High throughput screening of tubercle bacillus important antigens, and application of tubercle bacillus important antigens in tuberculosis diagnosis

A technology of Mycobacterium tuberculosis and antigen, applied in the direction of application, bacteria, fungi, etc., can solve the problems of false positive, failure to distinguish active and latent infection, and difficulty in distinguishing active TB and latent TB

Inactive Publication Date: 2013-12-25
TONGJI UNIV
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Molecular biology is based on the principle of nucleic acid amplification to detect MTB-specific nucleic acids. Although the specificity has been improved, the sensitivity varies greatly, and "false positives" are prone to occur.
At the end of 2010, WHO recommended Xpert MTB / RIF (Cepheid) technology as a new rapid nucleic acid amplification detection method for MTB. Its advantage is that the detection of sputum samples is simple and the detection time is shortened to 2 hours, but it is difficult to distinguish active TB from latent TB.
Using single, recombinant or fusion antigens to detect serum antibodies has a sensitivity of 1% to 60% and a specificity of 53% to 99%, failing to distinguish between active and latent infection

Method used

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  • High throughput screening of tubercle bacillus important antigens, and application of tubercle bacillus important antigens in tuberculosis diagnosis
  • High throughput screening of tubercle bacillus important antigens, and application of tubercle bacillus important antigens in tuberculosis diagnosis
  • High throughput screening of tubercle bacillus important antigens, and application of tubercle bacillus important antigens in tuberculosis diagnosis

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preparation example Construction

[0077] The preparation method of the antigen of the present invention has the following steps:

[0078] (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;

[0079] (2). Host cells cultured in a suitable medium;

[0080] (3). Isolate and purify the antigen of the present invention from culture medium or cells.

[0081] 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

[0112] High-throughput screening of important antigens of Mycobacterium tuberculosis

[0113] 1. Prediction of Mycobacterium tuberculosis Secretion and Transmembrane Proteins

[0114] 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.

[0115] Import the protein sequence into the signal peptide online prediction software (http: / / www.cbs.dtu.dk / services / TMHMM / ) for prediction. According to the prediction result, the sequence predicted to be the signal peptide protein is selected according to the p value greater than 0.1.

[0116] 2. Gene cloning

[0117] 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 available expression vector pGEX-4T-1, transform...

Embodiment 2

[0141] Comparison of Diagnostic Sensitivity of Strong Positive Clones

[0142] 1. Select 5 strong positive clones; respectively adsorb the 5 strong positive antigens to GSH-microwell plates, and set GST expressing bacteria liquid as negative control and PBS as blank control; wash with PBST five times;

[0143] 2. 200 μl 5% milk powder / PBST, block at room temperature for 2 hours; wash five times with PBST;

[0144] 3. The adsorbed serum was diluted 1:20 with 5% milk powder, and the final serum dilution was 1:1000; 100 μl / well, combined at 37°C for 1 hour; washed five times with PBST;

[0145] 4. Dilute HRP enzyme-labeled mouse anti-human IgG (H+L) antibody at 1:20,000, 100 μl / well, combine at 37°C for 1 hour; wash with PBST 5 times;

[0146] 5. Add 100 μl of commercially available Super Signal ELISA Femto for detection, read the fluorescence intensity value at 425 nm; calculate the R value.

[0147] 6. The specific results are shown in Table 2.

[0148] Table 2 Comparison of...

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Abstract

This invention relates to high throughput screening of tubercle bacillus important antigens, and an application of the tubercle bacillus important antigens in tuberculosis diagnosis. According to the present invention, specifically 46 positive antigens capable of being recognized by tuberculosis patient serum are screened with a GST fusion protein based high throughput function protein screening technology, wherein the 5 antigens present strong positive reactions, and serology detection results show that detection of tubercle bacillus by using the positive antigens has high sensitivity and specificity. The present invention further provides a corresponding tuberculosis diagnosis and monitoring method and a kit.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to the high-throughput screening of important antigens of Mycobacterium tuberculosis and its application in the diagnosis of tuberculosis. Background technique [0002] Tuberculosis (Tuberculosis, TB) is the most deadly disease caused by a single pathogen in the world. According to WHO estimates, about 2 billion people in the world are infected with Mycobacterium tuberculosis (MTB), and about 2 million people die from tuberculosis every year. China is one of 22 countries with a high burden of TB, accounting for 80% of global TB cases. At present, more than 1 / 3 of the country's population is infected with Mycobacterium tuberculosis, of which 4.5 million are active tuberculosis patients, and about 250,000 people die of tuberculosis every year, ranking first among various infectious diseases. [0003] In recent years, with the prevalence of drug-resistant tuberculo...

Claims

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Application Information

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IPC IPC(8): C07K14/35G01N33/68C12N15/31C12N15/63C12N1/21C12N1/19C12N5/10C12P21/02C07K16/12G01N33/569C12R1/32
Inventor 潘卫庆徐新东周方斌
Owner TONGJI UNIV
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