Applications of Mycobacterium tuberculosis antigen protein Rv0446c and T-cell epitope peptide thereof

A technology of Mycobacterium tuberculosis and rv0446c, which is applied in the fields of molecular biology and immunology, can solve the problems of poor sensitivity, high price, poor specificity of tuberculosis, etc., and achieves the effect of facilitating quality control, improving sensitivity and reducing false positives

Active Publication Date: 2016-12-21
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high requirements for the bacterial content in the sample, the sensitivity of this method is not high, resulting in a large number of smear-negative patients being undetected and turning positive, and smear-negative patients are contagious, which cannot be ignored. This method has no species specificity and poor sensitivity , affected by sputum specimen and disease condition
Sputum culture is the gold standard for the diagnosis of tuberculosis, but the culture time is too long. Now there are rapid culture systems such as the BACTECMGIT960 system that can isolate and culture Mycobacterium tuberculosis within 2 weeks. Inhibitors are expensive and cannot be widely used in developing countries, and rapid cultures have significantly higher contamina...

Method used

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  • Applications of Mycobacterium tuberculosis antigen protein Rv0446c and T-cell epitope peptide thereof
  • Applications of Mycobacterium tuberculosis antigen protein Rv0446c and T-cell epitope peptide thereof
  • Applications of Mycobacterium tuberculosis antigen protein Rv0446c and T-cell epitope peptide thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Cloning of Mycobacterium tuberculosis antigen gene Rv0446c and protein expression and purification

[0046] Rv0446c (GI: 15607587) is a conserved membrane protein encoded by the Mycobacterium tuberculosis H37Rv genome, containing 256 amino acids, and its amino acid sequence is shown in SEQ ID NO:5. According to its coding gene sequence, primers are designed, and the antigenic protein Rv0446c is obtained by expressing and purifying using a prokaryotic expression system (such as Escherichia coli).

Embodiment 2

[0047] Example 2 Synthesis of Mycobacterium tuberculosis antigen protein Rv0446c T cell epitope peptide

[0048] Based on the T cell IFN-γ release technology, the bioinformatics software TE predict and IEDB were used to predict the T cell epitope on the gene encoding Rv0446c, and the T cell epitope peptide was synthesized by the solid-state synthesis method, and then tested for tuberculosis by the T-SPOT method. Specific T lymphocytes in patients, patients with other lung diseases, and healthy people were detected, so as to evaluate the sensitivity and specificity of the antigen for tuberculosis detection.

[0049] The Mycobacterium tuberculosis antigenic protein Rv0446c T cell epitope peptide provided in this example is selected from P120, P121, P122 and P123, and its amino acid sequences are respectively shown in SEQ ID NO: 1-4.

Embodiment 3

[0050] Example 3 Preparation of Tuberculosis T-SPOT Detection Kit

[0051] The basic composition of the test kit is as follows:

[0052] ① The protein antigen Rv0446c prepared in Example 1 and / or the epitope peptide antigen synthesized in Example 2: the epitope peptide is selected from at least one of P120, P121, P122 and P123.

[0053] ② Primary antibody: mouse IgG monoclonal antibody against human or animal IFN-γ.

[0054] Enzyme-labeled reagent: another mouse IgG monoclonal antibody labeled with horseradish peroxidase against different epitopes of human or animal IFN-γ.

[0055] ③Standard product:

[0056] Culture plate: 96-well microwell reaction plate containing PVDF membrane or nitrocellulose membrane, the positive control well contains tuberculosis non-specific stimulating antigen (such as PHA, etc.), and the background control contains PBS or basal solution.

[0057] ④ Other reagents and consumables required for T-SPOT detection.

[0058] The primary antibody was i...

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Abstract

The present invention relates to applications of Mycobacterium tuberculosis antigen protein Rv0446c and a T-cell epitope peptide thereof in preparation of tuberculosis detection reagents, vaccines and medicines, wherein the amino acid sequences of the antigen protein Rv0446c and the T-cell epitope peptide thereof are respectively represented by SEQ ID NO:1-5. According to the present invention, the Mycobacterium tuberculosis antigen protein Rv0446c and the T-cell epitope peptide thereof are used as the stimulants for the specific T cell and B cell immune response caused by Mycobacterium tuberculosis infection, and the false positive caused by the impure antigen can be reduced compared with the use of the complete antigen in the prior art; and the detection reagents prepared from the antigen protein Rv0446c and the T-cell epitope peptide thereof can be widely used for assisted diagnosis of tuberculosis, epidemiological surveillance and other related fields, and the tuberculosis vaccines and the anti-tuberculosis drugs prepared from the antigen protein Rv0446c and the T-cell epitope peptide thereof can be used for prevention and treatment of tuberculosis.

Description

technical field [0001] The invention relates to the fields of molecular biology and immunology, in particular to the application of mycobacterium tuberculosis antigen protein Rv0446c and its T cell epitope peptide in the preparation of tuberculosis detection reagents, vaccines and medicines. Background technique [0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis. Survey results show that one-third of the world's population is latently infected, and 5% to 10% may develop active tuberculosis in future life. Since the World Health Organization declared tuberculosis a global crisis in 1993, the morbidity and mortality of tuberculosis have remained high. According to the WHO report, there are about 8 million new tuberculosis patients every year, and about 2 to 3 million people die of tuberculosis every year. my country ranks second among the 22 countries with a high burden of tuberculosis. The results of the fifth national epidemiological s...

Claims

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Application Information

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IPC IPC(8): G01N33/569C07K14/35A61K39/04A61K39/40A61P31/06
CPCA61K39/04A61K2039/505C07K14/35G01N33/5695G01N33/56972G01N2333/35
Inventor 万康林王雪枝刘海灿李马超蒋毅
Owner ICDC CHINA CDC
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