CN105861520B - Application of Rv3121 protein in detection of mycobacterium tuberculosis infection - Google Patents
Application of Rv3121 protein in detection of mycobacterium tuberculosis infection Download PDFInfo
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Abstract
The invention relates to an application of an Rv3121 protein in detecting mycobacterium tuberculosis infection, wherein an antigen amino acid sequence is shown as SEQ ID NO. 2. The preparation method of the protein antigen comprises the following steps: step (1) of
An entry vector for catalyzing Rv3121 (freely available from PFGRC under Craig Ventor Institute, USA) and
the vector recombinates and produces the expression vector of Rv 3121; transforming the expression vector of the Rv3121 in the step (1) into a target expression host, and expressing a target Rv3121 protein; and (3) crushing the cells to collect protein inclusion bodies and renaturing the target protein.
Description
Technical Field
The invention belongs to the technical field of medical products, and particularly relates to an application of an Rv3121 protein in detection of mycobacterium tuberculosis infection.
Background
The in vitro diagnostic industry in China is currently in a fast growing phase. However, the huge market demand is obviously different from the relatively backward independent research and development capability of the diagnostic reagent industry in China. How to develop independent innovation from the source and how to convert the scientific research achievements in the existing biological medicine field into products without being limited by individual overseas medicine huge on the supply of diagnostic reagent raw materials is a very much concerned hotspot in recent years in governments, scientific research institutions and medicine industries of China. And the market puts forward a rigid demand on a more sensitive and efficient diagnostic reagent nowadays, and the method can quickly and accurately diagnose tuberculosis patients and has important significance for medical workers and patients.
In recent years, data survey results show that 76.6% of patients with tuberculosis symptoms are subjected to relevant examination before tuberculosis, but only 35.8% of patients are diagnosed as tuberculosis patients, and the improvement of the patient discovery rate is still the key point of the current tuberculosis prevention and treatment work and is also difficult.
The in vitro diagnosis of pathogenic microorganism infection by using the cellular immune reaction of antigen-specific T cells is a new detection method developed in the present year. We isolated peripheral blood mononuclear cells from fresh whole blood and cultured in stimulation, and then tested the number of cells capable of secreting IFN-. gamma.using ELISPOT. The method is currently mainly applied to the diagnosis of mycobacterium tuberculosis infection. The current diagnosis of tuberculosis generally adopted in clinic mainly depends on clinical symptoms, influential diagnosis and etiological diagnosis, and is insensitive to the diagnosis of latent infection of mycobacterium tuberculosis. Meanwhile, in the tuberculosis screening process, the sensitivity and specificity for directly detecting pathogens or detecting mycobacterium tuberculosis antibodies are not ideal.
Disclosure of Invention
Aiming at the clinical practical working condition, the protein fragment of the known mycobacterium tuberculosis source is screened, the existing positive detection reference substance is combined, the mycobacterium tuberculosis marker antigen for improving the tuberculosis diagnosis accuracy is provided, and the antigen is used for in vitro detection of specific T cell immunoreaction and can be used as a reference for diagnosing patients with tuberculosis and used for diagnosing whether the patients are infected by mycobacterium tuberculosis.
Specifically, the invention firstly relates to a specific protein antigen Rv3121 of mycobacterium tuberculosis, the sequence of the antigen is shown as SEQ ID NO.2, and the sequence structure is as follows:
the DNA expression sequence of the antigen is shown as SEQ ID NO.1, and the sequence structure is as follows:
the invention also relates to a preparation method of the antigen, which comprises the following steps
Step (1) of
Enzyme mix catalysis
Rv3121And (c) an entry vector (freely available from PFGRC under the CraigVentor Institute, USA) and
pDEST
TM17, recombining the vector to generate an expression vector of the Rv 3121;
transforming the expression vector of the Rv3121 in the step (1) into a target expression host, and expressing a target Rv3121 protein;
and (3) crushing the cells to collect protein inclusion bodies and renaturing the target protein.
The specific method of the step (1) is as follows:
a. cloning reaction system: 2.5 μ L of the entry vector of Rv3121,
pDEST
TM17 the carrier is 1 mu L, and the carrier is,
II Enzyme mix 2.5. mu.L; reaction conditions are as follows: reacting at 25 ℃ overnight;
b. the transformation method comprises adding 100 μ L of Escherichia coli DH5 α competent (self-made), ice-cooling for 30min, heat-shocking at 42 deg.C for 90s, standing on ice for 10min, adding 200 μ L of LB culture medium for renaturation, spreading on LB solid culture medium containing ampicillin (100mg/L), and culturing at 37 deg.C for 20 h;
c. plasmid extraction: extracting the plasmid by using an N96 high-purity plasmid miniextraction kit (DP114) to obtain an expression vector of the Rv 3121.
The specific method of the step (2) is as follows:
a. adding 1 μ L of the plasmid solution obtained in step (1) into 100 μ L of Escherichia coli rosetta (DE3) for competence, ice-cooling for 30min, and heat-shocking at 42 deg.C for 90 s;
b. placing the system on ice for 10min, adding 200 μ L LB culture medium for renaturation, coating on LB solid culture medium containing ampicillin (100mg/L), culturing at 37 deg.C for 12 h;
c. rv3121 protein expression was induced with 0.75mM IPTG.
The specific method of the step (3) is as follows:
a. and (3) adding a heavy suspension: 60mM tris pH 9.0, 0.15M EDTA resuspended the bacteria, sonicated at 4 ℃ using a sonicator,
b.4 ℃, centrifuging at 10000rpm for 20min, and collecting the precipitate to obtain the solid inclusion body, wherein the result is shown in figure 2.
c. The inclusion bodies were solubilized with a solubilization solution (60mM Tris-HCl pH7.0, 10M urea, 15mM DTT, 1mM EDTA); first dialysis: dialyzing the protein by using a dialysis bag with the molecular weight cutoff of 3K overnight, wherein the components of dialysate are as follows: 20mM Tris-HCl pH 9.0, 1M Urea, 3mM L-Argine;
d. dialyzing for the second time, wherein the dialysate comprises the following components: 20mM Tris-HCl pH 9.0, 10mM NaCl, second dialysis: dialyzing the protein into dialysate by using a dialysis bag with the molecular weight cutoff of 3K overnight, and obtaining the protein which can be directly used for immunogen screening.
The invention also relates to application of the Rv3121 antigen as an immunogen for detecting mycobacterium tuberculosis. The application comprises that the Rv3121 protein antigen is used for detecting mycobacterium tuberculosis independently; or the Rv3121 antigen is used in combination with the existing Mycobacterium tuberculosis detection antigen for detecting Mycobacterium tuberculosis.
The invention also relates to application of the Rv3121 protein antigen in preparation of a mycobacterium tuberculosis detection kit, and the application specifically comprises that the Rv3121 protein antigen is independently used as a detection antigen to prepare the detection kit; or the Rv3121 protein antigen and the existing mycobacterium tuberculosis detection antigen are cooperatively combined, so that the accuracy of the existing antigen immunity detection is improved.
The existing antigen for detecting mycobacterium tuberculosis includes but is not limited to mycobacterium tuberculosis-derived ESAT-6 antigen, CFP10 antigen, PPD antigen, BCG antigen, LAM antigen and ES-31 antigen.
The invention also relates to a single antigenic type mycobacterium tuberculosis detection kit prepared from the Rv3121 protein antigen, which comprises,
(1) detecting an effective amount of Rv3121 protein antigen (preferably at a concentration of 60 ug/ml);
(2) necessary detection reagent and color reagent.
The invention also relates to a multi-antigen combined type mycobacterium tuberculosis detection kit containing the Rv3121 protein antigen, the kit contains,
(1) detecting an effective amount of Rv3121 protein antigen at a concentration (preferably 60ug/ml) and detecting an effective amount of other antigens;
(2) necessary detection reagent and color reagent.
The invention also relates to a detection method of the single antigen type or multi-antigen combined type mycobacterium tuberculosis detection kit prepared from the Rv3121 protein antigen, the detection method comprises the following steps,
(1) the detection sample is anticoagulated whole blood or PBMC cells prepared from a blood sample of a patient to be detected;
(2) the Rv3121 protein antigen as immunogen is incubated with anticoagulated whole blood or PBMC cells for 20-30 hours, preferably 20-24 hours
(3) Detecting the cytokine secreted by the Rv3121 protein antigen, comparing with a positive reference to determine the detection result, wherein the cytokine is IFN-gamma, IFN-alpha, TNF-alpha, IL-2, IL-13, IP-10, IL-1ra, GM-CSF, MIP-1 beta, IL-6, IL-8, MCP-1, IL-10 and IL-12, preferably IFN-gamma, TNF-alpha, IL-2, IL-13, IP-10, IL-1ra, GM-CSF, MIP-1 beta, further preferably IFN-gamma, TNF-alpha, IL-2, IL-13, most preferably IFN-gamma.
Drawings
FIG. 1 shows the results of enzyme digestion verification of the expression vector pDEST17-Rv 3121: lane B2 is a DNA fragment of Rv 3121.
FIG. 2 is a SDS-PAGE graph showing the results of protein purification, including A. an inclusion body sample, M. a protein Marker, B. an inclusion body solubilized sample, C. a primary dialyzed sample, and D. a secondary dialyzed sample.
Detailed Description
Example 1 basic procedure for construction of the Rv3121-pDEST17 expression vector
To be provided with
LR
Enzyme mix catalysis
Rv3121And (c) an entry vector (freely available from PFGRC under Craig Ventorinstitute, USA) and
pDEST
TM17 Table of recombinant production of Rv3121 by vectorsTo a carrier.
The specific method comprises the following steps:
A. cloning reaction system: 1.5 μ L of the entry vector of Rv3121,
pDEST
TM17 the carrier is 1 mu L, and the carrier is,
BP
II Enzyme mix 2.5. mu.L; reaction conditions are as follows: the reaction was carried out at 25 ℃ overnight.
B. The transformation method comprises adding 100 μ L Escherichia coli DH5 α competent (self-made) into reaction system, ice-cooling for 30min, heat-shocking at 42 deg.C for 90s, standing on ice for 10min, adding 200 μ L LB culture medium for renaturation, spreading on LB solid culture medium containing ampicillin (100mg/L), and culturing at 37 deg.C for 20 h.
C. Plasmid extraction: extracting the plasmid by using an N96 high-purity plasmid miniextraction kit (DP114) to obtain an expression vector of the Rv 3121.
D. Enzyme digestion verification: the restriction enzyme kit (R0575S NEB) with BsrG1 was used to check whether the cloning was completed, and the results are shown in FIG. 1, which shows that the restriction fragment size is about 1200bp, and the plasmid construction was successful.
Example 2 expression and renaturation of the target protein Rv3121
1. The expression vector of Rv3121 obtained in example 1 was introduced into rosetta (DE3) expression vector, the specific method was:
a. mu.L of plasmid solution was added to 100. mu.L of plasmid solution competent (in-house) by DE3, ice-cooled for 30min, heat-shocked for 90s at 42 ℃,
b. placing the system on ice for 10min, adding 200 μ L LB culture medium for renaturation, coating on LB solid culture medium containing ampicillin (100mg/L), culturing at 37 deg.C for 12 h;
c. rv3121 protein expression was induced with 0.75mM IPTG.
2. The method comprises the following steps of carrying out ultracentrifugation after ultrasonic disruption of bacteria, obtaining inclusion body solid precipitate, collecting protein and renaturing:
a. and (3) adding a heavy suspension: 60mM tris pH 9.0, 0.15M EDTA resuspension of the bacteria, sonication of the bacteria at 4 ℃ using a sonicator;
b.4 ℃, centrifuging at 10000rpm for 20min, and collecting the precipitate to obtain the solid inclusion body, wherein the result is shown in figure 2.
c. The inclusion bodies were solubilized with a solubilization solution (60mM Tris-HCl pH7.0, 10M urea, 15mM DTT, 1mM EDTA); first dialysis: dialyzing the protein by using a dialysis bag with the molecular weight cutoff of 3K overnight, wherein the components of dialysate are as follows: 20mM Tris-HCl pH 9.0, 1M Urea, 3mM L-Argine;
d. dialyzing for the second time, wherein the dialysate comprises the following components: 20mM Tris-HCl pH 9.0, 10mM NaCl, second dialysis: dialyzing the protein into dialysate by using a dialysis bag with the molecular weight cutoff of 3K overnight, and obtaining the protein which can be directly used for immunogen screening.
Example 3 immunogenicity testing of the target protein Rv3121
In order to detect the immunogenicity of the Rv3121 and the enhancement effect thereof on the existing antigen detection kit, further antigen detection screening was performed on a plurality of cases clinically confirmed from the Beijing thoracic hospital.
Testing blood samples: patients from Beijing thoracic hospital
Reagents and consumables: T-SPOT kit, Oxford immunotec (UK) product
T-SPOT experiment:
the cytokine was detected as IFN-. gamma.using the T-SPOT kit from Oxford immunotec.
1) Heparin anticoagulation, namely uniformly mixing an anticoagulation sample with RPMI1640 according to the volume of 1: 1; carefully adding the blood sample to the upper layer of the separating liquid of the Ficoll lymphocyte according to the proportion of 2-3: 1;
2) centrifuging at 1000g for 22 min; the horizontal rotor slowly rises and slowly falls;
3) transferring the mononuclear cell layer (which is located in the middle layer of the centrifuge tube and is in a thin white film shape) from the Ficoll separating tube to a sterile 15ml centrifuge tube added with 10ml of AIM-V culture solution, gently mixing the materials uniformly, and centrifuging the mixture for 7min at the room temperature of 600 g;
4) carefully removing the supernatant, adding 1ml of AIM-V, gently suspending the cells, adding AIM-V culture solution to 10ml, and centrifuging at 350g for 7 min;
5) carefully abandoning the supernatant, adding 1ml of AIM-V culture solution to resuspend the cells;
6) cells were counted and diluted, and 10. mu.l of cell suspension was added to 40. mu.l of 1% trypan blue to prepare 1: 5, counting living cells, calculating the volume required by cell dilution, adding corresponding AIM-V serum-free culture solution to prepare cell dilution solution, and detecting by using the kit, wherein the number of cells added into each hole (24-hole PVDF membrane plate) is 2.5 multiplied by 10
5；
7) The plates were removed from the aluminum sealed bags and added in order:
50 μ l of AIM V to negative control wells; 50 μ l of antigen ESAT-6 to antigen A well; 50 μ l antigen CFP10 to antigen B well; 50 μ l positive control to positive control wells; 100. mu.l of the prepared cell dilution medium was added to each of the 4 wells. Mu.l of Sample protein (initial concentration of 60ug/ml) obtained in example 2 was added to the Sample well, in which the final concentration of Sample protein was 1/3 of the initial concentration, followed by addition of the medium and the cells.
8) The plates were incubated at 37 ℃ in a 5% CO2 incubator for 16-20 hrs.
9) Fresh enzyme-labeled antibody working solution was prepared with sterile PBS at a ratio of 1: 200. The plate was removed from the incubator and the well liquid was discarded. Wash 4 times with 200. mu.l/well sterile PBS.
10) Add 50. mu.l/well enzyme-labeled antibody working solution and incubate the plate at 2-8 ℃ for 1 hour. Unbound enzyme-labeled antibody was removed by washing 4 times with PBS.
11) 50. mu.l of the substrate working solution equilibrated at room temperature was added to each well, and reacted at room temperature for 7 minutes. The reaction was stopped by rinsing with distilled water and the plates were dried at 37 ℃ for 2-3hrs or overnight at room temperature.
Immunogen test results and analysis:
immunogenicity testing was performed in the molecular biology laboratory at the thoracic hospital. A certain number of tuberculosis patients which are clinically diagnosed are selected for testing, blood samples of the tuberculosis patients are detected by using various protein antigens, and the immunogenicity and the application direction of the Rv3121 protein antigen are analyzed according to the results, the statistics of the specific test results are shown in the following table 1,
TABLE 1 Mycobacterium tuberculosis detection results of RV3121 antigen for hospitalized patients with confirmed tuberculosis
Injecting: T-SPOT double-antigen detection result, high-low response simultaneous existence
As can be seen from the data in the table above,
(1) when the Rv3121 protein antigen is used alone, the effect of detecting mycobacterium tuberculosis is achieved to a certain extent;
(2) aiming at the false negative detection result which cannot be diagnosed when ESAT-6 and CFP10 antigens are used for combined detection, the antigen of Rv3121 is matched for combined immunodetection, so that a more accurate detection result can be obtained, and the detection rate of diseases is improved;
(3) negative control (another randomly selected known protein fragment Rv2327 derived from Mycobacterium tuberculosis, and the nucleotide sequence and the amino acid sequence of the negative control are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4) can hardly be applied to clinical detection.
Finally, it should be noted that the above examples are only for those skilled in the art to understand the essence of the present invention, and should not be used as a limitation to the scope of the present invention.
Claims (3)
- The application of the Rv3121 protein antigen in the preparation of a mycobacterium tuberculosis detection kit is as follows:the Rv3121 protein antigen and the existing mycobacterium tuberculosis detection antigen are cooperatively combined, so that the accuracy of the existing antigen immunity detection is improved,the existing mycobacterium tuberculosis detection antigen is ESAT-6 antigen and CFP10 antigen which are derived from mycobacterium tuberculosis,the amino acid sequence of the Rv3121 protein antigen is shown in SEQ ID NO. 2.
- 2. A multi-antigen combined type Mycobacterium tuberculosis detection kit containing an Rv3121 protein antigen, the kit comprising,(1) detecting an effective amount of Rv3121 protein antigen, and detecting an effective amount of other antigens;(2) necessary detection reagents and color reagents;the other antigens are ESAT-6 antigen and CFP10 antigen derived from mycobacterium tuberculosis,the amino acid sequence of the Rv3121 protein antigen is shown in SEQ ID NO. 2.
- 3. The kit according to claim 2, wherein the Rv3121 protein antigen at a detectably effective amount concentration is an Rv3121 protein antigen at a concentration of 60 ug/ml.
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| CN109406776B (en) * | 2017-08-18 | 2022-02-11 | 中国科学院生物物理研究所 | Protein Rv2818c for specific detection of mycobacterium tuberculosis infection |
| CN109406777B (en) * | 2017-08-18 | 2022-02-18 | 中国科学院生物物理研究所 | Protein Rv2824c for specific detection of mycobacterium tuberculosis infection |
| CN111574600A (en) * | 2020-05-08 | 2020-08-25 | 华南农业大学 | Mycobacterium tuberculosis PPE10 protein and preparation method and application thereof |
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