Application of c5orf24 protein in tuberculosis diagnostic reagent

Abstract

Provided is an application of a C5orf24 recombinant protein in tuberculosis diagnosis, belonging to the field of tuberculosis diagnosis. On the basis that the C5orf24 protein can simultaneously capture various tuberculosis antigens in a tuberculosis patient specimen, the recombinant C5orf24 protein can be expressed and purified, so as to detect and capture binding antigens. The prepared C5orf24 recombinant protein can specifically and efficiently capture various tuberculosis antigens.

Description

Application of C5orf24 protein in tuberculosis diagnostic reagents Technical Field

[0001] The present invention belongs to the field of tuberculosis diagnosis, and specifically relates to an application of C5orf24 protein in a tuberculosis diagnostic reagent. Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease caused by the intracellular bacterial pathogen Mycobacterium tuberculosis. Due to its unique pathogenicity, prevalence, and transmission patterns, particularly the recent emergence of drug-resistant strains and cross-infection with the human immunodeficiency virus (HIV), TB is on the rise again. M. tuberculosis is an intracellular pathogen and one of the most successful pathogens, having evolved numerous strategies to adapt to host cells and evade immune surveillance. After entering the human body through the respiratory tract, M. tuberculosis primarily colonizes macrophages, employing a variety of strategies to evade host immune responses, allowing it to survive and slowly proliferate within these cells for extended periods. When host immunity is compromised, dormant M. tuberculosis can reactivate, leading to the development of active TB. Following infection with M. tuberculosis, the pathogen can remain dormant in the host for extended periods or cause persistent infection, creating significant challenges for the treatment and prevention of the disease. Furthermore, the current challenges in TB treatment include the emergence of multidrug-resistant and extensively drug-resistant TB. The lack of new tuberculosis drugs and insufficient basic research on Mycobacterium tuberculosis are the main reasons for the low cure rate of drug-resistant tuberculosis. Therefore, there is an urgent need to find new drug targets and develop new anti-tuberculosis drugs to treat tuberculosis.

[0003] According to the World Health Organization's 2022 Global Tuberculosis Report, approximately 10 million new cases of tuberculosis (TB) are diagnosed globally each year, and approximately 1.4 million people die from TB each year. Currently, TB diagnosis faces numerous technical challenges, primarily the following: 1) TB bacteria grow slowly, requiring a long time to culture. 2) Detection techniques are insensitive and unreliable. 3) Detection costs are high and susceptible to environmental influences. 4) Diagnosis is complex, making timely and accurate diagnosis difficult. TB antigen detection methods have been developed over many years and encompass a variety of approaches, including immunochromatographic strips, enzyme-linked immunosorbent assay (ELISA), chemiluminescence, and fluorescent immunoassay (FIA). These antigen detection methods, all based on the detection of TB antigens in patient fluids or tissues, offer rapid, accurate, and sensitive characteristics. However, these methods still have significant limitations. The sensitivity and specificity of some test kits still exhibit significant errors, while others are expensive, require imported equipment and technical training, and have limited clinical applicability. Therefore, tuberculosis antigen detection technology needs to be further improved and developed, and it is urgent to develop a tuberculosis antigen detection method with high sensitivity, good specificity, ease of use and low price.

[0004] C5orf24, a gene located on human chromosome 5q31.1, encodes a protein consisting of approximately 188 amino acids. C5orf24 is a cellular transcriptional regulator that plays diverse roles in different cell types, including processes such as cell proliferation, cell polarization, migration, and tumor development. C5orf24 plays a variety of important roles in various physiological and pathological conditions. C5orf24's ability to specifically and efficiently capture multiple tuberculosis antigens has led to the development of ligand protein capture assays for detecting tuberculosis antigens. Therefore, modulating C5orf24 activity could have significant impacts on the body and may play a crucial role in the prevention or treatment of certain diseases. One important pathway for regulating C5orf24 activity is through proteins that directly interact with C5orf24. Therefore, proteins that directly interact with C5orf24 could have significant pharmaceutical potential and could be developed as protein or peptide drugs for the prevention or treatment of certain diseases.

[0005] Summary of the Invention

[0006] The present invention utilizes the ability of C5orf24 protein to simultaneously capture multiple tuberculosis antigens in tuberculosis patient specimens, and detects the ability to capture tuberculosis antigens by recombining, expressing and purifying the C5orf24 protein.

[0007] In a first aspect, the present invention provides a C5orf24 recombinant protein having the ability to capture Mycobacterium tuberculosis antigens. The amino acid sequence of the C5orf24 recombinant protein is shown in SEQ ID NO. 1.

[0008] SEQ ID NO.1:

[0009] In a second aspect, the present invention provides a method for preparing a C5orf24 recombinant protein, the method comprising the following steps:

[0010] S1. Obtain the nucleotide sequence of C5orf24 protein, the nucleotide sequence is shown in SEQ ID NO.2,

[0011] S2. Load the nucleotide sequence obtained in step S1 into a specific vector to construct a recombinant plasmid.

[0012] S3. The recombinant plasmid constructed in step S2 is transfected into the strain for expression to obtain the recombinant protein of the present invention;

[0013] The C5orf24 recombinant protein of the present invention has the function of capturing tuberculosis antigens.

[0014] SEQ ID NO.2:

[0015] Furthermore, the vector includes but is not limited to one or more of pET-28a, pET-30a, pET-22b, pET-24a, and pET-32c, preferably pET-28a.

[0016] Furthermore, the strain is selected from E. coli BL21 (DE3).

[0017] In a third aspect, the present invention provides a kit for detecting Mycobacterium tuberculosis, comprising a reagent for detecting Mycobacterium tuberculosis and instructions, wherein the reagent further comprises a C5orf24 recombinant protein, and the C5orf24 recombinant protein has the ability to capture Mycobacterium tuberculosis antigens.

[0018] In a fourth aspect, the present invention provides a use of the C5orf24 recombinant protein as described in the first aspect in preparing a kit for detecting tuberculosis antigens, wherein the C5orf24 recombinant protein has the ability to capture tuberculosis antigens.

[0019] Furthermore, the tuberculosis antigen is selected from one or more of Rv0277c, Rv1838c, Rv1982c, Rv2098c, Rv3423c, and Rv3759c. Beneficial effects:

[0020] The C5orf24 recombinant protein prepared by the present invention has the characteristics of being able to capture multiple tuberculosis antigens specifically and efficiently, and can be used to detect tuberculosis antigens by establishing a ligand protein capture method. DETAILED DESCRIPTION

[0021] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below may be combined with each other as long as they do not conflict with each other.

[0022] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0023] Capture: When proteins interact directly, they can be firmly bound together, which can also be described as one protein capturing another. When such proteins interact, they are mainly bound together by the following non-covalent forces: (1) Electrostatic interaction: The interaction between charged amino acid residues. (2) Van der Waals force: The short-range attraction between non-polar amino acid residues. (3) Hydrophobic interaction: Non-polar residues aggregate in water to form a hydrophobic core, forming hydrophilic and hydrophobic interactions. (4) Hydrogen bond: The hydrogen bond interaction between polar residues.

[0024] Recombinant protein: The recombinant protein is the subject to be protected - the ligand protein. If it is to be used in practice, a corresponding ligand protein is necessary. There is no ready-made ligand protein in nature, so it can only be prepared (by recombinant expression to obtain the relevant ligand protein), which is an important part of practical application.

[0025] Tuberculosis antigen: It can be used as direct evidence of the etiology of tuberculosis and for the diagnosis and confirmation of tuberculosis. At the same time, after the patient's body is infected with Mycobacterium tuberculosis, tuberculosis antigen will first appear in the body and exist in the patient's body as the disease progresses. Tuberculosis antigen detection is not affected by the host's immune function status and can be used as direct evidence of the existence of Mycobacterium tuberculosis for diagnosis and confirmation, avoiding false negatives in humoral immunity detection or cellular immunity detection due to weak antigenicity of Mycobacterium tuberculosis or low immune response of tuberculosis patients. The amount of tuberculosis antigen in the patient's body can also directly reflect the extent of his disease.

[0026] Example 1: Synthesis of C5orf24 protein encoding gene and construction of recombinant plasmid

[0027] The nucleotide sequence of C5orf24 protein was obtained and loaded into the vector pET-28a. Sequencing analysis confirmed that the pET-28a-C5orf24 protein expression plasmid was successfully constructed. The nucleotide sequence is shown in SEQ ID NO. 2.

[0028] SEQ ID NO.2:

[0029] Example 2: Expression and purification of C5orf24 recombinant protein

[0030] The successfully constructed recombinant plasmid pET-28a-C5orf24 was transfected into E. coli BL21(DE3) to express the recombinant protein. Under IPTG induction, the recombinant plasmid successfully expressed the C5orf24 recombinant protein. After affinity purification using a Ni-NTA-His column, a highly pure protein with a relative molecular weight of approximately 25 kDa was obtained, meeting the expected size. The amino acid sequence of the C5orf24 recombinant protein is shown in SEQ ID NO. 1.

[0031] SEQ ID NO.1:

[0032] The BCA assay determined the C5orf24 concentration to be 1.506 mg / mL. The details are as follows:

[0033] Expression and purification of C5orf24 recombinant protein

[0034] 1) Inoculate E. coli BL21 (DE3) strain containing the pET-28a-C5orf24 expression plasmid into 5 ml of LB liquid medium containing the corresponding resistance and culture at 37°C and 200 rpm.

[0035] 2) The next day, the strain was inoculated into 500 mL of LB liquid medium (kanamycin concentration: 50 μg / mL) at a ratio of 1:100 and cultured at 37°C to an OD of 0.4-0.6. 0.8 mM IPTG was added and expression was induced at 25°C for 12 h. The cells were centrifuged at 12,000 rpm and 4°C for 15 min, and the cells were collected.

[0036] 3) Centrifuge the induced bacterial solution at 6000 rpm for 10 min, discard the supernatant, and resuspend the pellet in 200 ml of 500 mM NaCl 20 mM PBS pH 8.0 buffer.

[0037] 4) The resuspended bacteria were disrupted by ultrasonication, and then centrifuged at 12000 rpm for 10 min. The supernatant was collected for later use.

[0038] 5) Equilibrate the treated Ni column (GE) with 5 times the volume of Binding buffer (500mM NaCl 20mM PBS pH 8.0) at a flow rate of 1-1.5ml / min and adjust the baseline of the UV detector.

[0039] 6) Load the treated sample at a flow rate of 1 ml / min. Collect the flow-through. After loading, use Binding buffer (500 mM NaCl, 20 mM PBS, pH 8.0) to wash the UV detector absorbance back to baseline.

[0040] 7) Elute with washing buffer (500 mM NaCl, 20 mM PBS pH 8.0, 20 mM imidazole) and collect the elution peak.

[0041] 8) Elute with Elution buffer I (500 mM NaCl 20 mM PBS pH 8.0 60 mM imidazole) and collect the elution peak.

[0042] 9) Elute with Elution buffer II (500 mM NaCl 20 mM PBS pH 8.0 300 mM imidazole) and collect the elution peak.

[0043] 10) The imidazole eluted fraction was dialyzed against 20 mM PBS pH 8.0 buffer to obtain the purified recombinant protein C5orf24, and its protein concentration was determined using BCA.

[0044] Example 3 Identification of Tuberculosis Antigens Captured by C5orf24 Recombinant Protein by Mass Spectrometry

[0045] Leveraging the ability of the C5orf24 recombinant protein to specifically and efficiently capture multiple TB antigens, a recombinant protein capture assay was developed to detect TB antigens in patient samples. The C5orf24 recombinant protein specifically and efficiently captured multiple TB antigens, which were then identified using biomass spectrometry.

[0046] The specific method for identifying tuberculosis antigens captured by C5orf24 recombinant protein is as follows:

[0047] (1) Coating of C5orf24 recombinant protein: Dilute the C5orf24 recombinant protein to 4 μg / ml using coating solution (0.05 mol / L carbonate buffer, pH 9.6). Add 100 μl of the diluted coating solution to each well of the ELISA plate. Place the plate in a humidified chamber and coat overnight at 4°C.

[0048] (2) Wash the plate to remove the liquid in the wells of the enzyme-labeled plate, then fill all the wells with washing buffer (0.15 mol / L pH 7.4 PBS containing 0.05% Tween-20), remove the washing solution, wrap it with absorbent paper, and dry it. Wash the plate twice.

[0049] (3) Blocking: Add 250 μl of blocking solution (pH 9.6, 0.05 mol / L carbonate buffer containing 2.0% BSA) to each well of the ELISA plate, place the ELISA plate in a humidified chamber, and incubate at 37°C for 2 h.

[0050] (4) Wash the plate to remove the blocking solution and wash twice using the same procedure as above.

[0051] (5) Add TB patient samples to the sample wells on the ELISA plate. First, add 70 μl of sample diluent, then add 30 μl of the sample to be tested. Add the sample to the bottom of the wells, avoiding contact with the well walls. Incubate at 37°C for 1.5 h.

[0052] (6) Wash the plate to remove the sample solution from the wells of the ELISA plate and wash three times using the same procedure as above.

[0053] (7) Preparation of samples for mass spectrometry identification: An appropriate amount of mass spectrometry grade trypsin solution was added to the wells of the ELISA plate and digested overnight at 37°C. After overnight, FA (formic acid) was added to a final concentration of 0.1% to terminate the digestion.

[0054] (8) 10 μl of sample was loaded onto a liquid chromatography-tandem mass spectrometry (LC-MS / MS, BGI Protein Research Center Co., Ltd.) machine for detection. The mass spectrometry data obtained from the detection were searched and analyzed using the MASCOT database (http: / / www.matrixscience.com / ). The tuberculosis antigens identified were: Rv0277c, Rv1838c, Rv1982c, Rv2098c, Rv3423c, and Rv3759c.

[0055] Example 4 Method for detecting Mycobacterium tuberculosis antigens

[0056] Taking advantage of the ability of C5orf24 recombinant protein to specifically and efficiently capture a variety of tuberculosis antigens, a ligand protein capture method was created to detect tuberculosis antigens. That is, the C5orf24 recombinant protein is used to coat the enzyme-labeled plate, which is blocked and then eluted; then the tuberculosis patient sample is added, incubated and eluted. At this time, the C5orf24 recombinant protein captures the specific tuberculosis antigen in the sample; tuberculosis antibodies (HRP enzyme labeled) are then added, incubated and eluted; then HRP enzyme substrate is added for color development. If the color development result is positive, it indicates tuberculosis infection, otherwise it is tuberculosis negative. This method is a method for detecting Mycobacterium tuberculosis infection, but it is not limited to this detection method. Other detection methods such as fluorescent test strip detection method or colloidal gold test strip detection method can also be used. The detection method of tuberculosis antigens in specific samples is as follows:

[0057] 1. Detection reagents for Mycobacterium tuberculosis antigens:

[0058] Antigen coating solution (0.05 mol / L carbonate buffer, pH 9.6), washing buffer (0.15 mol / L PBS, pH 7.4, containing 0.05% Tween-20), blocking solution (0.05 mol / L carbonate buffer, pH 9.6, containing 2% BSA), diluent (washing buffer, containing 0.1% BSA), 1:1000 diluted HRP-labeled rabbit anti-tuberculosis IgG antibody (catalog number ab21189, purchased from Shanghai Union Biotechnology Co., Ltd.), positive serum, negative serum, TMB-H2O2 substrate development solution [sodium hydrogen phosphate-citrate buffer (pH 5.5) containing TMB (tetramethylbenzidine)], and 2 mol / L sulfuric acid stop solution.

[0059] 2. Detection methods for Mycobacterium tuberculosis antigens

[0060] (1) Antigen Coating: Dilute the C5orf24 recombinant protein to 4 μg / ml using coating solution. Add 100 μl of the diluted antigen solution to each well of the ELISA plate. Place the ELISA plate in a humidified chamber and coat overnight at 4°C.

[0061] (2) Wash the plate to remove the liquid in the wells of the enzyme-labeled plate, then fill all the wells with washing solution, remove the washing tower liquid, wrap it with absorbent paper and dry it, and wash the plate twice.

[0062] (3) Blocking: Add 250 μl of blocking solution (pH 9.6, 0.05 mol / L carbonate buffer containing 2.0% BSA) to each well of the enzyme-linked plate, place the plate in a humidified chamber, and incubate at 37°C for 2 h.

[0063] (4) Wash the plate to remove the blocking solution and wash twice using the same procedure as above.

[0064] (5) Add sample and incubate. First, add 90 μl of sample diluent to the sample wells on the ELISA plate, then add 10 μl of the sample to be tested (the final sample dilution is 10-fold). When adding the sample, add it to the bottom of the ELISA plate wells, avoiding touching the well walls as much as possible. Incubate at 37°C for 1.5 h.

[0065] (6) Wash the plate to remove the sample solution from the wells of the ELISA plate and wash three times using the same procedure as above.

[0066] (7) Incubation with enzyme-labeled antibody: Dilute the enzyme-labeled antibody with antibody diluent (1:1000 dilution). Add 100 μl of the diluted enzyme-labeled antibody to each well, excluding the blank well. Incubate in a 37°C wet chamber for 1 h.

[0067] (8) Wash the plate to remove the enzyme-labeled antibody solution and wash three times using the same procedure as above.

[0068] (9) Color development: Add 100 μl of color development solution to each well, gently shake to mix, and develop the color at room temperature in the dark for 10 to 20 minutes.

[0069] (10) After color development is complete, add 50 μl of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0070] (11) Rapidly measure the absorbance (OD value) of each well of the microplate using a microplate reader at a wavelength of 450 nm (Note: Use the blank well as background for background subtraction and zero adjustment). The measurement should be performed within 20 minutes after adding the stop solution.

[0071] Example 5: Detection of Mycobacterium tuberculosis infection

[0072] The detection reagent for Mycobacterium tuberculosis prepared by the above method was used to test sera from 110 active tuberculosis patients and 37 healthy subjects according to the above detection method; 102 samples from active tuberculosis patients were tested by Mycobacterium tuberculosis nucleic acid (PCR) detection as a comparison. The test results are shown in Table 1.

[0073] Table 1 Positive rates of tuberculosis patients detected by different detection methods

[0074] As can be seen from the results in Table 1, the sensitivity of the detection method for Mycobacterium tuberculosis antigen in the serum of tuberculosis patients of the present invention is 74.55%, which is significantly higher than the 54.9% of the Mycobacterium tuberculosis nucleic acid (PCR) detection method. The specificity of this detection method is 94.6% (1-5.4%=94.6%).

Claims

1. A kit for detecting Mycobacterium tuberculosis, the kit comprising a reagent for detecting Mycobacterium tuberculosis and instructions, the reagent further comprising a C5orf24 recombinant protein, the C5orf24 recombinant protein having the ability to capture Mycobacterium tuberculosis antigens, the amino acid sequence of the C5orf24 recombinant protein being as shown in SEQ ID NO.1, SEQ ID NO.1: MMHPVASSNPAFCGPGKPSCLNEDAMRAADQFDIYSSQQSKYSHTVNHKPMVCQ RQDPLNETHLQTTSGRSIEIKDELKKKKNLNRSGKRGRPSGTTKSAGYRTSTGRPLGTTKAAGFKTSPGRPLGTTKAAGYKVSPGRPPGSIKALSRLADLGYGCGTAAFPYPMMHGRAVHGVEETSSEVKPPNE.

2. Use of a C5orf24 recombinant protein in preparing a kit for detecting tuberculosis antigens, wherein the C5orf24 recombinant protein has the ability to capture tuberculosis antigens, and the tuberculosis antigens are selected from one or more of Rv0277c, Rv1838c, Rv1982c, Rv2098c, Rv3423c, and Rv3759c; the amino acid sequence of the C5orf24 recombinant protein is shown in SEQ ID NO.1, SEQ ID NO.1: MMHPVASSNPAFCGPGKPSCLNEDAMRAADQFDIYSSQQSKYSHTVNHKPMVCQ RQDPLNETHLQTTSGRSIEIKDELKKKKNLNRSGKRGRPSGTTKSAGYRTSTGRPLGTTKAAGFKTSPGRPLGTTKAAGYKVSPGRPPGSIKALSRLADLGYGCGTAAFPYPMMHGRAVHGVEETSSEVKPPNE.

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