Mycobacterium tuberculosis T cell antigen epitope polypeptide and application thereof

A technology of mycobacterium tuberculosis and cell antigens, applied in the field of T cell antigen epitope polypeptides, can solve the problems of reduced secretion, limited promotion, and insufficient sensitivity of detection, etc., to achieve increased sensitivity, guaranteed quality, and low cost Effect

Active Publication Date: 2017-07-11
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the above-mentioned ESAT-6 and CFP-10 antigens belong to the early secretory proteins of Mycobacterium tuberculosis, which are mainly secreted during the rapid growth period of Mycobacterium tuberculosis, and the secretion amount decreases during persistent tuberculosis infection, resulting in insufficient detection sensitivity, which limits the promotion of this reagent
Another study showed that the effect of the above two IGRAs reagents in developing countries is not satisfactory, which may be related to the higher infection rate of non-tuberculous mycobacteria in developing countries
In addition, the two IGRAs reagents are expensive and diffi...

Method used

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  • Mycobacterium tuberculosis T cell antigen epitope polypeptide and application thereof
  • Mycobacterium tuberculosis T cell antigen epitope polypeptide and application thereof
  • Mycobacterium tuberculosis T cell antigen epitope polypeptide and application thereof

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0032] [Example 1] Screening of Mycobacterium tuberculosis antigen dominant T cell epitope

[0033] We first artificially synthesized polypeptide fragments of different Mycobacterium tuberculosis antigens through peptide synthesis technology. According to the difference in sex, it is divided into PP1-PP13 peptides (as shown in Table 1), using peptide synthesis technology to artificially synthesize polypeptide fragments of different sizes (PP1-PP13 peptides) covering the full length of the antigenic protein, and comparing the T cell immunity of different peptide fragments. Effect, thereby identifying a novel dominant strong T cell epitope.

[0034] Table 1. Some different polypeptide fragments of the artificially synthesized RD13 region of Mycobacterium tuberculosis.

[0035]

[0036] The screening of T cell dominant epitopes is to detect the specific gamma interferon secreted by different peptides stimulating T cells by ELISA method, so as to measure the immune effect of d...

Embodiment 2

[0041] [Example 2] ELISPOT detects the release level of gamma interferon in tuberculosis patients

[0042] Using the dominant T cell epitope polypeptide PP4 to stimulate, 45 tuberculosis patients (ATB, no immunocompromised patients, with TB clinical symptoms and X-ray examination found abnormalities, tuberculosis culture confirmed), 20 medical staff (LTBI , without clinical symptoms of TB and abnormalities found in X-ray examination, without other abnormalities such as immunodeficiency, long-term exposure history of tuberculosis working in a tuberculosis hospital) and 57 healthy controls (HD, without clinical symptoms of TB and abnormalities found in X-ray examination, without immunodeficiency, etc. Other abnormalities, no history of tuberculosis contact) for testing, the specific steps are as follows:

[0043] (1) Coating: γ-interferon monoclonal antibody (100 μl 1:60 dilution) coated 96-well microplate plate (ELISA plate): add 1:60 diluted γ-interferon monoclonal antibody 10...

Embodiment 3

[0051] [Example 3] The dominant T cell epitope polypeptide and the polypeptide combined with the CE polypeptide used in T-SPOT.TB were used to stimulate respectively, and ELISPOT was used to detect the release level of gamma interferon in tuberculosis patients.

[0052] Utilize the tuberculosis infection diagnosis enzyme-linked immunospot-ELISPOT kit of the present invention, respectively use described dominant T cell epitope polypeptide and this polypeptide to combine CE polypeptide used for T-SPOT.TB to stimulate, treat 50 tuberculosis patients (TB, There were TB clinical symptoms and abnormal X-ray examination findings, tuberculosis bacillus culture confirmed, including 2 patients with tuberculosis / AIDS co-infection and 3 elderly people over 75 years old (78, 80 and 82 years old respectively) and 56 healthy controls (HD , no TB clinical symptoms and abnormalities found in X-ray examination, no immunodeficiency and other abnormalities, no history of tuberculosis exposure) for...

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Abstract

The invention belongs to the field of biomedical examination, and concretely relates to a reagent and a method for detecting Mycobacterium tuberculosis infection, and an application of a Mycobacterium tuberculosis T cell antigen epitope polypeptide. The Mycobacterium tuberculosis detection agent disclosed in the invention is obtained through screening Mycobacterium tuberculosis specific T cell antigen epitopes, and contains a polypeptide PP4 represented by SEQ ID NO.1. The method is characterized in that the single polypeptide PP4 or a combination of the PP4 and a CE polypeptide used in T-SPOT. TB test (Oxford Immunotec, Oxford, UK) is in contact with T cells of a Mycobacterium tuberculosi-infected individual in order to detect cytokines released from the T cells. Active tuberculosis or latent tuberculosis infection can be effectively detected without the interference of BCG vaccination. The method has the characteristics of high detection sensitivity, high specificity, fast speed, and simplicity in operation, and can be used for the assisted diagnosis of pulmonary tuberculosis, extrapulmonary tuberculosis and latent infection.

Description

technical field [0001] The present invention relates to Mycobacterium tuberculosis specific antigen epitope polypeptide, especially T cell antigen epitope polypeptide of antigen RD13 region. Background technique [0002] Tuberculosis is a chronic infectious disease that seriously endangers human health. Nearly one-third of people in the world are currently infected with Mycobacterium tuberculosis. The number of deaths due to tuberculosis reaches 130,000 per year, exceeding the total number of deaths from other infectious diseases. [0003] Early diagnosis of tuberculosis is of great significance in controlling the progression of the disease in patients and the spread of the disease. At present, the clinical diagnosis of tuberculosis still lacks high-sensitivity and high-specificity means. The antigen tuberculin used in the traditional tuberculin skin test (TST / PPD) can produce a high rate of false positives because of its ability to cross-react with BCG vaccination, and the...

Claims

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Application Information

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IPC IPC(8): C07K14/35G01N33/68G01N33/569
CPCC07K14/35G01N33/5695G01N33/6866G01N2333/57
Inventor 章晓联
Owner WUHAN UNIV
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