Tuberculosis immunodiagnosis molecular marker and vaccine use thereof

A technology for tuberculosis and Mycobacterium tuberculosis, applied in the fields of immunology and cell biology, can solve the problems of confusion of lung diseases, high false positive rate, easy false positive rate, etc., and achieve the effect of good coincidence rate and response intensity

Active Publication Date: 2016-03-09
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is easy to be confused with other lung diseases and needs to be confirmed by a professional doctor
(3) Immunological diagnosis of pulmonary tuberculosis: 1. The tuberculin pure protein derivative (PPD) test is commonly used. A positive test is one of the evidences of tuberculosis infection, but the false positive rate is high and it is easy to misdiagnose
2. Positive tuberculosis antibody tests in blood and sputum are also helpful for diagnosis, and false positive rates are also prone to occur
4. Polymerase chain reaction (PCR), the advantage is that the sensitivity can reach 98%-100%, and the disadvantage is that the specificity is poor
(4) Other examinations: can only be used as an auxiliary diagnosis, not as a basis for diagnosis

Method used

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  • Tuberculosis immunodiagnosis molecular marker and vaccine use thereof
  • Tuberculosis immunodiagnosis molecular marker and vaccine use thereof
  • Tuberculosis immunodiagnosis molecular marker and vaccine use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1: ELISPOT analysis

[0072] Elispot analysis visualizes single-cell secretion products. These assays are exceptionally sensitive because it is captured directly around the secreting cell, and before the surface is diluted, or captured by receptors on adjacent cells, or degraded. Autoimmune detection, organ transplantation, research and development and testing of vaccines and drugs, T cell function research, tumor research, research and detection of infectious diseases, virus research, etc. The specific principle is as follows: cells are stimulated by antigens to produce cytokines, and the cytokine antigens are captured by specific monoclonal antibodies. After cell dissociation, the captured cytokines are bound to a biotin-labeled secondary antibody, followed by alkaline phosphatase-labeled avidin. After incubation with BCIP / NBT substrate, "purple" spots appeared on the PVDF plate, indicating that the cells had produced cytokines, and the results were obtain...

Embodiment 2

[0105] Embodiment 2: Preparation of animal immunity and lymphocyte samples

[0106] In order to verify the results of in vitro cellular immunity tests, identification of the antigenicity of the target protein in vivo is also a very important verification method, so the inventors conducted animal experiments again, and the animal selected was C57BL / 6MarkDoherty, which is a model animal for tuberculosis research [T. Mark Doherty. Tropical Medicine and International Health. New vaccines against tuberculosis. 2004(9): 818-826.].

[0107] 1. Experimental Animals and Grouping

[0108] A total of 50 C57BL / 6 mice (female, 6-8 weeks old) were randomly divided into groups of 6, and the number of the mice was marked by ear cutting. In addition, the remaining 2 mice were used as full negative controls, without immunization, and kept for observation.

[0109] Group 1: PBS, as negative control.

[0110] Group 2: OVA, used as other protein control.

[0111] Group 3: Rv1811, as IFN-γ ne...

Embodiment 3

[0127] Example 3: Flow cytometric detection experiment of intracellular factors IFN-γ and IL-2

[0128] 1. Experimental samples and reagents

[0129] Cell samples: lymphocytes from the 8 groups of mouse samples in Example 2 above (8 copies in one batch).

[0130] Kit: CellTrace from invitrogen TM CFSECell Proliferation Kit (C34554).

[0131] 2. Experimental method steps

[0132] (1) A batch of 8 parts of lymphocytes collected in Example 2 was divided into 2×10 6 For dilution, use a 96-well plate, 100 μl per well, and set 3 wells for each lymphocyte. The excess cells were set as a control hole (no stimulator, no staining antibody). A total of 25 holes.

[0133] Wherein, the stimuli are respectively Rv0773c, Rv0875c, Rv1115, Rv2553c, PBS, OVA, negative protein control Rv1811, positive control Rv3875; and the stimuli added to each well correspond to the stimuli used for immunization in the previous Example 2, For example, if the original animal was immunized with Rv0773c...

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Abstract

The invention belongs to the fields of immunology and cytobiology, relates to a tuberculosis immunodiagnosis molecular marker and vaccine use thereof, and in particular relates to use of any one or more of proteins, selected from amino acid sequences shown as SEQ ID NO: 1-4, in preparing a tuberculosis diagnostic agent; preferably, the tuberculosis is phthisis. The protein shown by any sequence in SEQ ID NO: 1-4 can serve as a tuberculosis (such as phthisis) immunodiagnosis molecular marker, and has good coincidence rate and reaction intensity; besides, the tuberculosis immunodiagnosis molecular marker has the potential for being applied to preparation of a tuberculosis vaccine.

Description

technical field [0001] The invention belongs to the field of immunology and cell biology, and relates to a tuberculosis immunodiagnostic molecule marker and a vaccine application thereof. 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. After infecting the human body, Mycobacterium tuberculosis is mostly in a latent state or a persistent infection state. It can evade the host's defense in the infected host cells and reproduce in large numbers and achieve a delicate balance with the host. May be ill. Typically, 10% of infected people will develop active tuberculosis. An untreated active tuberculosis patient can infect 10-15 people a year. [0003] In the past 50 years, the theory and technology of tuberculosis control, clinical diagnosis and treatment level and researc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/569C07K16/12C12N5/0783A61K39/04A61P31/06
CPCA61K39/04C07K16/1289C12N5/0636C12N2501/998G01N33/5695G01N33/68
Inventor 金奇刘立国李海凤张笑冰张维佳
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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