Tuberculosis immunodiagnosis molecular marker and application thereof in preparation of vaccines

Abstract

Belonging to the field of immunology and molecular biology, the invention relates to a tuberculosis immunodiagnosis molecular marker and application thereof in preparation of vaccines. Specifically, the invention relates to use of protein with an amino acid sequence shown as SEQ ID NO:1 or SEQ ID NO:2 in preparation of anti-tuberculosis drugs or mycobacterium tuberculosis-inhibiting drugs, or use in preparation of drugs detecting tuberculosis or drugs detecting mycobacterium tuberculosis. As an antigen, the protein shown as SEQ ID NO:1 or SEQ ID NO:2 has good sensitivity and specificity, also has reaction intensity significantly higher than that of positive control 38kDa, and has the potential of application to preparation of antituberculosis drugs like anti-tuberculosis vaccines.

Description

technical field

[0001] The invention belongs to the field of immunology and molecular biology, and relates to a tuberculosis immunodiagnostic molecular marker and its application in preparing vaccines. 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 lev...

Examples

Embodiment 1

[0046] Embodiment 1: Mass spectrometry identification experiment

[0047] 1. Experimental Materials and Instruments

[0048]Target protein samples: prepared Rv0237 protein (SEQ ID NO:1) and Rv1111c protein (SEQ ID NO:2).

[0049] Instruments: Ultimate 3000Nano-LC system (Dionex, USA), linear ion trap multistage tandem mass spectrometer (linear trap quadruple, LTQ), Orbitrap Velos mass spectrometer (ThermoScientific, Germany).

[0050] Data processing software: MassLynx version 4.1 (Dionex, USA), Xcalibur software v2.2.6 (Thermo), MASCOT version 2.2 (Matrix Sciences, UK).

[0051] 2. Experimental method

[0052] (1) After the target protein is subjected to SDS PAGE, rinse the film with deionized water for 15 minutes; cut off the target fragment, 1-2 mm in size; put it in a centrifuge tube with low protein adsorption, and use 100 μL of ddH 2 O Rinse the pellets in the dish and repeat, discard the liquid and add 40 μL of (ACN) / ddH 2 O(50 / 50), discard the liquid, incubate for...

Embodiment 2

[0075] Embodiment 2: Humoral immunity experiment

[0076] Humoral immune antigen can specifically bind with the body produced, prevent it from infecting normal cells, and combine with macrophages, so that macrophages can phagocytize antigens to achieve the purpose of sterilization, so proteins with high humoral immune response intensity can also be Stimulate the body to produce antibodies to achieve immune protection.

[0077] 1. Experimental materials

[0078] Protein samples: Rv0237 protein and Rv1111c protein. Positive control 38kDa (Immuno Diagnostics Inc. USA).

[0079] Main reagents: mouse secondary antibody high-throughput kit, PS-MK15 Wes-Mouse 12-230 kDa master kit with split buffer for 200 data (Immuno Diagnostics Inc. USA). Streptomycin-labeled goat anti-human IgG, Thermo Scientific, Germany).

[0080] 2. Experimental principles and methods

[0081] Using Western Blot method. Western Blot technology is a classic protein detection technology. The traditional ...

technical field

[0001] The invention belongs to the field of immunology and molecular biology, and relates to a tuberculosis immunodiagnostic molecular marker and its application in preparing vaccines. 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 lev...

Examples

Embodiment 1

[0046] Embodiment 1: Mass spectrometry identification experiment

[0047] 1. Experimental Materials and Instruments

[0048]Target protein samples: prepared Rv0237 protein (SEQ ID NO:1) and Rv1111c protein (SEQ ID NO:2).

[0049] Instruments: Ultimate 3000Nano-LC system (Dionex, USA), linear ion trap multistage tandem mass spectrometer (linear trap quadruple, LTQ), Orbitrap Velos mass spectrometer (ThermoScientific, Germany).

[0050] Data processing software: MassLynx version 4.1 (Dionex, USA), Xcalibur software v2.2.6 (Thermo), MASCOT version 2.2 (Matrix Sciences, UK).

[0051] 2. Experimental method

[0052] (1) After the target protein is subjected to SDS PAGE, rinse the film with deionized water for 15 minutes; cut off the target fragment, 1-2 mm in size; put it in a centrifuge tube with low protein adsorption, and use 100 μL of ddH 2 O Rinse the pellets in the dish and repeat, discard the liquid and add 40 μL of (ACN) / ddH 2 O(50 / 50), discard the liquid, incubate for...

Embodiment 2

[0075] Embodiment 2: Humoral immunity experiment

[0076] Humoral immune antigen can specifically bind with the body produced, prevent it from infecting normal cells, and combine with macrophages, so that macrophages can phagocytize antigens to achieve the purpose of sterilization, so proteins with high humoral immune response intensity can also be Stimulate the body to produce antibodies to achieve immune protection.

[0077] 1. Experimental materials

[0078] Protein samples: Rv0237 protein and Rv1111c protein. Positive control 38kDa (Immuno Diagnostics Inc. USA).

[0079] Main reagents: mouse secondary antibody high-throughput kit, PS-MK15 Wes-Mouse 12-230 kDa master kit with split buffer for 200 data (Immuno Diagnostics Inc. USA). Streptomycin-labeled goat anti-human IgG, Thermo Scientific, Germany).

[0080] 2. Experimental principles and methods

[0081] Using Western Blot method. Western Blot technology is a classic protein detection technology. The traditional ...

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