CN106198971A - The application of antigen of mycobacterium tuberculosis albumen Rv2351c - Google Patents

Abstract

The invention provides the application of mycobacterium tuberculosis antigenic protein Rv2351c in the preparation of tuberculosis detection reagents, tuberculosis vaccines and anti-tuberculosis drugs. The invention provides a new tuberculosis detection antigen protein Rv2351c, which can reduce false negatives caused by single antigen-specific T cells and improve detection sensitivity compared with previous detection methods using a single antigen. The antigenic protein Rv2351c of the present invention is a factor related to tuberculosis virulence, and the high intensity of cellular immunity and humoral immunity shows that the antigen has strong immunogenicity, and can be used as a candidate antigen for a new type of tuberculosis vaccine.

Description

Application of Antigen Protein Rv2351c of Mycobacterium Tuberculosis

technical field

The invention relates to the fields of molecular biology and immunology, in particular to the application of mycobacterium tuberculosis antigenic protein Rv2351c in the preparation of tuberculosis detection reagents, tuberculosis vaccines and anti-tuberculosis drugs.

Background technique

Tuberculosis is a zoonotic infectious disease caused by Mycobacterium tuberculosis. According to the statistics of the World Health Organization, about 1/3 of the world's population is currently infected with Mycobacterium tuberculosis, and about 10% of them may develop active pulmonary tuberculosis. my country's tuberculosis infection rate is 44.5%, which is one of the 22 countries with severe tuberculosis epidemic in the world, and the total number of tuberculosis patients ranks second, second only to India. The 2014 WHO report showed that there were 9 million new cases and 1.5 million deaths from tuberculosis in 2013, of which 360,000 were HIV-positive patients, 480,000 were multi-drug-resistant tuberculosis patients (MDR-TB), Mycobacterium tuberculosis combined with human immunodeficiency Viral infection and the emergence of multidrug-resistant strains make tuberculosis a public health problem that seriously endangers human health.

At present, tuberculosis is still the most harmful disease among infectious diseases in my country. High specificity and high sensitivity early detection technology and reagents for tuberculosis can effectively cut off the transmission route and reduce the incidence of tuberculosis for early diagnosis of tuberculosis, timely isolation and treatment rates and mortality are critical. Diagnosis of tuberculosis generally relies on laboratory diagnosis, imaging examination and clinical diagnosis, etc. In bacteriological diagnosis, sputum smear staining and microscopy are simple and easy, but the concentration of the sample is relatively high, usually with a bacterial content of 5000- Only 10000/ml can be detected positive, so the positive rate is low, and it is easy to miss the test. Whether the sample is qualified or not directly determines whether the detection rate of sputum bacteria is positive or not. Sputum culture is the gold standard for tuberculosis diagnosis, but the cycle is long and the success rate of culture is only 80%, which is not conducive to rapid detection. The most commonly used clinically is the skin tuberculin test, but the skin test is interfered by Bacillus Calmette-Guerin (BCG) vaccination and environmental mycobacterial infection, resulting in false positives, making it less sensitive and requiring patients to seek medical attention again. Imaging examinations for tuberculosis, such as routine X-ray examinations, CT examinations, MRI examinations, and ultrasonography, are expensive, traumatic to the body, and have low specificity, making them unsuitable for routine examinations and diagnoses.

After Mycobacterium tuberculosis invades the human body, it resides in macrophages. The main immune response of the human body to Mycobacterium tuberculosis is cellular immunity, and the main cells involved are CD4+ and CD8+ T cells. T lymphocytes sensitized by pathogens will release gamma interferon after exposure to the same antigen again, and a high level of gamma interferon response can indicate the infection of the pathogen. The T cell-based ELISPOT detection method uses IFN-γ-specific antibodies to capture the IFN-γ produced by peripheral blood lymphocytes stimulated by tuberculosis antigens after culture, and presents them in the form of enzyme-linked spot color development, from the number of spots To determine the secretion of cytokines by cells, and evaluate cellular immune function from the single cell level. In vitro gamma interferon detection based on T cells is used in the auxiliary diagnosis of tuberculosis. This detection method can not only screen out active tuberculosis patients, but also detect patients in the latent period, so as to better prevent and control the latent period Tuberculosis, currently there are commercial IGRA kits that use the whole protein or polypeptide of the tuberculosis-specific antigens ESAT-6 and CFP-10 encoded by the RD1 region of the Mycobacterium tuberculosis genome as stimulators, such as QuantiFERON-TB Gold test and T- SPOT showed high sensitivity and specificity. Rv2351c (GI: 15609488) is a Mycobacterium tuberculosis-specific antigen encoded by the differential region RD7 of the Mycobacterium tuberculosis genome, also known as the membrane phosphatase plcA. This antigen protein contains 512 amino acids and is a possible related virulence factor. The protein was detected in all BCG (BCG-Danish, BCG-Prague, BCG-Glaxo, BCG-Frappier, BCG-Connauht, BCG-Phipps, BCG-Tice, BCG-Pateur, BCG-Moreau, BCG-Brikhaug, BCG-Sweden , BCG-Japan, BCG-Russian, BCG-Brazil) are all missing, and the antigen epitope prediction analysis of it is further carried out by bioinformatics software, and it is found that Rv2351c protein has more T cell epitopes, which has potential diagnostic efficiency. And Rv2351c only exists in Mycobacterium tuberculosis and a few environmental Mycobacteria, it can effectively distinguish tuberculosis patients, lung disease patients and healthy people, and is not affected by BCG vaccination.

The rapid and early diagnosis of tuberculosis provides a strong guarantee for the early treatment of tuberculosis, but if we want to curb tuberculosis at the source, we should start with the preparation of tuberculosis vaccine. At present, BCG vaccine is the most widely used and the only tuberculosis vaccine approved for use. BCG has a good protective efficiency against infantile meningitis, but the protection rate against adult tuberculosis varies. Currently, the transformation of tuberculosis vaccines is mainly divided into two categories. Aspects: First, the new subunit vaccine is used as a booster vaccine to enhance the immune response caused by BCG. The second is to recombine the immunodominant antigen lacking in BCG into the BCG vaccine by overexpressing the immunodominant antigen, thereby enhancing the protective immune response induced by BCG. BCG vaccine has been used for many years, and its safety has been verified by a large number of vaccinated people. Therefore, the above two vaccine transformation strategies are based on BCG and have great advantages. At present, antigens such as Ag85, Rv3425, HspX and other immunodominant antigens have been identified and are being used in the research of tuberculosis vaccines. Since Mycobacterium tuberculosis is an intracellular bacterium, it colonizes and proliferates in alveolar macrophages, and the cellular immune response plays an important role in clearing Mycobacterium tuberculosis. Bacillus disease course development, in which studies have confirmed that IgM, IgG1, IgG3 and IgA are protective antibodies, so cellular immunity and humoral immunity are crucial in tuberculosis infection.

Contents of the invention

The purpose of the present invention is to provide the application of Mycobacterium tuberculosis antigenic protein Rv2351c.

In order to achieve the purpose of the present invention, the present invention provides the application of Mycobacterium tuberculosis antigenic protein Rv2351c in the preparation of tuberculosis detection reagents. Wherein, the amino acid sequence of the antigenic protein Rv2351c is shown in SEQ ID NO: 1, or an amino acid sequence having the same immunogenicity and the same antigenicity formed by replacing, deleting or adding one or several amino acids to the sequence.

The present invention also provides a protein derived from the antigenic protein Rv2351c or an analogue thereof.

The present invention also provides a DNA molecule encoding the antigenic protein Rv2351c.

The invention also provides expression vectors and expression cassettes containing the DNA molecules.

The present invention also provides a transgenic cell line containing said DNA molecule.

The invention also provides recombinant bacteria containing said DNA molecule and recombinant protein expressed and purified. Preferably, the antigenic protein Rv2351c is expressed by a prokaryotic expression system, the preferred prokaryotic expression vector is pET-30a vector, and the preferred prokaryotic cell is Escherichia coli BL21 (DE3).

In a specific embodiment of the present invention, the target fragment of Rv2351c is amplified from the Mycobacterium tuberculosis H37Rv genome, the target fragment is recovered from the gel and connected to the T4 vector, transformed into Escherichia coli DH5α competent cells for cultivation, and the plasmid is extracted After the sequencing was correct, the plasmid was double-enzymatically digested and connected to the pET-30a vector and transformed into E. coli BL21 competent cells. The inclusion body protein was obtained after IPTG induction at 37°C, and the active Rv2351c protein was obtained after purification and renaturation.

The present invention also provides a tuberculosis detection reagent, which contains Mycobacterium tuberculosis antigen protein Rv2351c, or a DNA molecule encoding the antigen protein Rv2351c, or a recombinant protein produced by a recombinant bacterium containing the DNA molecule.

The present invention also provides a tuberculosis ELISPOT detection kit containing the above detection reagent.

Also included in the test kit:

① Primary antibody: mouse IgG monoclonal antibody against human or animal IFN-γ.

② Enzyme-labeled reagent: another mouse IgG monoclonal antibody labeled with horseradish peroxidase against different epitopes of human or animal IFN-γ.

③Standard product:

Culture plate: 96-well microwell reaction plate containing PVDF membrane or nitrocellulose membrane, the positive control well contains tuberculosis non-specific stimulating antigen (such as PHA, etc.), and the local control contains PBS or basal solution.

④ Other reagents and consumables required for ELISPOT detection.

Preferably, the primary antibody is immobilized on the aforementioned microwell reaction plate.

The invention also provides the application of the Mycobacterium tuberculosis antigenic protein Rv2351c in preparing tuberculosis vaccine.

The present invention also provides a tuberculosis vaccine, the active ingredient of which is Mycobacterium tuberculosis antigenic protein Rv2351c, or a DNA molecule encoding the antigenic protein Rv2351c, or a recombinant protein produced by a recombinant bacterium containing the DNA molecule.

The invention also provides the application of the Mycobacterium tuberculosis antigenic protein Rv2351c in the preparation of anti-tuberculosis drugs.

Use Mycobacterium tuberculosis antigenic protein Rv2351c as immunogen, supplemented with adjuvant to immunize experimental animals to prepare polyclonal antibody; or use Mycobacterium tuberculosis antigenic protein Rv2351c as immunogen, supplemented with adjuvant to immunize experimental animals, using hybridoma technology and DNA recombination technology to prepare a humanized monoclonal antibody that recognizes Mycobacterium tuberculosis antigenic protein Rv2351c.

The present invention also provides an anti-tuberculosis drug, the active ingredient of which is a polyclonal antibody prepared by using Mycobacterium tuberculosis antigenic protein Rv2351c as an immunogen, supplemented with an adjuvant to immunize experimental animals, or using Mycobacterium tuberculosis antigenic protein Rv2351c as an immunogen. The immunogen is supplemented with an adjuvant to immunize experimental animals, and the hybridoma technology and DNA recombination technology are used to prepare a humanized monoclonal antibody that recognizes the antigenic protein Rv2351c of Mycobacterium tuberculosis.

The present invention further provides the application of the antigen protein Rv2351c in detecting specific T cell and B cell immune responses caused by Mycobacterium tuberculosis infection. It detects cytokines secreted by T cells or B cells after human or animal lymphocytes are stimulated by antigenic protein Rv2351c.

Among them, the cytokines secreted by tuberculosis-specific T cells include: interferon gamma (IFN-γ), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 10 (IL-10 ), tumor necrosis factor alpha (TNF-α), etc. Cytokines secreted by B cells include antibodies.

Detection methods for cytokines secreted by tuberculosis-specific T cells include enzyme-linked immunospot assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA), immunocolloidal gold assay, cytokine internal staining, and T cell proliferation assay. The detection methods of cytokines secreted by B cells include enzyme-linked immunosorbent assay (ELISA) and the like.

The lymphocytes come from peripheral blood, venous blood, cerebrospinal fluid, pleural effusion or pleural effusion of humans or animals.

The present invention has the following advantages:

(1) The present invention provides a new tuberculosis detection antigen, which can reduce false negatives caused by single antigen-specific T cells and improve detection sensitivity compared with previous detection methods using a single antigen.

(2) The antigen protein Rv2351c of the present invention exists in the RD7 region, and is missing in all Mycobacterium tuberculosis and Mycobacterium bovis, so that the detection is not affected by BCG inoculation, which is conducive to improving the specificity of detection.

(3) The present invention uses a prokaryotic expression system to express the purified protein Rv2351c, which is suitable for large-scale commercial production and has low cost.

(4) The antigenic protein Rv2351c of the present invention is a factor related to tuberculosis virulence, and the high intensity of cellular immunity and humoral immune response shows that the antigen has strong immunogenicity, and can be used as a candidate antigen for a new type of tuberculosis vaccine.

Description of drawings

Fig. 1 is the agarose gel electrophoresis detection result of the target gene Rv2351c in Example 1 of the present invention.

Fig. 2 is the SDS-PAGE electrophoresis detection result of protein Rv2351c in Example 1 of the present invention; Wherein, 1 is the BL21 (DE3) bacterium that contains empty plasmid, 2 is the induced BL21 (DE3) that contains recombinant plasmid, 3 is purified Rv2351c protein.

Fig. 3 is an ELISPOT test dot diagram of Mycobacterium tuberculosis antigenic protein Rv2351c in Example 3 of the present invention.

Fig. 4 is an ELISPOT dot diagram of Rv2351c tuberculosis patients and healthy volunteers in Example 3 of the present invention.

Fig. 5 is the detection result of serum antibody titer of each group in Example 4 of the present invention.

detailed description

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Experiment Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions.

Example 1 Cloning of Mycobacterium tuberculosis antigen gene Rv2351c and protein expression and purification

1. Primer design: According to the genome sequence of Mycobacterium tuberculosis H37Rv on NCBI, use the software Primer5 to design primers, upstream primer: GCGCGGATCCATGTCACGTCGAGAGTTTTTG (including BamH1 restriction site), downstream primer: ATATAAGCTTTCAGCTGCACAGCCCGCTGG (including HindⅢ restriction site).

2. Amplification of the target gene: Utilize the CTAB method to extract the genomic DNA of Mycobacterium tuberculosis H37Rv, and use the DNA as a template to amplify the target gene. The PCR reaction system is as follows:

PCR reaction program: hot start at 95°C for 5 min; denaturation at 94°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 1 min, 30 cycles; and finally incubation at 72°C for 10 min.

3. Identification of the target gene: Take 7 μl of the PCR product for electrophoresis in 1% agarose gel, use DL1000DNAMarker as the molecular weight standard, use the gel imaging system to view the results and take pictures for preservation.

4. Recovery and purification of PCR products:

The PCR product was subjected to 1% agarose gel electrophoresis, the target band was cut with a sterilized scalpel, and the agar block containing the target segment was placed in a 2ml EP tube.

A(1) Add 3 times the volume of Buffer PG to 100 mg of the gel, incubate at 50°C for 10 minutes, and gently invert the centrifuge tube every 2 to 3 minutes to ensure that the gel is completely dissolved.

A (2) Add 1 times the volume of isopropanol, mix up and down.

A(3) Column balance: Add 200μl Buffer PS to the adsorption column loaded into the collection tube, centrifuge at 16200g for 2 minutes, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

A(4) Add the solution obtained in step (1) to the adsorption column with a collection tube, place it at room temperature for 2 minutes, centrifuge at 16200g for 1 minute, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube middle.

A(5) Put the adsorption column into a new 1.5ml centrifuge tube, add 50μl Buffer EB dropwise to the middle of the adsorption membrane, place at room temperature for 2 minutes, centrifuge at 16200g for 1 minute, collect the DNA solution, and store the DNA at -20°C.

5. Amplification of the plasmid:

Transform the empty plasmid pET-30a into Escherichia coli DH5α, after overnight at 37°C on an LB solid plate containing ampicillin, pick a single colony, culture it overnight in ampicillin-resistant liquid LB, and use reagents the next day Kit (the endotoxin-free plasmid medium extraction kit produced by Beijing Kangwei Century Biotechnology Co., Ltd.) to extract the plasmid, the steps are as follows:

B (1) Take 5-15ml of overnight culture bacteria solution, add it to a centrifuge tube, centrifuge at >6200g for 3 minutes to collect bacteria, and discard all supernatant as much as possible.

B(2) Add 500 μl Buffer P1 to the centrifuge tube with the bacterial pellet left, and use a pipette or vortex shaker to mix well to suspend the bacterial pellet.

B(3) Add 500μl Buffer P2 to the centrifuge tube, and mix it upside down gently for 6-8 times to fully lyse the bacteria. Leave it at room temperature for 3-5 minutes, add 500μl Buffer E3 to the centrifuge tube, and mix it upside down immediately. Homogenize 6-8 times, at this time, white flocculent precipitate appears, place at room temperature for 5 minutes, centrifuge at 16200g for 10 minutes, absorb the supernatant, add the supernatant to the filter column, centrifuge at 16200g for 1 minute to filter, transfer the filtrate in the collection tube to in a centrifuge tube. Add 450 μl of isopropanol to the filtrate and mix by inverting.

B(4) Column balance: add 200μl Buffer PS to the adsorption column loaded into the collection tube, centrifuge at 16200g for 2 minutes, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

B(5) Transfer the mixed solution of filtrate and isopropanol in step B(3) to the well-balanced adsorption column. Centrifuge at 16200ⅹg for 1 minute, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

B(6) Add 750μl Buffer PW to the adsorption column, centrifuge at 16200g for 1 minute, and discard the waste liquid in the collection tube. Put the adsorption column back into the collection tube, centrifuge at 16200g for 2 minutes, discard the waste liquid, and dry the adsorption column at room temperature for 5 minutes.

B(7) Put the adsorption column in a new centrifuge tube, add 100~300μl Endo-Free Buffer EB to the middle part of the adsorption membrane, let it stand at room temperature for 2~5 minutes, centrifuge at 16200g for 2 minutes, and store the plasmid at -20℃.

6. The obtained DNA product and plasmid were subjected to double enzyme digestion respectively.

The enzyme digestion reaction system of the DNA product is:

The plasmid digestion reaction system is:

After incubating at 37°C for 1 hour, the digested product was recovered with a DNA product purification kit (Tiangen Biochemical Technology Co., Ltd.), the steps are as follows:

C(1) Column equilibration step: add 500 μl of equilibrium solution BL to the adsorption column CB2, centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column CB2 back into the collection tube.

C (2) Add 5 times the volume of binding solution PB to it, and mix well. Add the obtained solution into an adsorption column CB2, place it at room temperature for 2 minutes, centrifuge at 12000 rpm for 30-60 sec, discard the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube.

C (3) Add 600 μl of rinse solution PW to the adsorption column CB2, centrifuge at 12000 rpm for 30-60 sec, discard the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube.

C(4) Repeat the above steps.

C(5) Put the adsorption column CB2 back into the collection tube, centrifuge at 12,000 rpm for 2 minutes, remove the rinse solution as much as possible, and place the adsorption column CB2 at room temperature for several minutes, and dry it thoroughly.

C(6) Put the adsorption column CB2 into a clean centrifuge tube, add 30-50 μl of elution buffer EB dropwise to the middle of the adsorption membrane, place at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 2 minutes to collect the DNA solution.

7. Obtaining recombinant plasmids and recombinant bacteria:

Ligation: Ligate the purified DNA obtained after enzyme digestion and the pET-30a plasmid with T4 DNA ligase. The ligation system is as follows:

Ligate overnight at 16°C.

Transformation: Transform the ligated plasmid into Escherichia coli DN5α, add 5 μl of the plasmid into a centrifuge tube containing 50 μl of competent cells, pipette evenly with a gun, bathe in ice for 30 min, bath in water at 42°C for 90 sec, bath in ice for 2 min, add 400 μl of 37 ℃ preheated LB medium without antibiotics, cultured in a shaker at 37 ℃ for 45min, 150r/min, to restore the drug resistance of the cells, centrifuged at 4000r for 2min. Discard 400 μl of supernatant, mix the remaining 55 μl, and apply to LB plates. After placing at room temperature for 4-5 minutes, invert the plate and incubate at 37°C for 12-16 hours.

Sequencing: Pick a single colony and perform bacterial liquid PCR (Figure 1). The positive clones are cultured in liquid LB medium for 12 hours, and then sent to Beijing Qingke Xinye Biotechnology Co., Ltd. for sequencing.

The sequenced correct bacterial species was expanded and cultured again to extract the plasmid and transformed into Escherichia coli BL21(DE3) (the steps were the same as above). After the colony PCR of the transformed BL21(DE3) was confirmed to be positive, some of them were stored in glycerol at -70°C as strains, and some of them were expanded to obtain proteins.

8. Induced expression of Mycobacterium tuberculosis antigen in Escherichia coli:

Add 1ml of fresh bacterial solution to 300ml of ampicillin-resistant LB medium and cultivate overnight on a shaker at 37°C, then add 300ml of LB medium to expand the culture for 3 hours the next day, and measure the OD value at 0.6-0.8. Add IPTG (isopropyl-β-D-thiogalactopyranoside) to make the final concentration 1mmol/ml, after induction at 37°C for 3 hours, collect the bacteria by centrifugation at 12000rpm for 5 minutes, and use 1% Triton X- Resuspend the bacteria in 100% PBS, mix well, use a 300W ultrasonic instrument, work for 5s, stop for 5s, and sonicate for 30min. Centrifuge at 12000rpm for 5min, save the supernatant, resuspend the pellet with PBS, and perform SDS-PAGE electrophoresis. It was determined that the protein was expressed in the cell pellet in the form of inclusion body.

9. Purification and renaturation of Rv2351c protein: Collect the culture medium, centrifuge at 8000 rpm for 10 minutes, collect the precipitated bacteria, add 4ml of breaking buffer (pH8.550mM Tris-HCL, 2mM EDTA, 100Mm NaCl, 0.5% Triton X-100, 1mg/ml lysozyme), mixed on ice for 45 minutes, mixed cells in ice water with a 300W ultrasonic instrument for 5s, stopped for 5s, ultrasonicated for 10 minutes, then centrifuged at 12000g for 10 minutes, discarded For precipitation, resuspend the precipitate with 50mM phosphate buffer (containing 0.5M NaCl, 8M urea, and 10mM imidazole) at pH 7.4, and filter with a 0.45μm filter membrane to avoid column plugging. The specific operation is as follows:

(1) Take 1ml nickel NTA agarose gel prepacked column, equilibrate with 10ml equilibration buffer, load the broken supernatant at 0.5ml/min, and then aliquot 2ml/tube.

(2) Wash away unadsorbed samples with 15ml of equilibration buffer at a flow rate of 1-2ml/min and collect in 2ml/tube.

(3) Use 5ml of washing buffer (containing 0.5M NaCl, 8M urea, and 25mM imidazole) to wash away unadsorbed samples at a flow rate of 1-2ml/min and collect in 2ml/tube.

(4) Use 5ml of washing buffer (containing 0.5M NaCl, 8M urea, and 40mM imidazole) to wash away the adsorbed foreign proteins at a flow rate of 1-2ml/min and collect in 2ml/tube.

(5) Wash away the target protein with 5ml washing buffer (containing 0.5M NaCl, 8M urea, 60mM imidazole) at a flow rate of 1-2ml/min and collect in 2ml/tube.

(6) Wash away the target protein with 5ml of elution buffer (containing 0.5M NaCl, 8M urea, and 300mM imidazole) at a flow rate of 1-2ml/min and collect in 2ml/tube.

(7) Equilibrate the column with 5ml equilibration buffer, fill it with 20% ethanol, and seal it.

After the collected protein samples were subjected to SDS-PAGE electrophoresis (Figure 2), the target protein was refolded with a protein refolding kit, and the refolded protein Rv2351c was quantified with a BCA protein quantification kit. The amino acid sequence of the protein Rv2351c is shown in SEQ ID NO:1.

Example 2 Preparation of Tuberculosis ELISPOT Detection Kit

The basic composition of the test kit is as follows:

① The protein antigen Rv2351c prepared in Example 1.

② Primary antibody: mouse IgG monoclonal antibody against human or animal IFN-γ.

Enzyme-labeled reagent: another mouse IgG monoclonal antibody labeled with horseradish peroxidase against different epitopes of human or animal IFN-γ.

③Standard product:

Culture plate: 96-well microwell reaction plate containing PVDF membrane or nitrocellulose membrane, the positive control well contains tuberculosis non-specific stimulating antigen (such as PHA, etc.), and the local control contains PBS or basal solution.

④ Other reagents and consumables required for ELISPOT detection.

The primary antibody was immobilized on the above-mentioned microwell reaction plate.

The kit is designed based on the principle of double-antibody sandwich. The ELISPOT method is used to detect the antigen. The experimental process is as follows: the primary antibody coated on the PVDF membrane can bind to the IFN-γ in the cell supernatant as the capture antibody, and the IFN-γ can Captured by enzyme-labeled secondary antibody and developed color. The two antibodies are monoclonal antibodies that recognize different epitopes of IFN-γ.

Example 3 Antigen protein Rv2351c is used for clinical detection of tuberculosis infection

1. Isolation of Peripheral Blood Lymphocytes

1.1 Subjects

Screening criteria for volunteer cases:

Pulmonary tuberculosis patients diagnosed with clinical manifestations, symptoms, signs and chest imaging examinations, and with positive sputum culture.

Screening criteria for patients with pulmonary disease:

Other lung diseases with negative sputum culture and sputum smear, such as pneumoconiosis, chronic obstructive pulmonary disease and other lung diseases.

Screening criteria for healthy volunteers:

No clinical symptoms of tuberculosis, no history of close contact with tuberculosis patients, no other diseases or infections.

The enrolled TB patients and volunteers, aged 15-80, were randomly selected from a continuous-time sample of visits to the TB ward. A total of 60 tuberculosis volunteers, 33 lung disease patients and 60 healthy volunteers collected blood samples. When collecting blood, heparin anticoagulated vacuum blood collection tubes without endotoxin were used to collect peripheral venous blood. Each volunteer collected about 5ml~ 10ml.

1) The samples were separated from PBMCs with Ficoll-Hypaque separation solution within 4 hours.

2) First dilute the whole blood with RPMI-1640 medium 1:1 and mix well, add a certain volume of separation liquid into the centrifuge tube, spread the diluted blood sample above the liquid surface of the separation liquid, and keep the interface between the two liquid surfaces clear , separation solution, anticoagulated undiluted whole blood, and RPMI 1640 medium in a volume ratio of 1:1:1, at room temperature (18-26°C), centrifuged at 800g for 20 minutes.

3) After centrifugation, the bottom of the tube is red blood cells, the middle layer is the separation solution, the uppermost layer is the plasma layer, and between the plasma layer and the separation solution layer is a layer of white cloudy mononuclear cells (including lymphocytes and monocytes). Use a pipette to absorb the white cloudy cell layer and transfer it to a 15ml sterile centrifuge tube, add RPMI 1640 medium to 10ml, and centrifuge at 800g for 10 minutes at room temperature.

4) Discard the supernatant, add 7ml RPMI 1640 medium after resuspension, and centrifuge at 700g for 10 minutes.

5) Discard the supernatant and add 0.5ml AIM-V medium to resuspend the pellet.

6) The cells were counted by an automatic cell counter, and 500 μL of a cell suspension with a cell concentration of 2.5×10 ⁶ /ml was prepared with AIM-V medium.

2. ELISPOT detection of antigenic protein Rv2351c-specific T cells

Using the kit in Example 2, add the following reagents to the microwell plate pre-coated with the primary antibody, and set 4 detection wells for each patient: positive control wells (add 100 μL phytohemagglutinin PHA as a positive stimulus), Negative control wells (add 100 μL PBS as negative control), detection wells (add 100 μL of Rv2351c with a final concentration of 20 μg/ml), add 100 μL of the above-mentioned diluted PBMCs to each well, so that the number of PBMCs in each well reaches 250,000, Place the antigen and PBMC cells in a 37°C, 5% _CO2 incubator for 20 hours.

3. ELISPOT plate washing and result judgment

Wash away PBMC cells and antigen stimulators, add 100 μL of primary antibody and incubate at room temperature for 1 hour, wash 5 times with PBS, add secondary antibody and incubate at room temperature for 1 hour, then wash 5 times with PBS, add substrate to avoid light and develop color for 7 minutes , use purified water to stop color development, place the culture plate at the air vent to dry and observe the number of spots on the plate.

Judgment of results: the number of spots in blank control wells = N, the number of spots in test wells = T, the number of spots in positive control wells = P. The results are shown in Table 1, Figure 3 and Figure 4.

Table 1 Judgment criteria for ELISPOT results

The results of 60 tuberculosis patients, 33 patients with other lung diseases and 60 healthy people are shown in Table 2. Among them, 5 of the 60 healthy people were excluded because the results could not be judged, and a total of 55 healthy people, 60 tuberculosis patients and 33 patients with other lung diseases were included in the statistical criteria.

Table 2 Rv2351c population detection results

Positive diagnosis of tuberculosis Negative diagnosis of tuberculosis total test positive 37 8 45 test negative twenty three 80 103 total 60 88 148

The calculated sensitivity of the antigen protein Rv2351c to detect tuberculosis patients is 61.67%, and the specificity is 90.91%.

Detection sensitivity = (positive number of tuberculosis patients / total number of tuberculosis patients) × 100%

Detection specificity = 1-(positive number of pulmonary tuberculosis patients + positive number of healthy volunteers)/(total number of lung disease patients + total number of healthy volunteers)

Example 4 Detection of immunogenicity of Rv2351c

1. Immunization of mice

30 6-week-old female BALB/c mice were screened and divided into 5 groups, PBS negative control group (PBS), adjuvant control group (DP), Mycobacterium tuberculosis antigenic protein Ag85B 20μg low-dose group (Ag85b-L ), Ag85B 50μg high-dose group (Ag85B-H), Rv2351c 20μg low-dose group (2351c-L), Rv2351c 50μg high-dose group (2351c-H). Poly(I:C) 50 μL and dioctadecyldimethylammonium bromide (DDA) 100 μL were fully emulsified and immunized mice by subcutaneous inoculation, once every 3 weeks, for a total of three times, and the last immunization was 4 Tested after a week.

2. Separation of mouse serum

500 μL of blood was collected from the mouse eyeball, and the blood was placed in a 37°C incubator for 2 hours, and then transferred to a 4°C refrigerator overnight. On the next day, the blood was centrifuged at 3000 rpm for 5 minutes and the supernatant was aspirated.

3. Detection of serum antibody titer

1) Coat the ELISA plate with 2 μg/ml Rv2351c protein overnight at 4°C, and wash 5 times with PBST the next day.

2) Block with PBS solution containing 2% BSA at 37° C. for 2 hours, and wash 5 times with PBST.

3) The sera of each group were diluted 20,000, 40,000, 80,000, 160,000, 320,000, 640,000, 1,280,000, and 2,560,000 times with PBS, and 100 μL of diluted serum was added to each well, incubated at 37°C for 1 hour, and washed 5 times with PBST.

4) Add 5000-fold diluted HRP-labeled IgG, IgG1, IgG2a antibodies respectively, 100 μL per well, incubate at 37° C. for 1 hour, and wash 5 times with PBST.

5) Add TMB and develop color at 37°C for 15 minutes, then add 2M sulfuric acid as a stop solution.

6) Microplate reader detects the luminosity value of the pipette with a wavelength of 450nm.

7) Judgment criteria: OD≥2.1×OD _{(negative control)} is judged as positive.

8) Result analysis: the antibody titers of serum in each group are shown in Fig. 5 .

The results showed that the IgG, IgG1, and IgG2a antibody titers produced by the Rv2351c high-dose and low-dose groups were all high, and Rv2351c-L and Rv2351c-H induced higher concentrations of IgG than the Ag85b-L and Ag85B-H groups respectively, and Rv2351c-L The IgG2a/IgG1 ratios of the Rv2351c-H group and the Rv2351c-H group were 2.5 and 3.5 respectively, and Rv2351c was more inclined to induce a Th1-type immune response, which could stimulate the body to produce an effective humoral immune response.

Example 5 Detection of cellular immunogenicity of Rv2351c

1. After the mice immunized in Example 4 were killed, soak them in 75% alcohol for 5 minutes, fix the mice on the foam board in the ultra-clean table, cut the peritoneum, separate the spleen, and place it in a 1640 in a Petri dish.

2. On a 200-mesh nylon mesh, gently grind the mouse spleen with the inner core of the syringe, and filter the ground cells through the filter. Centrifuge at 1000r/min for 5 minutes and discard the supernatant to obtain cells.

3. Gently oscillate the cells to loosen them, add red blood cell lysate at 2mL/only, mix well, and incubate in a 37°C incubator for 10 minutes, then add 2 times the volume of 1640 medium in the red blood cell lysate to stop the reaction , centrifuge at 1000r/min for 5 minutes, discard the supernatant, and obtain splenocytes.

4. Resuspend the splenocytes with 2ml of 1640 medium, centrifuge at 1000r/min for 5 minutes and discard the supernatant to obtain splenocytes. Determine the cell concentration with a cell counter.

5. Dilute splenocytes with 1640 medium containing 10% FBS to make the concentration 2×10 ⁶ /mL. Take 500 μL of splenocytes from each group and place them in a 24-well culture plate. The splenocytes in the PBS group and the DP adjuvant group were co-stimulated with 5 μg/mL of Ag85B and 5 μg/mL of Rv2351c antigen, respectively, and the Ag85B-L and Ag85B-H groups were respectively stimulated with 500 μL of 5 μg/mL Ag85B antigen was co-incubated, 2351c-L and 2351c-H groups were incubated with 500 μL of 5 μg/mL Rv2351c antigen, and each group was added with 500 μL of sterile PBS and 500 μL of 5 μg/mL phytohemagglutinin (PHA) As a negative control and a positive control, each detection well was done in duplicate. After incubating splenocytes with corresponding stimulators for 72 hours at 37°C in a 5% CO ₂ incubator, the cell supernatant was collected, and the IFN-γ, IL-2, IL-4, IL- 10.

6. Use carbonic acid coating solution (pH=9.6) for IFN-γ monoclonal antibody, IL-2 monoclonal antibody, IL-4 monoclonal antibody, etc., and carbonic acid buffer solution (pH=6.5) for L-10 monoclonal antibody according to the ratio of 1:500 After doubling dilution, add 100 μL/well into a 96-well plate, and coat overnight at 4°C.

7. After washing 3 times with PBTS, pat dry, add 10% FBS in PBS to block at room temperature for 1 hour.

8. After washing 5 times with PBST, the standard products of IFN-γ, IL-2, IL-4, and IL-10 were diluted to corresponding concentrations according to the corresponding proportions, and added to each ELISA plate respectively as a standard curve. Add 100 μL of the cell supernatant to be detected to the rest, and incubate at room temperature for 2 hours.

9. After diluting 5 times with PBST, add 100 μL of IFN-γ, IL-2, IL-4, IL-10 detection antibody + HRP-labeled secondary antibody respectively, and incubate at room temperature for 1 hour.

10. After washing 7 times with PBST, add 100 μL of TMB chromogenic solution, incubate at room temperature for 30 minutes, then add 50 μL of stop solution to terminate the reaction.

11. Measure the absorbance value at a wavelength of 450nm with a microplate reader, and simultaneously detect the absorbance value at a wavelength of 570nm as a control.

12. Result analysis: Make a standard curve based on the cytokine standard, and then substitute the OD value of the detection well into the formula to calculate the final concentration of various cytokines in each group of mice. The results are shown in Table 3.

Table 3 The detection results of four cytokines (pg/mL)

Grouping/Cytokine IFN-γ IL-2 IL-4 IL-10 PBS group 29.6±11.8 11.9±4 7.8±2.4 22.1±6 DP adjuvant group 40.3±11.4 17.5±11 12.1±3.4 34.6±10.2 Ag85B-L 4098.6± ^593.5ab 45± ^10.3ab 20.8± ^6ab 1590.3± ^438.1ab Ag85B-H 5257.4± ^818.5ab 43.4± ^19.6ab 38.4± ^19.4ab 3884.6± ^972.4ab 2351c-L 3805.3± ^1061.2ab 31.4±12.9 36.1± ^8.9ab 2860.9± ^485.2ab 2351C-H 4978.7± ^596.7ab 33±12.1 68.3± ^15.5ab 4733.7± ^629.2ab

Note: a indicates that there is a significant difference between the experimental group and the PBS group, b indicates that there is a significant difference between the experimental group and the DP adjuvant group.

The results showed that compared with the PBS group and the adjuvant group, the Rv2351c high and low dose groups could produce higher concentrations of IFN-γ (P<0.001), IL-4 (P<0.001), IL-10 (P<0.001), IFN-γ is a Th1 cytokine that can activate macrophages to promote the clearance of Mycobacterium tuberculosis by macrophages. IL-4 and IL-10 are Th2-type cytokines that promote antibody production during tuberculosis infection. Plays an immunomodulatory role in tuberculosis infection.

In summary, Rv2351c can be used as a diagnostic reagent for the diagnosis and detection of tuberculosis, and Rv2351c can induce strong cellular and humoral immune responses, and is an immunodominant antigen that can be used in the construction and preparation of tuberculosis vaccines.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (10)

1. The application of Mycobacterium tuberculosis antigenic protein Rv2351c in the preparation of tuberculosis detection reagent; wherein, the amino acid sequence of said antigenic protein Rv2351c is as shown in SEQ ID NO: 1, or this sequence is replaced, deleted or added one or more The amino acid sequence formed by amino acids has the same immunogenicity and the same antigenicity. 2. A tuberculosis detection reagent, characterized in that, the detection reagent contains Mycobacterium tuberculosis antigenic protein Rv2351c, or a DNA molecule encoding the antigenic protein Rv2351c, or a recombination produced by a recombinant bacterium containing the DNA molecule protein. 3. the tuberculosis ELISPOT detection kit that contains detection reagent described in claim 2. 4. test kit according to claim 3, is characterized in that, also comprises in the test kit: ① Primary antibody: mouse IgG monoclonal antibody against human or animal IFN-γ; ②Enzyme-labeled reagent: another mouse IgG monoclonal antibody labeled with horseradish peroxidase against different epitopes of human or animal IFN-γ; ③Standard product: Culture plate: 96-well microwell reaction plate containing PVDF membrane or nitrocellulose membrane, the positive control well contains tuberculosis non-specific stimulating antigen, and the local control contains PBS; ④ Other reagents and consumables required for ELISPOT detection; Preferably, the primary antibody is immobilized on the aforementioned microwell reaction plate. 5. The application of Mycobacterium tuberculosis antigen protein Rv2351c in the preparation of tuberculosis vaccine. 6. A tuberculosis vaccine, characterized in that its active ingredient is Mycobacterium tuberculosis antigenic protein Rv2351c, or a DNA molecule encoding said antigenic protein Rv2351c, or a recombinant protein produced by a recombinant bacterium containing said DNA molecule. 7. The application of Mycobacterium tuberculosis antigen protein Rv2351c in the preparation of anti-tuberculosis drugs. 8. The application according to claim 7, characterized in that the polyclonal antibody is prepared by using Mycobacterium tuberculosis antigenic protein Rv2351c as an immunogen and supplemented with an adjuvant to immunize experimental animals. 9. The application according to claim 7, characterized in that, the Mycobacterium tuberculosis antigenic protein Rv2351c is used as the immunogen, supplemented with an adjuvant to immunize experimental animals, and the hybridoma technology and DNA recombination technology are used to prepare the tuberculosis branch recognition Humanized monoclonal antibody to Bacillus antigenic protein Rv2351c. 10. An anti-tuberculosis drug, characterized in that its active ingredient is the polyclonal antibody prepared by using Mycobacterium tuberculosis antigenic protein Rv2351c as an immunogen, supplemented with an adjuvant to immunize experimental animals, or the Mycobacterium tuberculosis antigenic protein Rv2351c is used as an immunogen, supplemented with an adjuvant to immunize experimental animals, and a humanized monoclonal antibody recognizing Mycobacterium tuberculosis antigen protein Rv2351c is prepared by using hybridoma technology and DNA recombination technology.