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Protein for specifically detecting Mycobacterium tuberculosis infection

A Mycobacterium tuberculosis, specific technology, applied in the field of medical products, can solve the problems of unsatisfactory sensitivity and specificity of Mycobacterium tuberculosis antibody, insensitivity of latent infection diagnosis of Mycobacterium tuberculosis, etc.

Active Publication Date: 2017-05-31
TB HEALTHCARE BIOTECHNOLOGY (GUANGDONG) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the diagnosis of tuberculosis commonly used in clinical practice mainly relies on clinical symptoms, impact diagnosis and etiology diagnosis, and is not sensitive to the diagnosis of latent infection of Mycobacterium tuberculosis.
At the same time, in the process of TB screening, the sensitivity and specificity of direct detection of pathogens or detection of Mycobacterium tuberculosis antibodies are not ideal

Method used

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  • Protein for specifically detecting Mycobacterium tuberculosis infection
  • Protein for specifically detecting Mycobacterium tuberculosis infection
  • Protein for specifically detecting Mycobacterium tuberculosis infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Rv3899c-pDEST17 expression vector construction basic process

[0041] by LR Enzyme mix catalysis Rv3899c The entry carrier (provided free of charge by PFGRC under the Craig VentorInstitute in the United States) and pDEST TM 17 vectors were recombined to generate the expression vector of Rv3899c.

[0042] specific method:

[0043] A. Cloning reaction system: 1.5 μL of entry vector of Rv3899c, pDEST TM 17 Vehicle 1 μL, BP II Enzyme mix 2.5μL; Reaction conditions: 25℃, react overnight.

[0044] B. Transformation method: Add 100 μL Escherichia coli DH5α competent (self-made) to the reaction system, ice bath for 30 minutes, heat shock at 42°C for 90 seconds, place the system on ice for 10 minutes, add 200 μL LB medium for renaturation, and spread it on ampicillin-containing On LB solid medium of penicillin (100mg / l), culture at 37°C for 20h.

[0045] C. Plasmid extraction: The plasmid was extracted with N96 high-purity plasmid mini-extraction kit...

Embodiment 2

[0048] Example 2. Expression and renaturation of target protein Rv3899c

[0049] 1. Protein expression

[0050] a) LB plates were activated by streaking (adding kan), and left at 37°C overnight (about 16 hours).

[0051] b) Inoculate the Escherichia coli host cells transformed with the Rv3899c-integrated expression vector prepared in Example 1 in 5ml liquid LB medium (5ul kan added) at 37°C and 200rpm, and cultivate until the OD is greater than 0.6-0.8 (about 3h).

[0052] c) Inoculate 5 ml of the inoculum into 300 ml of LB liquid medium, culture at 37° C. and 200 rpm until OD600 ≈ 0.6-0.8 (about 2 hours). Cool down to 16°C, add final concentration 0.04mM IPTG

[0053] d) Cultivate at 16° C. at 200 rpm for 16 hours, then centrifuge at 4000 rpm for 10 minutes at 4° C. to collect bacteria.

[0054] 2. Protein purification

[0055]a) Bacterial resuspension: Resuspend the collected bacteria with about 40ml of lysis buffer per 1L of culture medium (add 1% protease inhibitor PMS...

Embodiment 3

[0069] Example 3. Immunogenicity test of target protein Rv3899c

[0070] In order to detect the immunogenicity of Rv3899c and its enhancement effect on the existing antigen detection kits, further antigen detection screening was carried out on multiple cases of clinical diagnosis from Beijing Chest Hospital.

[0071] Test blood samples: from patients in Beijing Chest Hospital

[0072] Positive control reagents and consumables: T-SPOT kit (ESAT-6 and CFP 10 double antigen kit), Oxford immunotec (UK) product,

[0073] Negative control: Rv2327 protein antigen, another randomly selected piece of known protein fragment Rv2327 derived from Mycobacterium tuberculosis, its nucleotide sequence and amino acid sequence are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively

[0074] SEQ ID No.3

[0075]

[0076]

[0077] SEQ ID No.4

[0078]

[0079] The experimental methods and evaluation methods are as follows:

[0080] 1) Heparin anticoagulant blood, anticoagulant blood sam...

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Abstract

The present invention relates to a protein for specifically detecting Mycobacterium tuberculosis infection, wherein the protein is derived from Mycobacterium tuberculosis, has the amino acid sequence represented by SEQ ID NO.1, can be used as the Mycobacterium tuberculosis marker antigen for tuberculosis detection, can be adopted as the reference for tuberculosis patient diagnosis, and is used for diagnosing whether the patient is infected with Mycobacterium tuberculosis.

Description

technical field [0001] The invention belongs to the technical field of medical products, and in particular relates to a protein for specifically detecting Mycobacterium tuberculosis infection. Background technique [0002] my country's in vitro diagnostic industry is now in a period of rapid growth. However, there is an obvious gap between the huge market demand and the relatively backward independent research and development capabilities of my country's diagnostic reagent industry. How to carry out independent innovation from the source, how to not be restricted by individual overseas pharmaceutical giants in the supply of raw materials for diagnostic reagents, and how to transform existing scientific research achievements in the field of biomedicine into products have become the key issues for the Chinese government, scientific research institutes and the pharmaceutical industry in recent years. hot spots of great concern. Moreover, there is a rigid demand for more sensi...

Claims

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Application Information

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IPC IPC(8): C07K14/35C12N15/31G01N33/569A61K39/04A61P31/06C12R1/32
CPCA61K39/00C07K14/35G01N33/5695G01N2469/20
Inventor 邓教宇毕利军张先恩朱国峰陶生策王雅果侯剑
Owner TB HEALTHCARE BIOTECHNOLOGY (GUANGDONG) CO LTD
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