Preparation method of recombinant antigen of mycobacterium tuberculosis capable of being used for diagnosing tuberculosis infection

A technology of Mycobacterium tuberculosis and recombinant antigen, applied in the field of Mycobacterium tuberculosis recombinant antigen and preparation thereof

Inactive Publication Date: 2014-01-22
NANJING UNIVERSTIY SUZHOU HIGH TECH INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] ESPC antigen is a specific secreted antigen component produced by Mycobacterium tuberculosis, similar to ESAT6 antigen and CFP‐10 antigen, ESPC antigen is also regulated by the RD1 gene region; ESPC antigen has multiple important epitopes, It can be recognized by CD4+ and CD8+ T lymphocytes. Compared with other Mycobacterium tuberculosis antigens, ESPC antigen has stronger antigen specificity. Studies have found that ESPC antigen and BCG antigen have no obvious common antigenic components

Method used

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  • Preparation method of recombinant antigen of mycobacterium tuberculosis capable of being used for diagnosing tuberculosis infection
  • Preparation method of recombinant antigen of mycobacterium tuberculosis capable of being used for diagnosing tuberculosis infection
  • Preparation method of recombinant antigen of mycobacterium tuberculosis capable of being used for diagnosing tuberculosis infection

Examples

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Effect test

Embodiment 1

[0042] Example 1: Using ESPC recombinant antigen to establish a method for detecting cellular immune function of tuberculosis infection

[0043] (1) The required reagents are as follows:

[0044] Lymphocyte separation medium: Ficoll-diatrizoate premix solution with a specific gravity of 1.077;

[0045] IFN-γ detection antibody: including mouse anti-human IFN-γ coating antibody and HRP-labeled mouse anti-human IFN-γ detection antibody;

[0046] IFN-γ standard protein: recombinant human IFN-γ recombinant protein;

[0047] Medium: RPMI1640 medium containing double antibody and 10% fetal bovine serum;

[0048] Chromogenic agent: TMB chromogenic solution;

[0049] Stop solution: 1N HO 2 SO 4 solution;

[0050] (2) Operation method:

[0051] First separate the peripheral blood mononuclear cells, add an appropriate amount of lymphocyte separation solution (polysucrose-diatrizoate meglumine premixed solution, the specific gravity of the layered solution is 1.077) in the test tu...

Embodiment 2

[0057] Embodiment 2: Utilize ESPC recombinant antigen to establish the serological detection method of tuberculosis infection

[0058] (1) The required reagents are as follows:

[0059] Sample diluent: 0.01M PBS, pH7.4

[0060] Washing buffer: containing 0.01M PBS and 0.05% Tween20, pH7.4

[0061] Blocking solution: 0.01M PBS buffer containing 1% bovine serum albumin BSA, pH7.4

[0062] Enzyme-labeled antibody: HRP-labeled rabbit anti-human IgG antibody, diluted 1:10000

[0063]Substrate buffer: one-component TMB chromogenic solution, containing 10ug / ml tetramethylbenzidine (tetramethylbenzidine, TMB)

[0064] Stop solution: 1N H 2 SO 4

[0065] (2) Operation method:

[0066] Add 100ul ESPC antigen solution (1ug / ml) diluted with sample diluent to the 96-well ELISA plate, coat overnight at 4°C, wash the culture plate 5 times with washing buffer, and then add 1% bovine serum albumin ( BSA) blocking solution for 2 hours, tuberculosis plasma / or serum samples to be tested w...

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Abstract

The invention provides a recombinant DNA (Deoxyribonucleic Acid) which codes a recombinant antigen of mycobacterium tuberculosis, an expression carrier containing the recombinant DNA, the recombinant antigen of mycobacterium tuberculosis which is coded by the recombinant DNA as well as preparation method and use of the recombinant DNA and the recombinant antigen. The invention further provides a reagent, a composition and a kit containing the recombinant antigen, which can specifically detect infection of mycobacterium tuberculosis. The method is free from cross reaction and can be used for detecting an antigen-specific serum IgG (immunoglobulin G) antibody of the mycobacterium tuberculosis, thereby providing an immunodetection method which is convenient and high in sensitivity, accuracy and stability for development of other kits. The method is simple in using instruments, and the technical operation is relatively easy to master.

Description

technical field [0001] The invention provides a Mycobacterium tuberculosis recombinant antigen and a preparation method thereof. The invention also provides a diagnostic kit for Mycobacterium tuberculosis, which can specifically detect Mycobacterium tuberculosis infection, and involves genetic engineering technology and immune diagnostic techniques. Background technique [0002] Tuberculosis is still a disease that seriously endangers human health in the 21st century, among which immigration, HIV infection and drug resistance are the three major problems facing the global tuberculosis control in the 21st century. According to the estimates of the World Health Organization (WHO), there are currently 2 billion people infected with Mycobacterium tuberculosis (MTB) in the world, with an average of 1 new infection every 1 second, and 8 to 10 million new tuberculosis patients each year , 3 million people died of tuberculosis, the infectious disease with the highest mortality rate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/70C12N15/10C07K14/35C12Q1/68C12Q1/04G01N33/68G01N33/569
Inventor 王宏伟高千张侠苏忠兰易龙丁一冰田曼
Owner NANJING UNIVERSTIY SUZHOU HIGH TECH INST
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