CN115873805A - Mycobacterium tuberculosis secretory protein LprA monoclonal antibody and application thereof - Google Patents
Mycobacterium tuberculosis secretory protein LprA monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention discloses a mycobacterium tuberculosis secretory protein LprA monoclonal antibody and application thereof. The hybridoma cell strain is a mouse hybridoma cell ZYS-CS-01, the mouse hybridoma cell ZYS-CS-01 secretes a monoclonal antibody specifically binding with LprA protein, and the preservation number of the monoclonal antibody in the China general microbiological culture Collection center is CGMCC No.18884. The invention also discloses a monoclonal antibody secreted and generated by the hybridoma cell strain. The invention discovers that the LprA is a potential secretory protein for identifying mycobacterium strains for the first time, and utilizes the LprA protein as an antigen to prepare a monoclonal antibody aiming at the LprA protein and further establishes a double-antibody sandwich ELISA method based on the LprA to identify the strains of mycobacterium positive culture solution, thereby realizing the rapid identification of NTM.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a mycobacterium tuberculosis secretory protein LprA monoclonal antibody and application thereof.
Background
Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium Tuberculosis (MTB), is the first cause of death of a single infectious source, and is also the 13 th leading cause of death worldwide. According to the WHO estimation, the latent tuberculosis infection population in the world is nearly 20 hundred million. In 2020, 987 thousands of tuberculosis patients are newly added all over the world.
Nontuberculous Mycobacteria (NTM) refers to a general term for other Mycobacteria in the genus Mycobacterium except for Mycobacterium Tuberculosis Complex (MTC) and Mycobacterium leprae, and is also called environmental Mycobacteria. Nontuberculous mycobacteria disease (NTM disease) refers to a disease that causes a lesion of a relevant tissue or organ due to infection with NTM. NTM is an environmental pathogenic bacterium, is ubiquitous all over the world, is most common in soil and water, and is most closely related to human infection. According to the epidemiological development law, with the increasing control degree of TB in one country or region, the proportion of NTM diseases in mycobacteria-related diseases will gradually increase. In recent years, NTM disease is rapidly increasing and has become one of the important public health problems threatening human health.
The NTM disease is difficult to diagnose, and the breakthrough of the laboratory diagnosis technology is the key. The existence and the increasing incidence of NTM diseases increase the difficulty and the complexity of tuberculosis disease control. Clinical symptoms and pathological changes and imaging characteristics of the NTM disease have many similarities with TB, and the clinical symptoms and pathological changes and imaging characteristics of the NTM disease are usually difficult to identify and are always in an underestimated or even overlooked state. Clinicians often diagnose tuberculosis based on the smear "acid-fast bacillus positive", ignoring differential diagnosis for NTM disease. 15% -20% of smear positive tuberculosis patients in the southeast coastal region of China are identified as NTM diseases in the follow-up. Moreover, most NTM strains are naturally resistant to common antituberculosis drugs, so that the NTM disease is easy to be misdiagnosed as drug-resistant tuberculosis clinically. Despite the problems described above, techniques for identification of mycobacterial species are very poor at home and abroad and the prior art relies on the presence of bacteria in specimens. Some techniques require that the bacteria be identified after they have been cultured, and are therefore very time consuming, requiring at least one week for fast growing Mycobacteria (RGM) and 1-2 months for Slow Growing Mycobacteria (SGM). For the 'bacteria negative' patients with no bacteria found in the specimen, no technology can realize the differential diagnosis of tuberculosis and NTM diseases at present, and the identification of the strains of mycobacteria is more impossible. In clinical practice, NTM patients who have not developed a strain identification or who are waiting for identification results, and who are myconegative, are treated in most cases as TB and are treated with an ineffective anti-tuberculosis regimen.
Therefore, the differential diagnosis method which has higher specificity, is rapid to research and develop and can distinguish the NTM disease and the tuberculosis has important significance and wide application prospect for the screening, diagnosis and treatment of the NTM disease, the control of the tuberculosis and the avoidance of the interference of the NTM to the diagnosis of the tuberculosis.
Disclosure of Invention
The technical problem to be solved by the invention is how to identify the mycobacterium tuberculosis and/or the nontuberculous mycobacteria, distinguish the nontuberculous mycobacteria from the mycobacterium tuberculosis and/or detect the LprA protein. The technical problem to be solved is not limited to the technical subject described, and other technical subject not mentioned herein may be clearly understood by those skilled in the art through the following description.
In order to solve the technical problems, the invention firstly provides a hybridoma cell strain which is a mouse hybridoma cell ZYS-CS-01, wherein the mouse hybridoma cell ZYS-CS-01 secretes a monoclonal antibody specifically binding with LprA protein, and the preservation number of the hybridoma cell strain in China general microbiological culture Collection center is CGMCC No.18884.
The invention also provides application of the LprA protein as an antigen of an antibody which is specifically combined with the LprA protein in identifying mycobacterium tuberculosis and/or nontuberculous mycobacteria, identifying and distinguishing nontuberculous mycobacteria and mycobacterium tuberculosis, identifying and distinguishing nontuberculous mycosis and tuberculosis, diagnosing or screening tuberculosis, and diagnosing or screening nontuberculous mycosis, wherein the amino acid sequence of the LprA protein is SEQ ID No.1.
The lprA protein is derived from Mycobacterium Tuberculosis (MTB).
The LprA protein can be a fusion protein with a His tag, namely recombinant protein LprA.
In the above application, the application may include any one of the following:
c1 Immunizing a mouse with the LprA protein as an immunogen, fusing splenocytes of the immunized mouse with mouse myeloma cells, and screening hybridoma cells capable of secreting a monoclonal antibody that specifically binds to the LprA protein;
c2 Preparing a monoclonal antibody specifically binding to the lprA protein by using the hybridoma cells of C1);
c3 Using the LprA protein as immunogen to immunize animals, and detecting to obtain polyclonal antibody capable of specifically binding to the LprA protein.
The animal may be a rabbit (e.g., a Japanese white rabbit), a mouse (e.g., a Balb/c mouse), a sheep, or a horse, but is not limited thereto.
The invention also provides a monoclonal antibody (namely the monoclonal antibody specifically binding with the LprA protein) secreted and generated by the hybridoma in the hybridoma cell strain ZYS-CS-01 or C1).
The monoclonal antibody secreted by the hybridoma cell strain ZYS-CS-01 is an anti-H37 Rv LprA mouse monoclonal antibody named as LprA-07.
The invention also provides any one of the following applications of the monoclonal antibody:
a1 Use for the identification of Mycobacterium tuberculosis or for the preparation of a product for the identification of Mycobacterium tuberculosis;
a2 Use in the identification of non-tuberculous mycobacteria or for the preparation of a product for identifying non-tuberculous mycobacteria;
a3 Use in or for the manufacture of a product for the differential discrimination between non-tuberculous mycobacteria and mycobacterium tuberculosis;
a4 Use in or for the manufacture of a product for the differential discrimination between nontuberculous mycobacteriosis and tuberculosis;
a5 Use in the diagnosis or screening of tuberculosis or for the preparation of a product for the diagnosis or screening of tuberculosis;
a6 Use in the diagnosis or screening of non-tuberculous mycobacteria disease or in the manufacture of a product for use in the diagnosis or screening of non-tuberculous mycobacteria disease;
a7 Application in detecting LprA protein or preparing a product for detecting the LprA protein;
a8 Use) in the manufacture of a product for binding LprA protein.
The product may be a reagent, kit, chip or strip.
The nontuberculous mycobacterium described herein may be a Slow Growing Mycobacterium (SGM) or a fast growing mycobacterium (RGM).
Further, the slow growing Mycobacterium may be Mycobacterium marinum (Mycobacterium marinum), mycobacterium thuringiensis (Mycobacterium szulgarii), mycobacterium gordoniae (Mycobacterium gordonae), or Mycobacterium kansasii (Mycobacterium kansasii).
The Mycobacterium fast-growing may be Mycobacterium abscessus (Mycobacterium abscessus) or Mycobacterium fortuitum (Mycobacterium fortuitum).
The invention also provides a reagent or a kit containing the monoclonal antibody, wherein the reagent or the kit has at least one of the following purposes:
b1 Identification of Mycobacterium tuberculosis and/or nontuberculous mycobacteria;
b2 To identify and distinguish between non-tuberculous mycobacteria and tuberculous mycobacteria;
b3 To identify and distinguish between nontuberculous mycobacteriosis and tuberculosis;
b4 Diagnosis or screening for tuberculosis;
b5 Diagnosis or screening for nontuberculous mycobacterial disease;
b6 Detecting the LprA protein;
b7 Bind LprA protein.
The kit can be a double-antibody sandwich ELISA kit.
Further, the kit also comprises a polyclonal antibody which specifically binds to the LprA protein.
The polyclonal antibody specifically binding to the LprA protein can be an anti-H37 Rv LprA rabbit polyclonal antibody.
The preparation method of the anti-H37 Rv LprA rabbit polyclonal antibody can comprise the following steps:
(1) Immunizing a rabbit: for the first immunization, lprA protein (immunogen) as an antigen and an equal volume of complete Freund's adjuvant are prepared into an emulsifier, and the emulsifier is injected into the back by subcutaneous multiple points, wherein each immunization dose is 500 mu g. The boosting immunization is carried out once every 2 to 3 weeks, the immunogen (500 mu g) with the same dose and incomplete Freund's adjuvant with the same volume are prepared into an emulsifier, and the back is injected subcutaneously in multiple points. Serum titers were determined after four immunizations. Killing rabbits to obtain blood for standby if the serum titer is qualified (the serum ELISA titer is more than or equal to 1,000); if the titer is not qualified, the immunization is carried out again to determine the serum titer, and the rabbit is killed to take blood for standby.
(2) One week after the last immunization, blood is taken from the marginal veins of the rabbit ears to determine the antibody titer, and serum is separated to obtain the rabbit polyclonal antibody against H37Rv LprA.
The immunogen LprA protein can be purified recombinant protein LprA (His-tagged LprA protein).
Further, the kit can also comprise an enzyme-labeled secondary antibody (such as goat anti-rabbit IgG-HRP), a confining liquid, a diluent, a washing liquid, a developing liquid and/or a stop solution.
The invention also provides a conjugate comprising an antibody according to any of the invention, and a detectable label linked to the antibody; in particular, the detectable label may be selected from enzymes (e.g. horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g. acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (e.g. fluorescein or fluorescent protein), radionuclides or biotin.
The invention also provides a method for identifying mycobacterium tuberculosis and/or nontuberculous mycobacteria, which comprises identifying mycobacterium tuberculosis and/or nontuberculous mycobacteria using the monoclonal antibody described in any one of the text or the kit.
Further, the method can be a double-antibody sandwich ELISA method, and the method comprises the step of judging whether the sample to be detected contains mycobacterium tuberculosis or nontuberculous mycobacterium or whether the sample to be detected is mycobacterium tuberculosis or nontuberculous mycobacterium according to the detection result of the double-antibody sandwich ELISA method.
The invention also provides a method of detecting LprA protein, the method comprising detecting LprA protein using a monoclonal antibody as described herein or the kit.
The detection of the LprA protein can be the detection of whether the LprA protein exists in a sample to be detected and/or the detection of the content of the LprA protein in the sample to be detected.
In the above method, the method may be a double antibody sandwich ELISA method.
The double-antibody sandwich ELISA method is used for detecting whether a sample to be detected contains an antigen to be detected (namely LprA protein).
Further, the double antibody sandwich ELISA method comprises the following steps:
(1) Coating a monoclonal antibody described herein (i.e., a monoclonal antibody that specifically binds LprA protein) onto a solid support;
(2) Adding a sample to be detected;
(3) Adding a polyclonal antibody that specifically binds LprA protein as described herein;
(4) Adding enzyme-labeled secondary antibody;
(5) Adding a substrate for enzyme reaction to react;
(6) Judging whether the sample to be detected contains the LprA protein according to whether a positive reaction exists or not, or calculating the content of the LprA protein in the sample to be detected according to the amount of a reaction product.
Further, the monoclonal antibody may be monoclonal antibody lprA-07.
The sample to be detected can be a tissue sample or a cell sample, or a homogenate thereof, or a lysate thereof.
The enzyme-labeled secondary antibody can be goat anti-rabbit IgG-HRP secondary antibody.
The substrate of the enzyme reaction can be TMB color development liquid.
The positive reaction can be judged by measuring OD 450.
The coating concentration of the monoclonal antibody in the step (1) can be 1 mug/mL.
The final concentration of polyclonal antibody that specifically binds to lprA protein added in step (3) can be 0.5. Mu.g/mL.
The invention also provides a method for preparing the monoclonal antibody specifically binding to the LprA protein, which comprises the step of culturing the hybridoma cell strain ZYS-CS-01 or C1).
Further, the method for preparing the monoclonal antibody specifically binding to the LprA protein comprises the following steps: immunizing a Balb/c mouse by using the purified recombinant protein LprA as an antigen, fusing a spleen cell of the mouse with a myeloma cell SP2/0 cell to prepare a hybridoma cell, detecting a cell supernatant by using an indirect ELISA method, screening to obtain a positive clone, screening to obtain the hybridoma cell capable of secreting a monoclonal antibody specifically binding to the LprA after three times of subcloning, culturing the hybridoma cell, collecting the culture supernatant, and purifying to obtain the monoclonal antibody specifically binding to the LprA protein.
The monoclonal antibodies of the invention may also be prepared by various methods known in the art, for example, by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector, followed by transfection of host cells, culturing of the transfected host cells under specific conditions, and expression of the antibody of the present invention. The host cell, the expression vector, a method for introducing the expression vector into the host cell, and a method for isolating and purifying the antibody, which are conventional in the art, can be selected by those skilled in the art as needed.
In one embodiment of the present invention, the double antibody sandwich ELISA method comprises the steps of:
(1) Coating: mu.L of diluted anti-H37 Rv LprA mouse monoclonal antibody LprA-07 (final concentration of 1. Mu.g/mL) was added to each well, the plate was sealed, and the plate was coated overnight at 4 ℃.
(2) The next day, the plate was removed, washed 3 times with 1 XPBST, 300. Mu.L/well, and blotted dry the last time.
(3) And (3) sealing: add 200. Mu.L of 3% BSA blocking solution per well and block by shaking horizontally at room temperature for 1h.
(4) Wash 3 times with 1 XPBST, 300. Mu.L/well, and beat dry the last time.
(5) Sample adding: mu.L of concentrated culture filtrate was added to each well and 3 wells were repeated for each sample, while positive controls (i.e., 0.1. Mu.g/mL of H37Rv LprA immunogenic protein (recombinant protein LprA), negative controls (0.1. Mu.g/mL of BSA) and blank controls were set and incubated on a horizontal shaker at room temperature for 2H.
(6) Wash 3 times with 1 XPBST, 300. Mu.L/well, and beat dry the last time.
(7) mu.L of diluted anti-H37 Rv LprA rabbit polyclonal antibody (final concentration 0.5. Mu.g/mL) was added to each well and blocked by shaking horizontally at room temperature for 1H.
(8) After washing with 1 XPBST for 3 times and draining, 100. Mu.L of goat anti-rabbit IgG-HRP secondary antibody (Kyoto King Kogyo Biotech, ltd., product No. ZB-2301) was added to each well and incubated at room temperature for 1 hour.
(9) Washing with 1 XPBST for 3 times, drying by beating, adding 100 mu L of freshly prepared TMB developing solution into each hole (the volume ratio of the solution A to the solution B is 1.
(10) 2% by weight of 50. Mu.L per well 2 SO 4 Stopping reaction, measuring OD by enzyme-linked immunosorbent assay 450nm And (4) processing the light absorption value.
Further, the preparation method of the culture filtrate is as follows: freezing the test strain, and adding 1: inoculating to 5mL 7H10 medium (containing 10% of OADC) at a ratio of 100, and culturing to OD 600 And (5) centrifuging the bacterium liquid at 3000rpm for 10min, wherein the supernatant is culture filtrate, and is 0.6-0.8.
The double-antibody sandwich ELISA method based on LprA established by the invention can be used for identifying the strains of the positive culture solution of mycobacterium: by OD 450 Determined as a critical value, OD 450 >0.5 is the mycobacterium tuberculosis; OD 450 0.2-0.5 for Slow Growing Mycobacteria (SGM) including Mycobacterium marinum, mycobacterium sulcata, mycobacterium gordonii and Mycobacterium kansasii; since M.fastgrowth did not contain LprA, the OD value was similar to that of the negative control 3% BSA.
The uses and methods of the invention may be for disease diagnostic purposes, disease prognostic purposes and/or disease therapeutic purposes, as well as for non-disease diagnostic purposes, non-disease prognostic purposes and non-disease therapeutic purposes; their direct purpose may be to obtain information on the outcome of a disease diagnosis, prognosis of a disease and/or intermediate outcome of a disease treatment, and their direct purpose may be non-disease diagnosis, non-disease prognosis and/or non-disease treatment.
The secreted proteins in the culture filtrates of Mycobacterium Tuberculosis (MTB) and the most frequently isolated pathogenic NTM in clinic (including 3 SGM: M. Kansaii, M.avium, M.intracellulare strain; 2 RGM: M.fortuitum and M.abscessus) standard strains were identified by the Label free proteomics technique, and found: (1) Both MTB and m.kansasii (mycobacterium kansasii) express lipoprotein LprA at high levels; (2) The M.avium complex (including M.avium and M.intracellulare) does not express LprA; (3) Neither Mycobacterium fortuitum nor Mycobacterium abscessus secrete LprA. In the alignment of homologous sequences to lipoproteins, (1) the general absence of the LprA homologous sequence in RGM was found; (2) In SGM, the avian intracellular mycobacterial complex does not encode the LprA protein; (3) Common SGMs such as m.kanasaii, m.marinum (mycobacterium marinum), and m.gordonae (mycobacterium gordonae) have LprA homology proteins, and all of them have LprA homology of about 80% with MTB. However, the amino end of the LprA protein is close to 50 amino acids, and the difference is very obvious among different SGM strains, and the homology is only between 45% and 50%. Thus, lprA is a secreted protein identified as a potential mycobacterium species.
Currently, the MPT64 secretory protein is mainly clinically used as a target point to diagnose the mycobacterium tuberculosis in culture positive filtrate supernatant. However, some of the clinical isolates of M.tuberculosis were missed because MPT64 was deleted or mutated and could not be detected. Therefore, there is a need to develop a new specific secretory antigen of mycobacterium tuberculosis as a target in clinical practice to supplement the deficiencies of the existing diagnostic techniques. The inventor of the application finds that the LprA is a potential secretory protein for identifying mycobacterium species for the first time through extensive and intensive research, prepares a monoclonal antibody and a polyclonal antibody aiming at the LprA protein by using the LprA protein as an antigen, can be specifically combined with the LprA protein, has good specificity, further establishes a double-antibody sandwich ELISA method based on the LprA to identify the mycobacterium positive culture solution, and realizes the rapid identification of mycobacterium tuberculosis and nontuberculous mycobacterium.
Deposit description
Suggested classification nomenclature: mouse hybridoma cells
Reference biological material (strain): ZYS-CS-01
The preservation unit is as follows: china general microbiological culture Collection center
The preservation unit is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No.1 Hospital No.3 of Beijing market facing Yang district
The preservation date is as follows: 12 and 23 months in 2019
The registration number of the collection center: CGMCC No.18884
Drawings
FIG. 1 shows the SDS-PAGE result of the recombinant LprA protein purified in example 1. Left side: peak profile of LprA (m. tuberculosis) after purification on ion exchange column; right side: coomassie Brilliant blue chromosome of SDS-PAGE of LprA proteins of different Mycobacterium species: m. marker;1.m. tuberculosis;2.m. kanasaii; gordonae; marinum; szulgai, m.
FIG. 2 is an SDS-PAGE electrophoresis of anti-H37 Rv LprA rabbit polyclonal antibody in example 2. Wherein: 1 is marker;2 is 1# anti-H37 Rv LprA rabbit polyclonal antibody; 3 is 2# anti-H37 Rv LprA rabbit polyclonal antibody.
FIG. 3 is a SDS-PAGE analysis of the purification of an anti-H37 Rv LprA murine monoclonal antibody in example 3.
FIG. 4 shows the confirmation of the specificity of the polyclonal antibody by Western blot in example 4. Wherein: in FIG. 4, A is the verification result of the 1# polyclonal antibody; in FIG. 4, B is the result of the verification of the 2# polyclonal antibody.
FIG. 5 shows the confirmation of the specificity of the monoclonal antibody by Western blot in example 4. Wherein: FIG. 5A shows that anti-his-tag mouse monoclonal antibodies have demonstrated different SGM recombinant LprA protein specificities; in FIG. 5, B is an anti-H37 Rv LprA mouse monoclonal antibody (LprA-07) to verify the specificity of different SGM recombinant LprA proteins; in FIG. 5, C is the anti-CFP-10 antibody detection M.tuberbulosi H37Rv bacterial liquid protein; in FIG. 5, D is anti-H37 Rv LprA mouse monoclonal antibody to detect different SGM wild strain filtrate proteins.
Detailed Description
The secreted proteins in the culture filtrate of Mycobacterium Tuberculosis (MTB) and the most commonly isolated pathogenic NTM (including 3 SGM: M. Kansaii, M.avium, M.intracellulare, 2 RGM: M.fortuitum and M.abscessus) standard strain were identified by the Label free proteomics technique, and found that: (1) Both MTB and m.kansas ii (mycobacterium kansasii) express lipoprotein LprA at high levels; (2) The M.avium complex (including M.avium and M.intracellulare) does not express LprA; (3) Both Mycobacterium sporangium and Mycobacterium abscessus do not secrete LprA. In the alignment of homologous sequences to lipoproteins, it was found that (1) the RGM is generally deficient in the LprA homologous sequence; (2) In SGM, the avian intracellular mycobacterial complex does not encode the LprA protein; (3) Common SGMs such as m.kanasaii, m.marinum (mycobacterium marinum), and m.gordonae (mycobacterium gordonae) have LprA homology proteins, and all of them have LprA homology of about 80% with MTB. However, the amino end of the LprA protein is close to 50 amino acids, and the difference is very obvious among different SGM strains, and the homology is only between 45% and 50%. Thus, lprA is a secreted protein identified as a potential mycobacterium species. The invention aims to establish a double-antibody sandwich ELISA method based on LprA to carry out strain identification on the positive culture solution of mycobacterium and realize the rapid differential diagnosis of NTM.
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
The pET28a vector in the following examples is a Novagen product, cat # 69864-3CN.
The pMV261 vector in the following examples is a product of Shanghai Lingmei bioengineering, inc., cat No. LM-8098.
The white rabbit of Japanese big ear in the following examples is a product of Beijing Jinmuyang laboratory animal raising LLC, and has a body weight of about 2-2.5 kg.
Mycobacterium tuberculosis (Mycobacterium tuberculosis), mycobacterium kansasii (Mycobacterium kansasii), mycobacterium gordonii (Mycobacterium gordonae), mycobacterium marinum (Mycobacterium marinum), mycobacterium suger (Mycobacterium szullgai), mycobacterium Smegmatis (Mycobacterium Smegmatis), mycobacterium abscessus (Mycobacterium abscessus) and Mycobacterium fortuitum (Mycobacterium fortuitum) in the following examples are all strains of the American Type Culture center (American Type Culture Collection, ATCC) with the accession numbers of Mycobacterium tuberculosis: ATCC27294, and the accession numbers of Mycobacterium kansasii are: ATCC12478, accession number of Mycobacterium gordonii: ATCC14470, accession number of Mycobacterium marinum: ATCC927, accession numbers of Mycobacterium Sulgaricus are: ATCC35799, accession number of mycobacterium smegmatis: ATCC19420, accession number of mycobacterium abscessus is: ATCC19977, accession number of Mycobacterium fortuitum is: ATCC6841.
Example 1 expression and purification of recombinant protein LprA
The invention uses the LprA protein of mycobacterium tuberculosis standard bacteria H37Rv as antigen to prepare the monoclonal antibody aiming at the LprA protein for the first time, and the preparation method of the antigen protein (recombinant protein LprA) is as follows:
1. construction of recombinant plasmid pET28a-LprA
The amino acid sequence of the LprA protein (derived from mycobacterium tuberculosis standard bacteria H37 Rv) is shown as SEQ ID No.1. The nucleotide sequence of the gene (namely the LprA gene) for encoding the Mycobacterium tuberculosis LprA protein is shown as SEQ ID No. 2. The DNA fragment between recognition sites of restriction enzymes NcoI and XhoI of a pET28a vector is replaced by a DNA molecule with the nucleotide sequence shown in SEQ ID No.2 in a sequence table, a gene of a His tag (consisting of 6 histidine residues) is added to the 3' end of the DNA molecule, and other nucleotide sequences on the pET28a vector are kept unchanged to obtain a recombinant plasmid pET28a-LprA (M.tuberculosis) for expressing a fusion protein with the His tag, namely the recombinant protein LprA (M.tuberculosis).
Meanwhile, the LprA gene of Mycobacterium kansasii (SEQ ID No. 4), the LprA gene of Mycobacterium gordonii (SEQ ID No. 6), the LprA gene of Mycobacterium marinum (SEQ ID No. 8) and the LprA gene of Mycobacterium sulci (SEQ ID No. 10) were cloned into the pET28a vector, respectively, to obtain recombinant vectors pET28a-LprA (M.kansas), pET28a-LprA (M.gordonae), pET28a-LprA (M.marinaum) and pET28a-LprA (M.szulgai), according to the above method.
Wherein: the amino acid sequence and the nucleotide sequence of the LprA protein of the M.kansasii are respectively shown as SEQ ID No.3 and SEQ ID No. 4; the amino acid sequence and the nucleotide sequence of the Gordona (M.gordonae) LprA protein are respectively shown as SEQ ID No.5 and SEQ ID No. 6; the amino acid sequence and the nucleotide sequence of mycobacterium marinum (M.marinum) LprA protein are respectively shown as SEQ ID No.7 and SEQ ID No. 8; the amino acid sequence and the nucleotide sequence of the protein LprA of the mycobacterium thuringiensis (M.szullgai) are respectively shown as SEQ ID No.9 and SEQ ID No. 10.
2. Expression of recombinant protein LprA
Mycobacterium tuberculosis and 4 different SGM recombinant plasmids: coli BL21 (DE 3) was transformed with pET28a-LprA (pET 28a-LprA (M.tuboculosis), pET28a-LprA (M.kansasii), pET28a-LprA (M.gordonae), pET28a-LprA (M.marinum), and pET28a-LprA (M.szulgai)), to obtain recombinant bacteria, which were named BL21/pET28a-LprA (M.tuboculosis), BL21/pET28a-LprA (M.kansasii), BL21/pET28a-LprA (M.gordonae), BL21/pET28a-LprA (M.marinum), BL21/pET28a-LprA (M.szulzulgaii), and M.szulzulgai), respectively.
Mixing the recombinant bacteria in a proportion of 1:50 were transferred to 20mL of LB medium resistant to kanamycin and cultured overnight at 37 ℃ and 200 rpm.
On the following day, the ratio of 1: transferring the strain into 1L of LB culture medium with kanamycin resistance at a ratio of 100 to obtain the bacterial OD 600nm When the concentration is 0.8-1.0, IPTG (isopropyl-. Beta. -D-thiogalactoside) with a final concentration of 0.5mM is added, and induction is carried out at 16 ℃ for 16-18 hours, and cell precipitation is collected by centrifugation at 5000rpm for 20min.
3. Purification of recombinant proteins
(1) Nickel column affinity chromatography
(1) Each 1L of the suspension was finally resuspended in 80mL of protein purification buffer, and 1% of PMSF (phenylmethylsulfonyl fluoride) was added thereto and mixed well.
(2) And (3) carrying out ultrasonic crushing with the power of 200W, working for 5s and intermittent for 3s for 15min. The whole process is carried out on ice until the bacterial liquid becomes clear.
(3) After the ultrasonic treatment, the bacterial liquid is transferred to a 50mL centrifuge tube, centrifuged at 12000rpm and 4 ℃ for 80min. The protein supernatant was transferred to a new 50mL centrifuge tube and placed on ice until use.
(4) And adding the cracked protein into a nickel column, and reserving a sample in the middle.
(5) The heteroproteins were washed, with 20CV protein wash buffer (30 mM imidazole), leaving a sample in between.
(6) Eluting protein: eluting the target protein by 10CV of imidazole buffer solution (60mM, 100mM and 300mM) with gradient concentration to obtain the recombinant protein LprA solution.
Wherein the protein purification buffer comprises the following components: 25mM, pH 8.0Tris-HCl +150mM NaCl.
(2) Ion exchange chromatography
Further purifying the recombinant protein LprA solution obtained in the step (1), wherein the steps are as follows:
(1) equilibration of ion exchange chromatography: firstly, washing the ion exchange column with a high-salt buffer solution to remove the mixed protein bound on the column, after the conductance reaches 80-90%, balancing the ion exchange column with a low-concentration salt buffer solution, and when the conductance reaches 10%, starting protein loading.
(2) Loading and eluting the recombinant protein: selecting an anion exchange Q column, loading by adopting a low-salt buffer solution, and then gradually increasing the salt concentration for elution. The gradient range of the high-salt buffer solution is 20-80%, and the elution volume is 25-30CV.
(3) The individual proteins corresponding to the UV280 peak were collected: and (3) taking 10 mu L of sample from a 96-hole collection plate, adding 2 mu L of 5 Xprotein loading buffer solution, heating the solution in a metal bath at 100 ℃ for 5min, performing SDS-PAGE protein gel electrophoresis and Coomassie brilliant blue staining and decoloring, and determining the collection range of the sample according to the position of a protein strip on the protein gel.
(4) Selecting the position of the target protein band, adding a 3Kd concentration tube for centrifugal concentration to obtain the purified recombinant protein LprA, wherein the SDS-PAGE result of the purified recombinant protein LprA is shown in figure 1.
Respectively obtaining the purified recombinant protein LprA (M.tuberculosis), the recombinant protein LprA (M.kansas), the recombinant protein LprA (M.gordonae), the recombinant protein LprA (M.marinum) and the recombinant protein LprA (M.szulgai).
Recombinant protein lprA (M.tuberculosis) is abbreviated as recombinant protein LprA.
Example 2 preparation of anti-H37 Rv LprA Rabbit polyclonal antibody
1. Immunization of animals
(1) 2 Japanese big-ear white rabbits were immunized at a dose of 500. Mu.g per immunization, and the immunogen (purified recombinant protein LprA prepared in example 1) was first immunized with an equal volume of complete Freund's adjuvant as an emulsifier, and injected subcutaneously at multiple points on the back.
(2) Preparing the same dose of immunogen and the same volume of incomplete Freund's adjuvant into an emulsifier at intervals of 2-3 weeks, performing subcutaneous multi-point injection on the back to complete the second immunization, sequentially completing the third immunization and the fourth immunization according to a two-immunization method, and measuring the serum titer after the fourth immunization.
(3) If the serum titer is qualified (serum ELISA titer)>1, 64,000) killing rabbits and taking blood for later use; and if the titer is not qualified, immunizing again to determine the titer of the serum, killing the rabbits and taking blood for later use.
2. Blood sampling and potency detection
(1) One week after the last immunization, 3-4 mL of blood is taken from the vein of the rabbit ear edge, and after standing overnight at 4 ℃,1500g of blood is centrifuged for 15min to separate the upper layer serum for standby detection.
(2) An appropriate amount of the protein for detection (i.e., the purified recombinant protein lprA prepared in example 1) was diluted to 0.1. Mu.g/mL, 1. Mu.g/mL, and 5. Mu.g/mL with the coating buffer, 100. Mu.L was added to each well of the 96-well plate, the plate was tapped to mix the samples, the samples were sealed with a preservative film, and the samples were coated overnight at 4 ℃.
(3) Washing the ELISA plate 1 time by using a washing solution according to the volume of 300 mu L/hole, and patting the ELISA plate dry.
(4) Then the enzyme label plate is sealed by sealing liquid according to the ratio of 300 mu L/hole, and the enzyme label plate is sealed for 1 hour at room temperature.
(5) The microplate was washed 2 times with a washing solution at 300. Mu.L/well, and the sample diluted in a gradient (i.e., serum of an immunized animal) and a sample diluent were added to the sample at 100. Mu.L/well, and simultaneously 100. Mu.L/well of a detection antibody (IGL Co., ltd., cat.: GGFC-90P) were added, followed by incubation at room temperature for 2 hours.
(6) Washing the ELISA plate 3 times with a washing solution at 300. Mu.L/well, adding a developing solution at 200. Mu.L/well (volume ratio of solution A to solution B is 1).
(7) Adding stop solution into 50 mu L/hole to stop reaction; the detection is carried out by a microplate reader, and the measurement wavelength is 450nm.
3. Purification of anti-H37 Rv LprA rabbit polyclonal antibody
The purification process included 2 parts, proteinA affinity purification and antigen affinity purification.
3.1 affinity purification of ProteinA
(1) The ProteinA affinity column was rinsed with ultra pure water and then equilibrated with equilibration buffer.
(2) And (3) loading the treated rabbit serum into an affinity chromatographic column, and leaching with an equilibrium buffer solution after loading.
(3) Eluting with elution buffer, collecting elution peak, and neutralizing with Tris buffer.
3.2 affinity purification of antigen
(1) Washing the antigen affinity chromatographic column with ultrapure water for 5CV, and then balancing with a balancing buffer solution;
(2) Loading the protein sample subjected to protein A affinity purification to an antigen affinity chromatographic column, and leaching with a balance buffer solution after loading;
(3) Eluting with elution buffer, collecting the elution peak, and neutralizing with Tris buffer.
(4) The eluted sample was desalted into PBS (ph 7.4) to obtain a 1# anti-H37 Rv LprA rabbit polyclonal antibody (abbreviated as 1# polyclonal antibody) and a 2# anti-H37 Rv LprA rabbit polyclonal antibody (abbreviated as 2# polyclonal antibody), respectively, and the purification electrophoretogram of the rabbit polyclonal antibody is shown in fig. 2.
Example 3 preparation of anti-H37 Rv LprA monoclonal antibody
1. Immunization of animals
(1) 5 Balb/c mice (the body weight is about 16-20 g) with the age of 6-8 weeks are immunized, and the immunization dose of each mouse is 50 mu g of protein (recombinant protein LprA). First immunization the immunogen (purified recombinant protein lprA prepared in example 1) was emulsified with an equal volume of complete Freund's adjuvant and injected subcutaneously in multiple spots per abdomen. Blood serum is collected by cutting tail before immunization and is preserved at the temperature of minus 80 ℃ for later use.
(2) After 2-3 weeks of first immunization, cutting the tail and taking blood to precipitate serum, and storing at-80 ℃ for later use.
(3) And (3) secondary immunization: 50 mu g of protein immunogen and an equal volume of incomplete Freund's adjuvant are taken again to prepare an emulsifier, and subcutaneous injection is performed on the abdomen at multiple points. After three immunizations in the same manner, blood was obtained by cutting the tail and collecting the blood, and the serum was stored at-80 ℃ for later use to determine the serum titer.
(4) The mice with qualified titer are injected with 100 mug of protein in the abdominal cavity for boosting, and the spleens of the mice are taken for hybridoma fusion after 3 days of boosting.
2. Blood sampling and potency detection
(1) One week after each immunization, 50-60 mu L of blood is taken from orbital venous plexus of mice, and after standing overnight at 4 ℃, the upper serum is centrifugally separated for later detection.
(2) Coating: an appropriate amount of the test protein (i.e., purified recombinant protein lprA prepared in example 1) was diluted to 5. Mu.g/mL with the coating buffer, 100. Mu.L of the test protein was added to each well of the 96-well plate, the plate was gently tapped to mix the samples, the plate was sealed with a membrane and the plate was coated overnight at 4 ℃.
(3) Washing the ELISA plate with washing solution PBST at 300. Mu.L/well, and patting the plate dry for 2 times.
(4) Then the enzyme label plate is sealed by sealing liquid according to the ratio of 300 mu L/hole, and the enzyme label plate is sealed for 1 hour at room temperature.
(5) After washing the ELISA plate with a washing solution at a concentration of 300. Mu.L/well for 2 times, samples were added, and the samples diluted in a gradient (i.e., serum from an immunized animal) and a sample diluent were added at a concentration of 100. Mu.L/well, and at the same time, 100. Mu.L of a detection antibody (IGL Co., ltd., product number: GGFC-90P) was added to each well, and incubated at room temperature for 2 hours.
(6) The plate was washed with a washing solution at 300. Mu.L/well for 5 times, and a developing solution was added at 100. Mu.L/well and allowed to stand at room temperature for 15min.
(7) Stop solution was added at 50. Mu.L/well to stop the reaction.
(8) And measuring the light absorption value at the wavelength of 450nm by using a microplate reader.
3. Hybridoma cell fusion and monoclonal antibody screening
3.1 preparation of feeder cells
(1) One day prior to fusion, the blank mice were enucleated, bled and killed, and then placed in 75% alcohol for 10min.
(2) Cell acquisition: the 75% alcohol-sterilized mice were transferred to a biosafety cabinet, in an abdominal-up position, and then the skin was carefully cut open using sterile ophthalmic scissors to expose the peritoneum. And then replacing new sterile scissors forceps, cutting a small opening on the peritoneum, sucking the precooled HAT culture medium by using a sterile suction tube, injecting the precooled HAT culture medium into the abdominal cavity of the mouse, repeatedly and gently blowing and beating the culture medium for several times, sucking the culture medium in the abdominal cavity by using a syringe, placing the culture medium in a 50mL sterile centrifuge tube, and repeating the operation for 3-5 times. Centrifuging at 1000rpm for 10min, discarding supernatant, and adding HAT culture medium to resuspend cells.
(3) Plating and culturing: cell counting was performed, and the cell concentration was adjusted to about 5X 10 with HAT medium 5 Per mL and added to a 96 well cell culture plate at 100. Mu.L/well, at 37 ℃ C. 5% CO 2 Cultured overnight under the conditions.
(4) The feeder cells are observed to have a pollution-free condition on the next day, the growth state of the cells is good, the cells are full in shape and good in refractive index, and the requirement of subsequent tests is met.
3.2 preparation of mouse spleen lymphocytes
(1) Preparation of positive serum: after the immune enhancement, the mice with higher immune antibody level pick eyeballs and bleed to die, and meanwhile, whole blood is collected, and serum is collected by centrifugation at 4 ℃ and then stored for later use.
(2) Preparing a cell suspension: after the mice are sacrificed, the mice are soaked in 75% alcohol for 10min, and are placed in a biological safety cabinet upside down for aseptic operation to expose the left abdominal wall and peritoneum.
(3) Separating the spleen: carefully removing connective tissues on the surface of a spleen of a mouse in a visual field, taking out the spleen, placing the spleen in a sterile plate, shearing the spleen by using sterile shearing forceps, then gently grinding the spleen into single cells, standing for 10min, carefully sucking an upper cell suspension to prepare a single cell suspension, then centrifuging for 10min at 1000rpm, and discarding a supernatant to obtain splenic lymphocytes of the mouse for later use.
3.3 cell fusion
(1) SP2/0 myeloma cell preparation: collecting SP2/0 cells with good growth status in logarithmic growth phase before fusion in a 50mL sterile centrifuge tube, horizontal centrifugation at 1000rpm for 5min, adding RPMI 1640 medium containing 10-FBS, gently pipetting to sufficiently resuspend it, repeating the operation once, and finally resuspending into single myeloma cells using RPMI 1640 medium containing 10-FBS.
(2) Taking all splenic lymphocytes of the immunized mice, and carrying out the following steps: 1 ratio and mouse myeloma cells were mixed in a sterile centrifuge tube, centrifuged at 1000rpm for 10min.
(3) After carefully removing the supernatant, the tube was gently shaken to mix the precipitated splenic lymphocytes with mouse myeloma cells uniformly into a loose paste, which was then placed in a 37 ℃ water bath.
(4) To the cell mix pellet was added 1mL of 37 ℃ preheated PEG immediately along the tube wall of the centrifuge tube, while the centrifuge tube was slowly rotated.
(5) The tube was gently shaken for 1min and then allowed to stand for 90s.
(6) Termination of the fusion: slowly adding 1mL of DMEM medium along the tube wall of the centrifuge tube at a constant speed within 1 min; adding 4mL of DMEM culture medium into the centrifuge tube within 1 min; then 10mL of DMEM medium was added within 1min, and after standing at room temperature for 5min, the mixture was centrifuged at 1000rpm for 5min.
(7) Inoculating cells: after discarding the supernatant, adding 6mL HAT culture medium to resuspend the cells; after 5mL HAT culture medium is added into the tube, the tube is gently blown and beaten until the HAT culture medium is suspended to a single cell, and 20mL HAT culture medium is continuously added and fully and uniformly mixed. Inoculating 100. Mu.L/well into 96-well cell culture plates containing feeder cells, incubating at 37 ℃ and 5% CO 2 Culturing in a cell culture box.
(8) The growth of the fused cells was observed daily, and half-exchange was performed 3d after fusion with HAT medium.
3.4 screening of Positive hybridoma cells
(1) When the fused cell clone grows to reach about 1/4-1/3 of the bottom of the hole, detecting the supernatant of the fused cell (namely the culture supernatant of the hybridoma) by using an indirect ELISA method by using an antigen protein (recombinant protein LprA);
(2) And detecting the cell supernatant once every two days for 3 times, transferring the cell clone detected as positive into a 24-hole cell culture plate for amplification culture, and discarding the negative hole.
3.5 subcloning of Positive hybridoma cells
This study subcloned positive hybridoma cells by limiting dilution
(1) When the cell mass of the 24-well plate reaches 25% -50% of the bottom area of a single well, positive wells with good growth state and containing about 100 cells can be selected, HI culture medium (Beijing Yiqiao Shenzhou science and technology Co., ltd.) is supplemented and selected to the total volume of 10mL, 100 μ L per well of 100 μ L in 96-well cell culture plate according to the density of 0.5/100 μ L, 1/100 μ L and 2/100 μ L, and the plate is placed at 37 ℃ and 5% CO 2 Culturing in a cell culture box.
(2) Observing the cell growth state every day, culturing for 5 days, selecting cell hole containing only one cell clone, subcloning again for ELISA detection, subcloning the selected positive hole for at least 3 times to obtain stable passage positive hybridoma cell strain, expanding culture, and freezing and storing the positive hybridoma cell according to the method. Finally, 5 positive monoclonals (hybridoma cell strains) are obtained by screening: MM01, MM02, MM03, MM05 and MM07. The results of ELISA detection of cell culture supernatants after three subclonings are shown in Table 1.
TABLE 1 ELISA test results of cell culture supernatants after three subcloning
3.6 preservation of hybridoma cells
The obtained hybridoma cell strain MM07 capable of secreting monoclonal antibody (anti-H37 Rv LprA mouse monoclonal antibody) capable of specifically binding with LprA protein is named ZYS-CS-01, and the hybridoma cell ZYS-CS-01 is preserved in China general microbiological culture Collection center (CGMCC for short, the address is No.3 of the national institute of microbiology, china institute of sciences, china) in 12 months and 23 days in 2019, and the registration number is CGMCC No.18884.
The monoclonal antibody against H37Rv LprA secreted by the deposited cell strain was designated as LprA-07.
3.7 purification of anti-H37 Rv LprA murine monoclonal antibody
3.7.1 resuscitating and culturing hybridoma cell lines MM01, MM02, MM03, MM05 and MM07
(1) A15 ml sterile centrifuge tube was prepared and 9ml of RPMI 1640 medium was added for future use.
(2) The cells were rapidly thawed in a 37 ℃ water bath within 1 minute after removal from the liquid nitrogen and the cell fluid was transferred to the centrifuge tubes prepared in step (1).
(3) After centrifugation at 1000rpm for 5 minutes, the supernatant was discarded.
(4) The cell pellet was suspended in 3ml of complete medium (RPMI 1640 medium containing 10% fetal bovine serum) and transferred to a culture flask.
(5) Observing the cell state under a microscope, and if the cell covers more than 50% of the bottle bottom area, continuously supplementing 2ml of complete culture medium.
(6) The content of CO in the mouth of the culture flask is 8% at 37 ℃% 2 The cell culture is performed under conditions.
The culture supernatants of the hybridoma cells were collected, centrifuged, and filtered to obtain supernatants of five groups of hybridoma cells (MM 01, MM02, MM03, MM05, and MM 07).
3.7.2 Protein A affinity column purification of antibodies
Washing the Protein A affinity chromatographic column with ultrapure water, and then balancing with a balance buffer solution; the supernatants of the five groups of hybridoma cells (MM 01, MM02, MM03, MM05 and MM 07) were loaded onto the affinity column, and after loading, the column was rinsed with equilibration buffer. Eluting with elution buffer, collecting elution peak, and neutralizing with Tris buffer. Desalting into PBS (pH7.4) resulted in 5 purified anti-H37 Rv LprA murine monoclonal antibodies, the final concentrations and total amounts of anti-H37 Rv LprA murine monoclonal antibodies are shown in Table 2. The results of the purification of the murine monoclonal antibody are shown in FIG. 3.
TABLE 2 final concentration and Total amount of anti-H37 Rv LprA murine monoclonal antibody
Example 4 detection of Mono/polyclonal antibody specificity by Western blot
The different SGMs in this example are Mycobacterium tuberculosis (Mycobacterium tuberculosis), mycobacterium kansasii (Mycobacterium kansasii), mycobacterium gordonae (Mycobacterium gordonae), mycobacterium marinum (Mycobacterium marinum) and Mycobacterium sojae (Mycobacterium szulgai).
The LprA protein of the different SGMs is over-expressed in Mycobacterium smegmatis, and the steps are as follows:
genomic DNAs of the SGMs were extracted, respectively, and the LprA gene of the SGM was amplified. The amplification primer sequences are as follows:
TABLE 3 vector construction primer sequences
Cloning the amplified LprA genes of different SGMs into a pMV261 vector respectively, and transforming Mycobacterium Smegmatis (Mycobacterium Smegmatis) by the obtained recombinant vector to obtain recombinant Mycobacterium Smegmatis (collectively referred to as MS-pMV 261) which overexpresses the LprA genes of different SGMs.
(1) Preparation of different SGM recombinant MS-pMV261 culture filtrate proteins:
different SGM recombinant mycobacterium Smegmatis (Mycolibacillosis) (MS-pMV 261) grow to logarithmic growth phase, and culture supernatant is taken after centrifugation at 10000rpm for 30min at 4 ℃. Filtering with 0.22 μm filter membrane, concentrating with ultrafiltration tube with molecular weight cutoff of 10kDa by 50 times, adding 5 times protein sample buffer solution at a certain proportion, heating with 100 deg.C metal bath for 5min, centrifuging, and loading at 20 μ L/hole.
(2) Recombinant LprA protein of different SGMs: the proteins purified in example 1 (recombinant protein LprA (M.tuberculosis), recombinant protein LprA (M.kansas), recombinant protein LprA (M.gordonae), recombinant protein LprA (M.marinum), and recombinant protein LprA (M.szullgai)) were adjusted to 0.2mg/mL, respectively, 5 Xprotein loading buffer was added in proportion, heated at 100 ℃ for 5min in a metal bath, and loaded at 20. Mu.L/well after centrifugation.
(3) Culture supernatant filtrate proteins of different SGMs: different SGMs grow to logarithmic phase, and culture supernatant is taken after centrifugation at 10000rpm for 30min at 4 ℃. Filtering with 0.22 μm filter membrane, concentrating with ultrafiltration tube with molecular weight cutoff of 3kDa by 50 times, adding 5 × protein sample buffer solution, heating with 100 deg.C metal bath for 5min, centrifuging, and sampling at 20 μ L/well.
(4) Specificity was detected by western blot: different SGM recombinant MS-pMV261 culture filtrate proteins, different SGM recombinant LprA proteins and different SGM culture supernatant filtrate proteins are verified by using an Anti-His-tag mouse monoclonal antibody and a purified Anti-H37 Rv LprA monoclonal/Rabbit polyclonal antibody as primary antibodies and using a HRP-labeled Goat Anti-Rabbit/mouse IgG as secondary antibodies respectively. The anti-H37 Rv LprA monoclonal antibody as a primary antibody is a monoclonal antibody LprA-07 secreted by hybridoma MM07.
The detection results of the antibodies are shown in fig. 4 and 5, fig. 4 is the verification of the specificity of the polyclonal antibody, and it can be seen from fig. 4 that both the polyclonal antibody # 1 and the polyclonal antibody # 2 can recognize the LprA protein of SGM. FIG. 5 shows the monoclonal antibody specificity verification, and it can be seen from FIG. 5 that only M.tuberculosis can be specifically recognized, and other SGMs cannot be detected.
Example 5 detection of SGM culture filtrate specificity by double antibody Sandwich ELISA
The test strains in this example were SGM (slow growing Mycobacteria) and RGM (fast growing Mycobacteria). Among the different SGMs are Mycobacterium tuberculosis (Mycobacterium tuberculosis), mycobacterium kansasii (Mycobacterium kansasii), mycobacterium gordonae (Mycobacterium gordonae), mycobacterium marinum (Mycobacterium marinum) and Mycobacterium sojae (Mycobacterium szulgai). Different RGMs (fast growing Mycobacteria) are Mycobacterium abscessus (Mycobacterium abscessus) and Mycobacterium fortuitum (Mycobacterium fortuitum).
Preparation of culture filtrates for different SGM wild strains and different RGM wild strains: freezing the test strain, and mixing the frozen stock solution with a mixture of 1: inoculating at a ratio of 100% in 5mL 7H10Culture medium (containing 10% OADC). Except for culturing Mycobacterium marinum at 30 deg.C, culturing other strains at 37 deg.C to OD 600 =0.6-0.8 (2 weeks for SGM strain and about 5 days for fast growing mycobacterium RGM). Centrifuging the bacterial liquid at 3000rpm for 10min, and obtaining the supernatant as culture filtrate.
The invention establishes a double-antibody sandwich ELISA method based on LprA to carry out strain identification on the positive culture solution of mycobacterium, can realize the rapid diagnosis of NTM, and comprises the following specific steps:
(1) Coating: mu.L of diluted anti-H37 Rv LprA mouse monoclonal antibody LprA-07 (final concentration of 1. Mu.g/mL) was added to each well, the plate was sealed, and the plate was coated overnight at 4 ℃.
(2) The next day the plate was removed, washed 3 times with 1 XPBST, 300. Mu.L/well and finally blotted dry.
(3) And (3) sealing: add 200. Mu.L of 3% BSA blocking solution per well and block by shaking horizontally at room temperature for 1h.
(4) Wash 3 times with 1 XPBST, 300. Mu.L/well, and beat dry the last time.
(5) Sample adding: mu.L of concentrated culture filtrate was added to each well and 3 wells were repeated for each sample, while a positive control (i.e., 0.1. Mu.g/mL of H37Rv LprA immunogen protein (recombinant protein LprA prepared in example 1)), a negative control (0.1. Mu.g/mL of BSA), and a blank control were set and incubated in a horizontal shaker at room temperature for 2H.
(6) Wash 3 times with 1 XPBST, 300. Mu.L/well, and beat dry the last time.
(7) mu.L of the diluted anti-H37 Rv LprA rabbit polyclonal antibody (polyclonal antibody # 1, final concentration 0.5. Mu.g/mL) prepared in example 2 was added to each well and blocked by shaking horizontally at room temperature for 1H.
(8) After washing with 1 XPBST for 3 times, the cells were blotted dry, 100. Mu.L of goat anti-rabbit IgG-HRP secondary antibody (Kyoto China fir gold bridge Biotech Co., ltd., cat. No. ZB-2301) was added to each well and incubated at room temperature for 1 hour.
(9) Washing with 1 XPBST for 3 times, drying by beating, adding 100 mu L of freshly prepared TMB developing solution into each hole (the volume ratio of the solution A to the solution B is 1.
(10) 2% H of 50. Mu.L per well 2 SO 4 Terminating the reaction, the enzymeOD measurement by standard instrument 450nm The results are shown in Table 4.
The purified anti-H37 Rv LprA mouse monoclonal antibody (LprA-07) and rabbit polyclonal antibody are utilized to establish a double antibody sandwich ELISA method for detecting the specificity of different SGM culture filtrate proteins, wherein the double antibody sandwich ELISA method is used for detecting the anti-H37 Rv LprA mouse monoclonal antibody, detecting different SGM wild strain culture filtrates, namely anti-H37 Rv LprA rabbit polyclonal antibody-HRP labeled secondary antibody, and the result shows that the monoclonal antibody LprA-07 has better reaction with proteins in M.tubergulosis strain culture filtrates, the specificity of antigen-antibody reaction is higher, and the monoclonal antibody is basically not combined with weak binding signals of SGM wild strain culture filtrate proteins of mycobacterium kansasii, goden mycobacterium, mycobacterium marinum, mycobacterium thuringiensis and mycobacterium perusamii, and the like, and is basically not combined with common pathogenic fast-growing mycobacterium fortuitum (RGM) accidental mycobacteria and mycobacterium swertia. Thus, the method can achieve the discrimination between Mycobacterium tuberculosis and common NTM.
TABLE 4 detection of protein specificity of different SGM and RGM culture filtrates by ELISA method
The double-antibody sandwich ELISA method based on LprA established by the invention can be used for identifying the strains of the positive culture solution of mycobacterium: by OD 450 Determined as a critical value, OD 450 >0.5 is mycobacterium tuberculosis; OD 450 0.2-0.5 for Slow Growing Mycobacteria (SGM) including Mycobacterium marinum, mycobacterium sulcata, mycobacterium gordonii and Mycobacterium kansasii; the fast growing Mycobacteria did not contain LprA, so the OD values were similar to the negative control 3% BSA.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is made possible within the scope of the claims attached below.
Claims (10)
1. The hybridoma cell strain is a mouse hybridoma cell ZYS-CS-01, the mouse hybridoma cell ZYS-CS-01 secretes a monoclonal antibody specifically binding with LprA protein, and the preservation number of the hybridoma cell strain in China general microbiological culture Collection center is CGMCC No.18884.
The LprA protein is used as an antigen of an antibody which is specifically combined with the LprA protein and is used for identifying mycobacterium tuberculosis and/or nontuberculous mycobacteria, identifying and distinguishing nontuberculous mycobacteria and the mycobacterium tuberculosis, identifying and distinguishing nontuberculous mycosis and tuberculosis, diagnosing or screening tuberculosis and diagnosing or screening nontuberculous mycosis, wherein the amino acid sequence of the LprA protein is SEQ ID No.1.
3. The application according to claim 2, wherein the application comprises any of:
c1 Immunizing an animal with the LprA protein as an immunogen, fusing spleen cells of the immunized animal with myeloma cells, and screening hybridoma cells capable of secreting a monoclonal antibody that specifically binds to the LprA protein;
c2 Preparing a monoclonal antibody specifically binding to the lprA protein by using the hybridoma cells of C1);
c3 Using the LprA protein as immunogen to immunize animals, and detecting to obtain polyclonal antibody capable of specifically binding to the LprA protein.
4. A monoclonal antibody secreted by the hybridoma cell line of claim 1 or the hybridoma cell of claim 3.
5. The monoclonal antibody of claim 4 for any one of the following uses:
a1 Use in the identification of Mycobacterium tuberculosis or for the preparation of a product for the identification of Mycobacterium tuberculosis;
a2 Use in the identification of non-tuberculous mycobacteria or for the preparation of a product for identifying non-tuberculous mycobacteria;
a3 Use in or for the manufacture of a product for the differential discrimination between nontuberculous mycobacteria and tuberculous mycobacteria;
a4 Use in or for the manufacture of a product for the differential discrimination between nontuberculous mycobacteriosis and tuberculosis;
a5 Use in the diagnosis or screening of tuberculosis or for the preparation of a product for the diagnosis or screening of tuberculosis;
a6 Use in the diagnosis or screening of non-tuberculous mycobacteria disease or in the manufacture of a product for use in the diagnosis or screening of non-tuberculous mycobacteria disease;
a7 Application in detecting LprA protein or preparing a product for detecting the LprA protein;
a8 Use) for the preparation of a product for binding to LprA protein.
6. A reagent or kit comprising the monoclonal antibody of claim 4, wherein the reagent or kit has at least one of the following uses:
b1 Identification of Mycobacterium tuberculosis and/or non-Mycobacterium tuberculosis;
b2 To identify and distinguish between non-tuberculous mycobacteria and tuberculous mycobacteria;
b3 To identify and distinguish between nontuberculous mycobacteriosis and tuberculosis;
b4 Diagnosis or screening of tuberculosis;
b5 Diagnosis or screening for nontuberculous mycobacterial disease;
b6 Detecting the LprA protein;
b7 Bind LprA protein.
7. A method for identifying Mycobacterium tuberculosis and/or nontuberculous mycobacteria, comprising identifying Mycobacterium tuberculosis and/or nontuberculous mycobacteria using the monoclonal antibody of claim 4 or the kit of claim 6.
8. A method for detecting LprA protein, which comprises detecting LprA protein using the monoclonal antibody according to claim 4 or the kit according to claim 6.
9. The method according to claim 7 or 8, wherein the method is a double antibody sandwich ELISA method.
10. A method for producing a monoclonal antibody that specifically binds to LprA protein, comprising culturing the hybridoma cell line of claim 1 or the hybridoma cell of claim 3.
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| US20150260714A1 (en) * | 2012-10-12 | 2015-09-17 | Albert Einstein College Of Medicine Of Yeshiva University | Serologic test for the rapid diagnosis of active tuberculosis |
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| US20150260714A1 (en) * | 2012-10-12 | 2015-09-17 | Albert Einstein College Of Medicine Of Yeshiva University | Serologic test for the rapid diagnosis of active tuberculosis |
| CN109975543A (en) * | 2019-03-01 | 2019-07-05 | 中国疾病预防控制中心传染病预防控制所 | The application of mycobacteria Ku albumen |
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