Combined protein and detection reagent for the detection of Mycobacterium tuberculosis

A technology of mycobacterium tuberculosis and combined proteins, which is applied in the field of medical immunological detection, can solve the problems of delayed clinical diagnosis and treatment, long detection cycle, high price, etc., and achieve the effect of improving the cure rate, accurate diagnosis and treatment, and avoiding transmission

Active Publication Date: 2022-07-01
中国人民解放军总医院第八医学中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Based on this, the traditional separation and culture method inspection often requires a long detection period, which delays clinical diagnosis and treatment, and the test results of γ-interferon release test need to be further excluded, Xpert-MTB / RIF test is expensive and difficult to popularize technical problems , the present invention proposes a combination protein and detection reagent for the detection of Mycobacterium tuberculosis

Method used

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  • Combined protein and detection reagent for the detection of Mycobacterium tuberculosis
  • Combined protein and detection reagent for the detection of Mycobacterium tuberculosis

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Experimental program
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Effect test

Embodiment 1

[0039] The preparation method of the detection reagent for Mycobacterium tuberculosis is as follows:

[0040] 1), the synthetic gene fragments of the first combined protein of the present invention and the second combined protein are excised from the cloning vector with restriction endonucleases NdeI and XhoI, cloned into the expression vector PET28b, in Escherichia coli BL21 (DE3) After cultured to OD600 of 0.6, IPTG was added to the final concentration of 0.1M to induce expression, and the identification and analysis were carried out by gel electrophoresis and Western Blotting;

[0041] 2), adopt gel (Pharmacia company chelating sephrose fast flow production), purify expression protein through metal chelate chromatography;

[0042] 3), immunize rabbits with human IgG (purchased from Tiangen Biotechnology Co., Ltd.) using standard immunization procedures to prepare rabbit immunized human IgG antibodies (also including IgM, IgA);

[0043] 4), prepare colloidal gold by citric ...

Embodiment 2

[0053] The preparation method of the detection reagent for Mycobacterium tuberculosis is as follows:

[0054] 1), the synthetic gene fragments of the first combined protein of the present invention and the second combined protein are excised from the cloning vector with restriction endonucleases NdeI and XhoI, cloned into the expression vector PET28b, in Escherichia coli BL21 (DE3) After cultured to OD600 of 0.6, IPTG was added to the final concentration of 0.1M to induce expression, and the identification and analysis were carried out by gel electrophoresis and Western Blotting;

[0055] 2), adopt gel (Pharmacia company chelating sephrose fast flow production), purify expression protein through metal chelate chromatography;

[0056] 3), immunize rabbits with human IgG (purchased from Tiangen Biotechnology Co., Ltd.) using standard immunization procedures to prepare rabbit immunized human IgG antibodies (also including IgM, IgA);

[0057] 4), prepare colloidal gold by citric ...

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Abstract

The present invention provides a combined protein and detection reagent for the detection of Mycobacterium tuberculosis. The combined protein is a B cell composed of TBCG_03312 protein or partial amino acids of TBCG_03312 protein and the B cell antigenic determinant of Rv1681 antigen protein and Rv1932 antigen protein A combined protein formed by combining any two or more of the antigenic determinant, the B-cell antigenic determinant of the Rv1956 antigenic protein, and the B-cell antigenic determinant of Rv3368c through a soft linker. The formed combined protein has stronger sensitivity and specificity, and the application of the combined protein in the rapid detection method can more accurately and rapidly detect the serum of suspected active tuberculosis patients, so as to quickly and accurately assist the detection of active tuberculosis. Diagnose and treat tuberculosis patients, improve the cure rate of tuberculosis patients, and avoid the spread of tuberculosis.

Description

technical field [0001] The invention belongs to the technical field of medical immune detection, in particular to a combined protein and detection reagent for the detection of Mycobacterium tuberculosis. Background technique [0002] Despite 90 years of vaccinations and 60 years of chemotherapy, tuberculosis remains the world's leading cause of death from infection and has been the leading cause of death from a single infectious disease since 2007. The World Health Organization estimates that there are about 1.7 billion people with latent tuberculosis infection in the world, accounting for about 1 / 4 of the whole population. There were approximately 10.4 million new cases and 1.7 million deaths from tuberculosis in 2018. A third of these new cases, about 3 million people, are still unknown to the health system, and many are not receiving proper treatment. [0003] Almost all patients with drug-susceptible pulmonary TB can be cured with prompt diagnosis and proper treatment,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/35C12N15/31G01N33/569G01N33/543
CPCC07K14/35G01N33/5695G01N33/54346G01N2333/35
Inventor 张灵霞孙卫国李夏南熊志红朱琰黄华董恩军郝彦斐
Owner 中国人民解放军总医院第八医学中心
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