Mycobacterium tuberculosis ESAT6 antigen protein serial recombinant expression method and application in tuberculosis detection thereof

A Mycobacterium tuberculosis fusion protein technology, which is applied in the field of tandem recombinant expression of Mycobacterium tuberculosis ESAT6 antigen protein and its application in tuberculosis detection, can solve the problems of poor specificity and low sensitivity

Inactive Publication Date: 2013-06-12
郑州博赛生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the current methods focus on the expression of a single antigen, the expression of a single epitope, and the combined expression of multiple antigens to obtain tuberculosis-specific antigens. There are few studies on the tandem expression of a single antigen, and most tuberculosis-specific antigens are directly used in serum The traditional method for detecting tuberculosis infection has low sensitivity and poor specificity

Method used

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  • Mycobacterium tuberculosis ESAT6 antigen protein serial recombinant expression method and application in tuberculosis detection thereof
  • Mycobacterium tuberculosis ESAT6 antigen protein serial recombinant expression method and application in tuberculosis detection thereof
  • Mycobacterium tuberculosis ESAT6 antigen protein serial recombinant expression method and application in tuberculosis detection thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Preparation of tandem recombinant fusion protein of Mycobacterium tuberculosis ESAT6

[0027] Analysis of the nucleotide sequence encoding the tuberculosis-specific antigen ESAT6, and binding E. coli According to the codon preference of ESAT6, synonymous codons were optimized for the nucleotides of ESAT6, and designed restriction enzyme sites were added at both ends of its coding sequence, so as to realize the tandem expression of the sequence.

[0028] The optimized nucleotide sequence is as follows:

[0029] catatgggaattcccatggaggatccgatgacagagcagcagtggaatttcgcgggtatcgaggccgctgcaagcgcaatccagggtaatgtcacgtccattcattccctccttgacgaggggaagcagtccctgaccaagctcgcagcggcctggggcggtagcggttcggaggcgtaccagggtgtccagcaaaaatgggacgccacggctaccgagctgaacaacgcgttgcagaacctggcgcgtacgatcagcgaagccggtcaggcaatggcttcgaccgaaggcaacgtcactgggatgttcgcagcagatctggctaagcttcccgggtcgactcgagcggccgc

[0030] Add NcoI and BamHI upstream of the sequence; HindIII, BglII and SalI and other restriction ...

Embodiment 2

[0099] Example 2 Human tuberculosis detection kit based on tandem recombinant fusion protein of Mycobacterium tuberculosis ESAT6 (chemiluminescence method)

[0100] 【Packing specification】

[0101] 9 servings / box (48T) 21 servings / box (96T)

[0102] 【expected usage】

[0103] This kit uses the chemiluminescent double-antibody sandwich method to quantitatively determine the content of human interferon-γ (Interferon-γ, IFN-γ) secreted by T lymphocytes in human blood samples after being stimulated by Mycobacterium tuberculosis specific antigen polypeptide. Mycobacterium tuberculosis infection mainly causes a cell-mediated immune response. As part of the immune response, T lymphocytes are stimulated by Mycobacterium tuberculosis-specific antigen polypeptides to become activated effector T lymphocytes and secrete the cytokine IFN-γ. By detecting the content of IFN-γ in the sample, it can be inferred whether there are effector T lymphocytes in the body that respond to Mycobacter...

Embodiment 3

[0150] Example 3 Human tuberculosis TCELL-SPOT detection kit based on tandem recombinant fusion protein of Mycobacterium tuberculosis ESAT6

[0151] Experimental program:

[0152] Coating:

[0153] 1. Wet the bottom of the 96-well membrane plate with 15ul / well of 35% ethanol for 30 seconds;

[0154] 2. Wash the plate 5 times with 200ul sterile deionized water, 1min / time;

[0155] 3. Slightly control the residual moisture in the well, add 100ul IFN-γ monoclonal capture antibody (diluted to 15ug / ml with PBS) to each well, and freeze at 4°C overnight;

[0156] 4. Discard the coating solution, wash the plate twice with PBS, 1min each time, and pat dry;

[0157] 5. Add 200ul of PBS containing 2% casein and 1% trehalose to each well to block, and incubate at room temperature for 2 hours;

[0158] 6. Discard the blocking solution, rinse once with 1640 culture medium, and set aside.

[0159] Separation and counting of lymphocytes:

[0160] 1. Specimen collection: Take 4-5ml of...

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Abstract

The invention discloses mycobacterium tuberculosis ESAT6 serial recombinant fusion protein and application thereof. The invention provides a mycobacterium tuberculosis ESAT6 codon optimization method, a serial recombinant fusion method and a method for preparing the fusion protein, and also provides a method for manufacturing and applying the recombinant fusion protein in a whole-blood IFN-gamma tuberculosis diagnosis kit and a TCELL-SPOT tuberculosis infection diagnosis kit. The fusion protein has the advantages of high specificity, high sensitivity and the like, and the requirements of the tuberculosis (TB) infection clinical diagnosis are well met.

Description

technical field [0001] The present invention relates to the field of genetic engineering and medical immunology, and to a rapid and early specific detection method for tuberculosis patients and tuberculosis-infected patients, in particular to a Mycobacterium tuberculosis ESAT6 tandem recombination fusion protein and its application, especially a A tuberculosis antigen ESAT6 tandem recombinant fusion protein-specific whole blood IFN-γ tuberculosis diagnostic kit and a TCELL-SPOT tuberculosis infection diagnostic kit are produced and applied. Background technique [0002] Tuberculosis is caused by Mycobacterium tuberculosis ( Mycobacterium tuberculosis , MTB) is a human disease caused by infection, which is mainly transmitted through the respiratory tract, and the epidemic situation is becoming more and more serious. The prevention, early diagnosis and timely treatment of tuberculosis are of great significance. MTB standard strain H37Rv is widely used in TB-related biomedica...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/35C12N15/70C12N1/21G01N33/68C07K16/12C12R1/32
Inventor 杨艳坤白仲虎刘晓磊李昕朱国珍林兴兵曹成付建军
Owner 郑州博赛生物技术股份有限公司
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