Diagnostic Sensitivity of Strong Positive Clones

[0184] 1. Select 14 strong positive clones determined in Example 1, choose 42 active tuberculosis patient serum and 22 healthy serum samples, and verify these 14 strong positive antigens;

[0185] 2. The 14 antigens were respectively coated on polystyrene microwell plates, overnight at 4°C;

[0186] 3. 200ul 5% milk powder / PBST, block at room temperature for 2 hours; wash with PBST 3 times;

[0187] 4. Add the adsorbed serum at a ratio of 1:50, 100ul / well, combine at 37°C for 1h, wash with PBST 5 times;

[0188] 5. According to 1:10000, dilute HRP enzyme-labeled mouse anti-human IgG (H+L) antibody, 100ul / well, combine at 37°C for 1h, wash 5 times with PBST;

[0189]6. Add 100ul chromogenic substrate for detection, read the OD value at 450nm, and calculate the sensitivity and specificity.

[0190] 7. Data processing

[0191] The OD value of the well to be tested is greater than or equa...

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Abstract

The invention provides screening and application of active tuberculosis diagnosis molecules. In particular, the invention discloses high throughput screening of mycobacterium tuberculosis's important antigens and application thereof in active tuberculosis diagnosis. Based on a high throughput functional protein screening technology of GST fusion expression, 92 positive antigens recognizable by tuberculosis patients' serum are screened out, wherein 14 antigens present strongly positive reaction. Active tuberculosis serologic detection shows that the positive antigens have high sensitivity and specificity in detection of active tuberculosis. Specifically, TBGP1, TBGP2, TBGP3, TBGP4, TBGP5 and TBGP6 6 proteins form an antigen combination for tuberculosis serologic detection, and laboratory verification shows that the combination has high sensitivity and specificity in detection of mycobacterium tuberculosis, and has application value in tuberculosis diagnosis and monitoring.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to the screening and application of active tuberculosis diagnostic molecules. Background technique [0002] Tuberculosis (Tuberculosis) is caused by Mycobacterium Tuberculosis Complex (Mycobacterium Tuberculosis Complex) infection, mainly respiratory infection infectious disease. Due to the invention of Bacillus Calmette-Guerin (BCG) and the widespread use of effective drugs for the treatment of tuberculosis, tuberculosis was once effectively controlled. [0003] Since the 1990s, due to the large-scale population movement brought about by globalization, the abuse of antibiotics, and the slackness of people's awareness of tuberculosis prevention, tuberculosis has resurged and made a comeback, becoming an important infectious disease that seriously threatens human health. WHO and other international organizations formulated the Millennium Development Goals plan to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/35C07K19/00C07K16/12G01N33/569
CPCC07K14/35C07K16/1289C07K2319/00C07K2319/23G01N33/5695G01N2333/35G01N2469/10G01N2469/20
Inventor 潘卫庆徐新东周方斌
Owner TONGJI UNIV
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