WO2013075608A1 - Antigène recombiné chimère et application associée - Google Patents

Antigène recombiné chimère et application associée Download PDF

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WO2013075608A1
WO2013075608A1 PCT/CN2012/084865 CN2012084865W WO2013075608A1 WO 2013075608 A1 WO2013075608 A1 WO 2013075608A1 CN 2012084865 W CN2012084865 W CN 2012084865W WO 2013075608 A1 WO2013075608 A1 WO 2013075608A1
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Prior art keywords
sample
recombinant antigen
whole blood
chimeric recombinant
tuberculosis
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PCT/CN2012/084865
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English (en)
Chinese (zh)
Inventor
葛胜祥
熊君辉
符美娟
许荣均
乔杉
李芳�
罗文新
张军
夏宁邵
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厦门大学
北京万泰生物药业股份有限公司
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Publication of WO2013075608A1 publication Critical patent/WO2013075608A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)

Definitions

  • the present invention relates to the fields of molecular biology, immunology, and disease diagnosis.
  • the present invention relates to a chimeric recombinant antigen comprising three antigenic regions derived from M. tuberculosis specific proteins Rv3875, Rv3874 and TB7.7, respectively, and also to a combination comprising said chimeric recombinant antigen And the use of the chimeric recombinant antigen.
  • the present invention also provides a method of detecting a specific T cell immune response in vitro using the chimeric recombinant antigen.
  • the present invention also provides a method of diagnosing whether a patient is infected with Mycobacter um tuberculos i s, which comprises using the chimeric recombinant antigen of the present invention. Background technique
  • the in vitro diagnosis of pathogenic microorganism infection using the cellular immune response of antigen-specific T cells is a new detection method developed in recent years.
  • This method is currently mainly used for the diagnosis of Mycobacterium tuberculosis infection.
  • the commonly used diagnosis of tuberculosis mainly depends on clinical symptoms, imaging diagnosis and pathogenic diagnosis, and is not sensitive to the diagnosis of latent infection of Mycobacterium tuberculosis.
  • direct detection of pathogens or detection of tuberculosis The sensitivity and specificity of mycobacterial antibodies are also less than ideal.
  • the initial T lymphocytes in the latent infection of M. tuberculosis undergo amplification and differentiation after antigen stimulation and activation.
  • the initial T lymphocytes are mainly differentiated into effector T cells and memory cells (memory T cel l).
  • memory T cel l memory T cel l
  • memory T cells when memory T cells re-accept the same antigen presented by APC, they can rapidly proliferate and secrete cytokines, which contain a large number of cytokines such as IFN- ⁇ . Therefore, by using this process to stimulate T cells in vitro, and observe the secretion of cytokine IFN- ⁇ In other words, it can be determined whether the subject contains sensitized T lymphocytes, thereby diagnosing whether the subject is infected with M. tuberculosis.
  • Quant i-FERON Gold directly uses the blood collection tube to stimulate and culture whole blood.
  • the advantage is that it is easy to operate.
  • the disadvantage is that the blood collection volume cannot be accurately controlled during blood collection, which will have a greater impact on the stimulation effect, thus affecting the diagnosis result.
  • ELISpot requires separation and cultivation of PBMC, which is highly demanding for operators, equipment and the environment, and is not suitable for promotion in grassroots units.
  • chimeric recombinant antigen refers to a recombinant protein obtained by fusing different proteins or fragments thereof, particularly fragments comprising an epitope.
  • This class Recombinant proteins can be produced by genetic engineering or by chemical synthesis.
  • epitope refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope” is also known in the art as an "antigenic determinant”. Epitopes or antigenic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrates or sugar side chains and typically have specific three dimensional structural characteristics as well as specific charge characteristics. For example, an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be "linear” "or” conformation. See, for example, Epi tope Mapping Protocol in Methods in Mo lecular Biology, Vol. 66, G. E.
  • the term "fusion" may be direct fusion (ie, joining the C-terminus of one amino acid sequence to the N-terminus of another amino acid sequence by a simple covalent bond), or it may be fusion using a linker ( That is, the C-terminus of one amino acid sequence is linked to the N-terminus of another amino acid sequence through a linker).
  • the term “junction” refers to a short peptide used to join two molecules, such as proteins.
  • linkers are well known to those skilled in the art, such as the hinge region of IgG, or a polypeptide linker composed of small amino acids, such as glycine, serine, threonine or alanine, in various lengths and combinations.
  • the linker may be a polyglycine repeat interrupted by serine or threonine at intervals.
  • the joints may be Ser-Gly-Gly-Gly-Ser, Gly-Gly-Gly-Gly, Gly-Gly-Ser-Ser, Gly-Gly-Gly-Gly-Ser and
  • antigenic region derived from a protein refers to a fragment of the protein that is antigenic (ie, capable of eliciting an immune response and/or recognized by an antibody), which is typically a fragment comprising an epitope. .
  • M. tuberculosis specific protein Rv3875 or “Rv3875” refers to the early secretory antigen-6 (ESAT-6) derived from Mycobacterium tuberculosis, which is well known to those skilled in the art. See, for example, Andersen, P., et al., Lancet, 2000. 356 (9235): p. 1099-104 (which is incorporated herein by reference), and GENBANK accession number YP_178023.
  • ESAT-6 early secretory antigen-6
  • M. tuberculosis-specific protein Rv3874 or “Rv3874” refers to medium-filtered protein-10 (CFP-10) derived from Mycobacterium tuberculosis, which is a person skilled in the art.
  • CFP-10 medium-filtered protein-10
  • M. tuberculosis-specific protein TB7. 7 or “TB7. 7” refers to Ruhwa ld M, et al., 2007 Jun; 9 (7): 806-12 (which The M. tuberculosis specific antigen Rv2654C, which is reported to specifically stimulate T cell production of cytokines, is reported herein, the GENBANK accession number of which is described, for example, in P71951.
  • expression control sequence refers to a control sequence required to effect expression of a gene, which is well known in the art.
  • Expression control sequences include, but are not limited to, promoter sequences, transcription termination sequences, and enhancer sequences.
  • operably linked means that the molecules attached are connected in such a way as to achieve the desired function. For example, expression control sequences and gene coding control effects.
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector When the vector enables expression of the inserted polynucleotide-encoded protein, the vector is referred to as an expression vector.
  • the vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be amplified and/or expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phage; cosmid and the like.
  • the term "pharmaceutically acceptable carrier” refers to a carrier that is pharmacologically and/or physiologically compatible with the subject or cell and the active ingredient, as is well known in the art (see, for example, Remington s). Pharmaceut ica l Sc iences. Edi ted by Gennaro AR, 19 th ed. Pennsylvania: Mack Publ i shing Company, 1995), and includes, but is not limited to, pH adjusters and ionic strength enhancers.
  • pH Conditioning agents include, but are not limited to, phosphate buffers; ionic strength enhancers include, but are not limited to, sodium chloride.
  • subject refers to mammals, such as rodents (e.g., mice, rats) and primates (e.g., humans).
  • tuberculosis culture supernatant containing a plurality of antigens and having cross-reactivity between mycobacteria of different species Therefore, when using PPD for in vitro stimulation, the results obtained cannot distinguish between BCG vaccinates and M. tuberculosis infected individuals.
  • Mycobacterium tuberculosis possesses a gene region that is not found in BCG and other mycobacteria by comparative genomics, and found two proteins located in this region - early secretory antigen-6 (ESAT-6, Rv3875) and medium-filtered protein-10 (CFP-10, Rv3874) - have strong T cell epitopes (Andersen, P., et al., Lancet, 2000. 356 (9235) : p. 1099-104).
  • TB7. 7 (Rv2654C) is a M.
  • tuberculosis protein reported in recent years that specifically stimulates T cells to produce cytokines (Ruhwa ld M, et al., 2007 Jun; 9 (7): 806-12) threaten The antigens all have good T cell epitopes and can be used for in vitro detection of M. tuberculosis antigen-specific T cells.
  • TB7. 7 is too hydrophobic, and its solubility is not high when expressed alone. The stability is not good; and the endotoxin bound by CFP-10 antigen is not easy to remove, which is likely to cause high background stimulation and affect the reliability of the test results.
  • the inventors creatively put the above three antigens (Rv3875, Rv3874) In combination with TB7. 7), chimeric recombinant antigens were constructed using segments derived from these three antigens with strong T cell epitopes and strong sequence conservation.
  • the chimeric recombinant antigen is highly specific to M. tuberculosis and can be used to detect M. tuberculosis-specific T cell immune responses in vitro, and thus can be used to diagnose whether a subject is infected with M. tuberculosis.
  • the invention provides a chimeric recombinant antigen comprising three antigenic regions derived from Rv3875, Rv3874 and TB7.7, respectively.
  • the antigenic region derived from Rv3875 is a Rv3875 protein fragment comprising amino acid residues 1-80 of the Rv3875 (aa 1-80).
  • the antigenic region derived from Rv3874 is a Rv3874 protein fragment comprising amino acid residues 1-100 of the Rv3874 (aa 1-100).
  • the antigenic region derived from TB7. 7 is a TB7.7 protein fragment comprising amino acid residues 37-81 of TB7.7 (aa 37-81).
  • the chimeric recombinant antigen of the invention comprises amino acid residues 1-80 of the Rv3875 (aa 1-80), amino acid residues 1-100 of the Rv3874 (aa 1-100) And amino acid residues 37-81 of TB7. 7 (aa 37-81).
  • the three antigenic regions comprised by the chimeric recombinant antigen of the invention are joined together by direct fusion.
  • the three antigenic regions comprised by the chimeric recombinant antigen of the invention are joined by a linker.
  • linkers are well known to those skilled in the art, such as the hinge region of IgG, or a polypeptide linker composed of small amino acids (e.g., glycine, serine, threonine or alanine) in various lengths and combinations, for example, Ser. -Gly-Gly-Gly-Ser, Gly-Gly-Gly-Gly, Gly-Gly-Ser-Ser, Gly-Gly-Gly-Gly-Ser and (Gly-Gly-Gly-Gly-Ser) 3 .
  • the choice of such a linker is well within the abilities of those skilled in the art.
  • the chimeric recombinant antigen of the invention further comprises a tag at its N-terminus and/or C-terminus.
  • tags are well known to those skilled in the art, for example, 6 X Hi s tags, Myc tags, Flag tags, fluorescent protein tags, and the like. The use of such tags allows the chimeric recombinant antigen to be readily detected and purified.
  • metal can be used Chelation-type chromatography (eg, nickel column) for rapid purification of chimeric recombinant antigens carrying the 6 x Hi s tag; affinity chromatography (eg, antibodies) can be used to rapidly carry chimeric recombinant antigens carrying Myc tags or Flag tags Purification; chimeric recombinant antigens carrying fluorescent protein tags can also be conveniently detected using a fluorescence microscope. The selection of such labels is well within the capabilities of those skilled in the art.
  • the chimeric recombinant antigen of the present invention is recombinant protein EC5 having the amino acid sequence set forth in SEQ ID NO: 2.
  • the invention provides a polynucleotide encoding a chimeric recombinant antigen as defined above.
  • the polynucleotide of the present invention has a nucleotide sequence as shown in SEQ ID NO: 1.
  • constructs comprising such polynucleotides.
  • constructs also contain restriction endonuclease sites to facilitate genetic engineering operations.
  • constructs further comprise an expression control sequence operably linked to a coding sequence.
  • the invention provides a vector comprising a polynucleotide encoding a chimeric recombinant antigen as defined above or a construct comprising such a polynucleotide.
  • the vector of the present invention may be a cloning vector or an expression vector.
  • host cells comprising a polynucleotide or construct or vector of the invention are also provided.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, etc.).
  • the cells of the invention may also be cell lines, such as 293T cells.
  • the invention provides a composition comprising a chimeric recombinant antigen as defined above, optionally further comprising a pharmaceutically acceptable carrier, such as a buffer.
  • a pharmaceutically acceptable carrier such as a buffer.
  • the invention relates to a method of detecting a Mycobacterium tuberculosis-specific T cell immune response in a sample in vitro, comprising the steps of:
  • the increase in the level of the cytokine in the stimulated sample indicates that a Mycobacterium tuberculosis-specific T cell immune response exists in the sample, and the level of the cytokine is reflected in the sample.
  • the level of M. tuberculosis-specific T cell immune response in the sample is reflected in the sample.
  • sample is anticoagulated whole blood or peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • the cytokine is IFN- ⁇ and/or IL-2.
  • the control is the sample to be detected that is not stimulated with the chimeric recombinant antigen, and/or the sample of uninfected M. tuberculosis stimulated with the chimeric recombinant antigen or Sample population.
  • the anticoagulated whole blood is whole blood that is anticoagulated with an anticoagulant such as heparin.
  • the anticoagulated whole blood is freshly collected anticoagulated whole blood, for example, placed at room temperature for no more than 12 hours, preferably no more than 10, 8, 6, 4 or 2 after collection. Hours of anticoagulated whole blood.
  • the anticoagulated whole blood is diluted with a medium such as, but not limited to, RPMI-1640 medium and DMEM medium prior to performing step 1).
  • the chimeric recombinant antigen has been endotoxin removed.
  • the chimeric recombinant antigen is contained in a culture medium such as, but not limited to, RPMI-1640 medium and DMEM medium.
  • the stimulation of step 1) is carried out under standing or non-resting conditions, preferably under standing conditions. In another preferred embodiment, the stimulation of step 1) is carried out at 30 ° C to 39 ° C, preferably 35 ° C to 37 ° C, more preferably 37 ° C. In another preferred embodiment, the stimulation of step 1) is carried out in a device that provides a constant temperature, such as, but not limited to, a CO 2 incubator and a constant temperature water bath. In another preferred embodiment, the stimulation of step 1) is carried out for 11 to 39 hours, preferably 15 to 35 hours, preferably 20 to 30 hours, more preferably 20 to 24 hours.
  • the method also provides a positive control, which is a sample to be detected that is stimulated with non-specific T cells.
  • the non-specific T cell stimulating agent refers to a compound capable of non-specifically activating T lymphocytes, allowing them to proliferate and release cytokines, including but not limited to, plant lectin (PHA). And concanavalin A.
  • the non-specific T cell stimulator is contained in a culture medium such as, but not limited to, RPMI-1640 medium and DMEM medium.
  • the methods of the invention are useful for non-diagnostic and therapeutic purposes.
  • the method of the present invention can be used to study the infection mechanism of Mycobacterium tuberculosis, the immune mechanism of the body against Mycobacterium tuberculosis infection, the immune mechanism of the body against co-infection of Mycobacterium tuberculosis and other infectious bacteria/viruses, and the body resistance
  • the immune mechanism of tuberculosis and other non-communicable diseases provides a method of diagnosing whether a subject is infected with M.
  • tuberculosis comprising using a chimeric recombinant antigen as defined above to detect the presence or absence of tuberculosis in a test sample obtained from said subject Mycobacterial-specific T cell immune response, wherein the sample is anticoagulated whole blood or peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • the method comprises the steps of:
  • an increase in the level of the cytokine in the stimulated sample compared to the control indicates that the subject is infected with M. tuberculosis.
  • the cytokine is IFN- ⁇ and/or IL-2.
  • the control is the test sample that is not stimulated with the chimeric recombinant antigen, and/or the sample or sample population of uninfected M. tuberculosis stimulated with the chimeric recombinant antigen .
  • the anticoagulated whole blood is whole blood that is anticoagulated with an anticoagulant such as heparin.
  • the anticoagulated whole blood is freshly collected anticoagulated whole blood, for example, placed at room temperature for no more than 12 hours, preferably no more than 10, 8, 6, 4 or 2 after collection. Hours of anticoagulated whole blood.
  • the anticoagulated whole blood is diluted with a medium such as, but not limited to, RPMI-1640 medium and DMEM medium prior to performing step 2).
  • the chimeric recombinant antigen has been endotoxin removed.
  • the chimeric recombinant antigen is contained in a culture medium such as, but not limited to, RPMI-1640 medium and DMEM medium.
  • the stimulation of step 2) is carried out under resting conditions, preferably under standing or non-rest conditions. In another preferred embodiment, the stimulation of step 2) is carried out at 30 ° C to 39 ° C, preferably 35 ° C to 37 ° C, more preferably 37 ° C. In another preferred embodiment, the stimulation of step 2) is carried out in a device that provides a constant temperature, such as, but not limited to, a CO 2 incubator and a constant temperature water bath. In another preferred embodiment, the stimulation of step 2) is carried out for 11 to 39 hours, preferably 15 to 35 hours, preferably 20 to 30 hours, more preferably 20 to 24 hours.
  • the method also provides a positive control which is the test sample stimulated with non-specific T cells.
  • the non-specific T cell stimulating agent refers to a compound capable of non-specifically activating T lymphocytes, proliferating and releasing cytokines, including but not limited to, plant lectin and concanavalin A .
  • the non-specific T cell stimulator is contained in a culture medium such as, but not limited to, RPMI-1640 medium and DMEM medium.
  • the invention provides a method of evaluating an anti-tuberculosis treatment effect, comprising:
  • the M. tuberculosis-specific T cell immune response in whole blood of a subject receiving the anti-tuberculosis treatment is detected using the above-described method provided by the present invention
  • the invention provides a chimeric recombinant antigen as defined above for use in detecting a Mycobacterium tuberculosis-specific T cell immune response in a sample in vitro, or for diagnosing whether a subject is infected with Mycobacterium tuberculosis infection.
  • the invention provides the use of a chimeric recombinant antigen or composition as defined above for the preparation of a kit for the in vitro detection of a Mycobacterium tuberculosis-specific T cell immune response in a sample, Or to diagnose whether a subject is infected with M. tuberculosis, or to evaluate the effectiveness of anti-tuberculosis treatment.
  • the invention provides a kit comprising a chimeric recombinant antigen or composition as defined above.
  • the kit optionally further comprises one or more of the following reagents:
  • cytokine for example, IFN- ⁇ and/or IL-2
  • Blood collection devices such as pyrogen-free vacuum blood collection tubes
  • an anticoagulant such as heparin
  • a medium such as, but not limited to, RPMI-1640 medium and DMEM medium;
  • Non-specific T cell stimuli such as, but not limited to, lectin and concanavalin A.
  • the present invention is characterized by the establishment of a simple and practical method for quantifying released cytokines using a chimeric heavy and then using a cytokine quantitative detection reagent to determine whether the test sample is infected with M. tuberculosis infection.
  • the operation involved is simple and convenient: for example, by collecting whole blood, dispensing it into a closable centrifuge tube and adding a chimeric recombinant antigen, and then culturing it in a constant temperature incubator to complete the whole blood. Stimulate
  • Figure 1 is a schematic view showing the structure of the expression vector pTO-T7-EC5 containing the coding sequence of the recombinant protein EC5.
  • Figure 2 shows the results of SDS-PAGE analysis showing the recombinant protein EC5 purified by Ni-NTA column after high expression in E. coli (E co il) er2566 and two steps (Ni-NTA column purification and HPLC layer)
  • the purified recombinant protein EC5 was purified.
  • lane 1 represents the protein molecular weight standard
  • lane 2 represents the EC5 protein purified by the Ni-NTA column
  • lane 3 represents the EC5 protein purified in two steps.
  • Figure 3 shows changes in cellular immune levels after stimulation with different amounts of PHA. Among them, S1-S4 are randomly selected 4 healthy people.
  • Figure 4 shows the effect of the time of standing at room temperature after whole blood collection on the level of specific stimulation.
  • S5 and S6 are randomly selected two tuberculosis patients.
  • FIG. 5 shows the effect of different culture methods on the level of specific stimulation. The results showed that the stimulation intensity under static culture was significantly higher than that under shaking culture.
  • Figure 6 shows the effect of different culture temperatures on the level of specific stimulation. The results showed that the stimulation effect was optimal at a culture temperature of 37 °C. Among them, S7 and S8 are two other tuberculosis patients randomly selected.
  • Figure 7 shows the effect of different incubation times on the level of specific stimulation. The results showed that the stimulation effect basically reached the platform 20-22 hours (h) after the start of stimulation. Sequence information
  • FAATAAAAAAVDRGDPP Sequence 3 (SEQ ID NO: 3):
  • the target gene fragment was amplified by PCR using the following primers: For the gene fragment 1 encoding Rv3875 aa 1-80, use The following primers: F: 5' -t 11 tCATATgCACCACCACCACCACCACATGACAGAGCAG
  • the PCR amplification conditions were: pre-denaturation at 94 ° C for 5 minutes (min); denaturation at 94 ° C for 50 seconds (sec), annealing at 57 ° C for 50 sec, extension at 72 ° C for 1 min, a total of 25 cycles; Extend at 72 ° C for 10 min.
  • the PCR product was confirmed and isolated by agarose gel electrophoresis, and recovered and purified using a kit (Universal DNA Purification kit, TianGen).
  • the vector obtained in the previous step and the PCR product encoding TB7.7 aa 37-81 were double-digested with EcoRI/Hindlll, and the digested PCR product was ligated into the enzyme-cut vector.
  • the desired expression vector pTO-T7-EC5 was obtained.
  • a schematic representation of the structure of the expression vector pTO-T7-EC5 is shown in Figure 1.
  • the constructed PTO-T7-EC5 expression vector was transformed into E. coli ER2566, which was applied to solid LB medium containing kanamycin (Kan, final concentration 100 g/ml) (LB medium composition: 10 g/ L protein ⁇ , 5g / L yeast powder, 10g / L sodium chloride, the same below), and static culture at 37 ° C until the single colony is clearly identifiable. Single colonies were picked into liquid LB medium (containing 100 g/ml kanamycin), and cultured at 37 ° C, shaking at 180 rpm for 8 hours.
  • the bacterial solution was then transferred to a culture flask containing 500 ml of liquid LB medium (containing 100 ⁇ 8 / ⁇ kanamycin), and cultured at 37, 180 rpm with shaking.
  • liquid LB medium containing 100 ⁇ 8 / ⁇ kanamycin
  • IPTG was added to a final concentration of 0.2 mM/L and induction culture was continued for 4 hours at 37 ° C, 180 rpm.
  • the culture was then centrifuged at 5000 rpm for 10 min to collect the cells.
  • the recombinant protein EC5 Since the recombinant protein EC5 is used to stimulate whole blood, it is necessary to remove endotoxin from the protein product. Methods for purifying recombinant proteins from bacterial cells and removing endotoxins are known to those skilled in the art. In the present embodiment, the following exemplary methods are used.
  • the collected cells (obtained from 20 bottles of X 500 ml/culture flask) were suspended in 200 ml of a buffer containing 20 mM PB6.0 (phosphate buffer, pH 6.0, the same below), 500 mM NaCl, and placed. The mixture was crushed by ultrasonic waves in an ice bath, and then centrifuged at 12,000 rpm for 10 minutes to collect a precipitate.
  • PB6.0 phosphate buffer, pH 6.0, the same below
  • Ni-NTA column Purification of Ni-NTA column: The precipitate was dissolved in 200 ml of 0.2% sodium deoxycholate, PBS (phosphate buffer, 150 mM aCl, pH 7.4, the same below), and purified by Ni-NTA column (Qiagen). . Briefly, the sample was loaded onto a Ni-NTA column and washed with 100 ml of 0.2% sodium deoxycholate and 20 mM PBS to remove some of the endotoxin; then washed with 200 ml of PBS to remove sodium deoxycholate; The target protein was eluted with 100 ml of eluate (150 mM imidazole, 20 mM PBS). The eluted target protein was dialyzed to an equal volume of 20 mMPB7.0 (phosphate buffer, pH 7.0, the same below).
  • PBS phosphate buffer, 150 mM aCl, pH 7.4, the same below
  • the buffer used was prepared by using water for injection, and the experimental containers used were dry-baked at 200 ° C for more than 2 hours.
  • the purified recombinant protein EC5 was detected by SDS-PAGE, and the results are shown in Fig. 2. As a result, the molecular weight of the recombinant protein EC5 was about 26 kD, and after the above two steps of purification, the purity of the recombinant protein EC5 was over 95%.
  • the endotoxin in the purified recombinant protein EC5 was detected using a guanidine reagent. The results showed that after the above two steps of purification, the endotoxin in the recombinant protein EC5 was substantially removed, and its content was less than 30 EU/mg.
  • Example 2 Selection of non-specific T cell stimuli and determination of the concentration thereof used In the in vitro stimulation culture of anticoagulated whole blood, there are certain factors (such as operational errors, low cellular immunity levels of subjects, etc.), It can result in no change in cytokine levels after antigen stimulation, and there are certain factors (eg, excessively high stimulatory concentrations) that can cause immune-tolerant lymphocytes to also react. Therefore, in order to ensure the accuracy and reliability of the results of the method of the invention, it is preferred to provide a positive control during the experiment. Such controls may be whole blood stimulated with a non-specific stimuli.
  • Both plant lectin (PHA) and concanavalin A have strong ability to promote lymphocyte transformation, and can effectively stimulate and respond to T cells in peripheral blood. Therefore, both plant lectin (PHA) and concanavalin A can be used as non-specific stimuli to reflect the ability of peripheral blood T cells to respond.
  • phytohemagglutinin was exemplarily used as a non-specific stimulator, and its use concentration was determined.
  • concentration in 1ml whole blood 5 ⁇ g/mK 10 ⁇ g/mK 20 ⁇ g/mK 40 ⁇ g/mK 60 ⁇ g/mK 80 ⁇ g/ml, 100 ⁇ 8 / ⁇ 200 ⁇ g/ml
  • phytohemagglutinin to stimulate 1 ml of whole blood (from four healthy subjects, S1-S4) and allowed to stand at 37 ° C for 22 hours. Then, the stimulated whole blood plasma was collected by centrifugation.
  • the level of IFN- ⁇ in whole blood was measured using commercially available Human IFN- ⁇ El i sa ki t (BD company) according to the manufacturer's instructions.
  • the level of IFN- ⁇ produced by stimulating whole blood with phytohemagglutinin was plotted against the concentration (amount) of lectin, and the results are shown in Fig. 3.
  • the results show that the level of stimulation (i.e., the level of I FN- ⁇ ) increases with increasing stimuli concentration and varies linearly only over a range of stimuli concentrations.
  • the stimuli concentration at which the stimulation level is within the linear range and is approximately 30% of the maximum response value is the concentration at which the non-specific antigen PHA is used (see Figure 3).
  • Example 3 Selection of blood collection tubes
  • the blood collection tube used to obtain anticoagulated whole blood should contain an anticoagulant and a low endotoxin content.
  • a variety of commercially available blood collection tubes can be used to collect anticoagulated whole blood, such as lithium heparin vacuum blood collection tubes produced by BD.
  • heparin lithium vacuum blood collection tubes produced by BD Corporation were used to collect whole blood of healthy subjects (N1) and tuberculosis patients (T1). Then, the recombinant protein EC5 was used to stimulate the above two whole blood samples (each lml, the final concentration of recombinant protein EC5 was 2 ⁇ g/ml), and the cells were statically cultured at 37 °C for 22 hours. Whole blood without stimuli stimulation was used as a negative control. Whole blood plasma of each sample (including the negative control) was then collected by centrifugation, and IFN- ⁇ in whole blood plasma was detected using commercially available Human IFN- ⁇ El i sa ki t (BD) according to the manufacturer's instructions. s level.
  • BD Human IFN- ⁇ El i sa ki t
  • T1 tuberculosis patients 0. 1 05 2. 593
  • Example 4 Effect of placement time after whole blood collection
  • Fresh peripheral whole blood was collected from 2 tuberculosis patients (S5 and S6).
  • the collected whole blood was divided into 8 equal portions and allowed to stand at room temperature for different times (0, 1, 2, 4, 6, 8, 10 and 12 hours); then, the recombinant protein EC5 was used for stimulation ( Reorganization
  • the final concentration of protein EC5 was 2 ⁇ g/ml), and the cells were statically cultured at 37 ° C for 22 h.
  • the whole time of the non-stimulated whole blood stimulated at room temperature was used as a negative control; then, each sample was collected by centrifugation (including Negative control) whole blood plasma; Then, the level of IFN- ⁇ in whole blood plasma was measured using commercially available Human IFN- ⁇ El i sa ki t (BD) according to the manufacturer's instructions.
  • BD Human IFN- ⁇ El i sa ki t
  • the level of IFN- ⁇ in whole blood was plotted against the time of collection after whole blood collection, and the results are shown in Fig. 4.
  • the results showed that the whole blood-specific stimulation level (i.e., the level of IFN- ⁇ ) continued to decrease as the time of prolongation.
  • the stimulation level can be maintained at about 80% of the stimulation level (maximum stimulation level) without room temperature (ie, placed for 0 hours); and when room temperature is allowed for more than 2 hours At the time, the rate of decline in stimulation levels increased significantly, below 60% of the highest level of stimulation (see Figure 4).
  • the whole blood sample used is a whole blood sample within 2 hours after collection.
  • Figure 7 shows the average of the results for three independent experiments (i.e., peripheral blood samples from three patients).
  • the results showed that the level of stimulation (i.e., the level of IFN- ⁇ ) substantially reached the plateau after 20 hours of culture. Therefore, in the method of the present invention, the preferred incubation time is at least 20 hours, for example 20-24 hours.
  • Example 8 Diagnostic value of cytokine IL-2
  • IL-2 In the local skin reaction induced by Mycobacterium tuberculosis, the researchers observed high expression of IL-2 and IFN- ⁇ mRNA in Th1 cells by in situ hybridization, and observed increased secretion of IL-2 and IFN- ⁇ . .
  • levels of IL-2 in addition to IFN-[gamma], can also be used to reflect specific T cell immune responses in whole blood.
  • IL was confirmed using 6 fresh whole blood (T2-T7) from different M. tuberculosis infected persons and 3 fresh whole blood (N2-N4) from different healthy subjects.
  • T2-T7 6 fresh whole blood
  • N2-N4 3 fresh whole blood from different healthy subjects.
  • -2 has the same diagnostic value as IFN- ⁇ .
  • recombinant protein EC5 recombinant protein EC5 at a final concentration of 2 ⁇ g/ml
  • whole blood without stimuli was used as a negative Control.
  • whole blood plasma of each sample was collected by centrifugation; then, IFN- ⁇ in each plasma sample was detected using BD Human IFN- ⁇ Elisa kit Human IL-2 Elisa kit according to the manufacturer's instructions. The level of IL-2.
  • Table 3 Comparison of cytokines IL-2 and IFN- ⁇

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Abstract

La présente invention concerne un antigène recombiné chimère et son utilisation dans le diagnostic des infections à Mycobacterium tuberculosis. L'antigène recombiné chimère comprend 3 régions d'antigène dérivées de protéines spécifiques de Mycobacterium tuberculosis, respectivement Rv3875, Rv3874 et TB7.7, et peut être utilisé pour détecter une réponse immunitaire spécifique médiée par les lymphocytes T in vitro.
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KR20180104038A (ko) * 2016-01-20 2018-09-19 시아먼 유니버시티 활동성 결핵의 진단을 위한 방법 및 키트
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KR101923198B1 (ko) * 2017-09-25 2018-11-28 주식회사 엠디엡투스 Tb7.7에 특이적으로 결합하는 dna 압타머 및 이의 용도
CN108872610B (zh) * 2018-09-07 2021-03-09 首都医科大学附属北京胸科医院 结核感染诊断试剂盒、筛查系统及该试剂盒的应用

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