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Application of rv3121 protein in detection of Mycobacterium tuberculosis infection

A technology of Mycobacterium tuberculosis and rv3121, applied in the field of medical products, can solve the problems of unsatisfactory sensitivity and specificity of Mycobacterium tuberculosis antibody, insensitive diagnosis of latent infection of Mycobacterium tuberculosis, etc.

Active Publication Date: 2020-02-11
TB HEALTHCARE BIOTECHNOLOGY (GUANGDONG) CO LTD
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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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  • Application of rv3121 protein in detection of Mycobacterium tuberculosis infection
  • Application of rv3121 protein in detection of Mycobacterium tuberculosis infection
  • Application of rv3121 protein in detection of Mycobacterium tuberculosis infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. Rv3121-pDEST17 expression vector construction basic process

[0044] by LR Enzyme mix catalysis Rv3121 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 Rv3121.

[0045] specific method:

[0046] A. Cloning reaction system: 1.5 μL of entry vector of Rv3121, pDEST TM 17 Vehicle 1 μL, BP II Enzyme mix 2.5μL; Reaction conditions: 25℃, react overnight.

[0047] 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.

[0048] C. Plasmid extraction: The plasmid was extracted with N96 high-purity plasmid mini-extraction kit (...

Embodiment 2

[0050] Example 2. Expression and renaturation of target protein Rv3121

[0051] 1. the expression vector of the Rv3121 that embodiment 1 obtains is imported in the rosetta (DE3) expression vector, and concrete method is:

[0052] a. Take 1 μL of plasmid solution and add 100 μ rosetta (DE3) competent (self-made), ice bath for 30 minutes, heat shock at 42°C for 90 seconds,

[0053] b. Place the system on ice for 10 minutes, add 200 μL LB medium for renaturation, spread on LB solid medium containing ampicillin (100 mg / l), and incubate at 37°C for 12 hours;

[0054] c. Rv3121 protein expression was induced with 0.75mM IPTG.

[0055] 2. Ultracentrifuge the bacteria after sonication to obtain the inclusion body solid precipitation and collect the protein and renaturation steps. The specific method is as follows:

[0056] a. Resuspend bacteria with resuspension liquid: 60mM tris PH 9.0, 0.15M EDTA, and use an ultrasonic breaker to sonicate the bacteria at 4°C;

[0057] b. Centrifu...

Embodiment 3

[0060] Example 3. Immunogenicity test of target protein Rv3121

[0061] In order to detect the immunogenicity of Rv3121 and its enhancement effect on the existing antigen detection kits, further antigen detection screening was carried out on multiple clinically confirmed cases from Beijing Chest Hospital.

[0062] Test blood samples: from patients in Beijing Chest Hospital

[0063] Reagents and consumables: T-SPOT kit, Oxford immunotec (UK) product

[0064] T-SPOT experiment:

[0065] The T-SPOT kit of Oxford immunotec was used to detect the cytokine as IFN-γ.

[0066] 1) Heparin anticoagulant blood, anticoagulant blood sample is mixed with RPMI1640 according to volume 1:1; carefully add blood sample to the upper layer of Ficoll lymphocyte separation medium according to the ratio of 2-3:1;

[0067] 2) Centrifuge at 1000g for 22 minutes; horizontal rotor, slowly rising and falling;

[0068] 3) Transfer the mononuclear cell layer (located in the middle layer of the centrifug...

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Abstract

The invention relates to an application of Rv3121 protein for detecting mycobacterium tuberculosis infection. According to the invention, an antigen amino acid sequence is shown as a SEQ ID NO.2. A method for preparing the proteantigen comprises the following steps: 1) an entry vector (free provision by PFGRC of American Craig Ventor Institute ) of Gateway LR Clonase Enzyme Mix catalyzed Rv3121 and a Gateway pDEST carrier are recombined to generate an expression vector of Rv3121; 2) the expression vector of Rv3121 in the step 1) is conveyed to a target expression host for expressing the target Rv3121 protein; and 3) breaking cells and collecting a protein inclusion body, and performing renaturation on the target protein.

Description

technical field [0001] The invention belongs to the technical field of medical products, and specifically relates to an application of Rv3121 protein in 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 mo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C07K14/35C12N15/70G01N33/68G01N33/569
Inventor 毕利军张先恩朱国峰邓教宇陶生策侯剑王雅果
Owner TB HEALTHCARE BIOTECHNOLOGY (GUANGDONG) CO LTD
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