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IgM specific protein Rv2915c of mycobacterium tuberculosis and application of IgM specific protein Rv2915c

A technology of Mycobacterium tuberculosis and rv2915c, applied in the medical field, can solve the problems of insufficient specificity and low accuracy.

Inactive Publication Date: 2013-09-04
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The tuberculosis epidemic has aroused widespread concern and attention worldwide, but the only vaccine currently used in tuberculosis prevention, BCG, is only effective in preventing tuberculosis in children, but its protective effect on tuberculosis in adults is uncertain. In the early stage of tuberculosis There are defects such as low accuracy rate and insufficient specificity in serological diagnosis, which cannot well meet the clinical needs

Method used

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  • IgM specific protein Rv2915c of mycobacterium tuberculosis and application of IgM specific protein Rv2915c
  • IgM specific protein Rv2915c of mycobacterium tuberculosis and application of IgM specific protein Rv2915c
  • IgM specific protein Rv2915c of mycobacterium tuberculosis and application of IgM specific protein Rv2915c

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Biopanning of phage

[0026] Coat goat anti-human IgM (purchased from Beijing Boaosen Technology Co., Ltd.) (100μg / mL) on 2 wells of a 96-well plate, labeled as "screening well" and "reduction well", each well is 100μL, 4 ℃ overnight; blocked with 1% bovine serum albumin (BSA), washed 4 times with TBST (pH 7.6 tris-hydrochloric acid buffer (Tris-HCl 50mM, NaCl 150mM) with 0.05% Tween-20) ; One hole (screening hole) was added with serum from recovered tuberculosis patients (diluted 1:20), and the other hole (with subtraction hole) was added with healthy human serum (100μg / mL), overnight at 4°C; TBST (0.1% Tween-20) was used After washing 6 times, the heptapeptide library stock solution was diluted 1:20 and added to the subtraction well, gently shaken at room temperature for 30 minutes; transferred to the screening well, gently shaken at room temperature for 30 minutes; discarded unbound phage, TBST (0.1% Tween -20) Wash 10 times, add non-specific buffer 0.2M Glyc...

Embodiment 2

[0027] Example 2 Phage DNA extraction and sequencing

[0028] Take 500μL of the supernatant of the picked phage clone amplification product (positive clones after screening in Example 1), add 200μL of PEG / NaCl (PEG8000-polyethylene glycol 8000), mix upside down, place at room temperature for 10 minutes, and centrifuge for 10 minutes. Discard the supernatant. The pellet was thoroughly resuspended in 100 μL of iodide buffer and 250 μL of ethanol was added. Incubate at room temperature for 10 min. Centrifuge for 10 min and discard the supernatant. Wash the precipitate with 70% ethanol and vacuum dry briefly. The pellet was resuspended in 30 mL TE and sent to BGI for sequencing. The sequenced 7 peptide sequences were searched and aligned (BLAST) in NCBI (National Center for Biological Information) with Mycobacterium tuberculosis H37Rv (Mycobacterium tuberculosis H37Rv) protein. As a result, it was found that 73.3% of the sequence of the positive clones tested had the greatest de...

Embodiment 3

[0029] Example 3 Rv2915c protein cloning, expression and purification

[0030] Design primers based on the sequencing results. Use primers for Rv2915c sequence (upstream primer: 5’-TAG GAATTC GTGAAGCGGGTCG-3’, underlined is the restriction site EcoRI; Downstream primer: 5’-ACT AAGCTT GCCAAAGCTAGGGCC-3’, underlined is the restriction site Hind III) PCR amplified from the genomic DNA of Mycobacterium.tuberculosis H37Rv (please give the full name of the Latin name and the Chinese name of Mycobacterium tuberculosis H37Rv strain). The PCR products Rv2915c and pET28a(+) were digested with EcoR I and Hind III respectively, and after ligation using a ligation system, they were transformed into DH5α competent (CaCl 2 Method). The transformed product was spread on a solid medium containing 50 μg / ml kanamycin, and placed in a constant temperature biochemical incubator at 37°C overnight. After spotting culture, PCR verification of bacterial solution is carried out. After extracting recom...

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Abstract

The invention belongs to the technical field of medicine, and particularly relates to IgM (immunoglobulin M) specific protein Rv2915c of mycobacterium tuberculosis and an application of the IgM specific protein Rv2915c. The IgM specific protein Rv2915c and the application aim at providing a new choice for detecting tuberculosis. The protein Rv2915c adopts the technical scheme that an amino acid sequence of the protein Rv2915c of the mycobacterium tuberculosis is shown as SEQ ID No. 1 (sequence identifier number 1). The invention further provides the application of the protein Rv2915c of the mycobacterium tuberculosis in tuberculosis diagnosis. The invention further provides a diagnostic reagent for the tuberculosis. A main active ingredient of the diagnostic reagent is the protein Rv2915c of the mycobacterium tuberculosis. The invention further provides a diagnostic kit for the tuberculosis, which mainly comprises the protein Rv2915c of the mycobacterium tuberculosis. The IgM specific protein Rv2915c of the mycobacterium tuberculosis is applicable to the diagnosis of the tuberculosis.

Description

Technical field [0001] The invention belongs to the field of medical technology, and specifically relates to the Mycobacterium tuberculosis IgM specific protein Rv2915c and its use. Background technique [0002] Tuberculosis caused by Mycobacterium tuberculosis (MTB) infection is the chronic infectious disease with the highest mortality rate in the world. It is also the second most harmful infectious disease in the world after AIDS. It has now developed into a global epidemic. There are 9 million new cases worldwide every year, causing 2 million deaths. The co-infection of HIV and tuberculosis has become the first cause of death in AIDS patients. The emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) has reduced the cure rate of tuberculosis. -TB) The cure rate is low, usually ranging from about 50% to 70%. The tuberculosis epidemic has attracted widespread attention and attention worldwide, but the only vaccine BCG curre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/35C12N15/32G01N33/68
Inventor 谢建平赵宇中
Owner SOUTHWEST UNIVERSITY
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