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WO2022148491A1 - Élément protéique assemblé et utilisation associée - Google Patents

Élément protéique assemblé et utilisation associée Download PDF

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WO2022148491A1
WO2022148491A1 PCT/CN2022/075886 CN2022075886W WO2022148491A1 WO 2022148491 A1 WO2022148491 A1 WO 2022148491A1 CN 2022075886 W CN2022075886 W CN 2022075886W WO 2022148491 A1 WO2022148491 A1 WO 2022148491A1
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protein
complementary
detection
complementary fragment
fgl1
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耿勇
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Shanghai Yongjian Biotechnology Co Ltd
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Shanghai Yongjian Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • 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
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • 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
    • 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
    • G01N33/56983—Viruses
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • 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
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the invention relates to the detection of various biomolecules in the fields of biomedicine, agriculture, animal husbandry, fishery and aquaculture. Specifically, it relates to an assembleable protein element and its use.
  • Complementary fragments of biological enzymes are linked to interacting proteins to observe the dynamic properties of interacting proteins.
  • the object of the present invention is to connect different single domain antibodies or scFV or Fab fragments of antibodies of the biological substances to be detected with the complementary fragments of biological enzymes to detect and analyze the content of biological substances.
  • a protein assembly that can be assembled with each other, the protein assembly comprising: n protein elements, wherein n is 2, 3 or 4, and each protein element has the structure of formula I,
  • i is a positive integer from 1 to n;
  • Pi is the ith complementary fragment of an active protein
  • Li is none, key or linker
  • NBi is the i-th anchoring element connected to Pi through Li;
  • n 1-3;
  • each Pi is a complementary fragment from the same active protein, and each Nbi is independently targeted and bound to a predetermined detection object;
  • each protein element binds to the detection object through their respective anchoring elements, so that all Pi can together form a reconstructed complementary fragment composite structure (that is, the reconstructed activity protein), the complementary fragment composite structure has or retains the activity of the active protein;
  • NBi ie, NB1, NB2, . or a combination thereof.
  • NBi is a single domain antibody (or nanobody).
  • NBi includes: (a) a single domain antibody, scFV, Fab fragment, or (b) 2 or more (such as 2, 3 or 4) tandem single domain antibodies, scFV , Fab fragment.
  • Li ie, L1, L2, . . . , Ln
  • L1, L2, . . . , Ln is each independently none, a bond or a linker.
  • the joint includes a flexible structure.
  • the linker is a peptide linker.
  • the peptide linker comprises 1-50 amino acids, preferably 3-40 amino acids, more preferably 5-30 amino acids.
  • the protein component when n is 2, includes: 2 protein elements, wherein the first protein element has the structure of formula Ia, and the second protein element has the structure of formula Ib,
  • P1 is the first complementary fragment of an active protein
  • L1 is none, key or connector
  • NB1 is the first anchoring element connected to P1 through L1;
  • P2 is the second complementary fragment of an active protein
  • L2 is none, key or joint
  • NB2 is the second anchoring element connected to P2 through L2;
  • n is each independently 1-3.
  • NB1 and NB2 are independently selected from Nanobodies targeting FGL1.
  • the protein component includes: 3 protein elements, wherein the first protein element has the structure of formula Ia, the second protein element has the structure of formula Ib, and the third protein element has the structure of formula Ic structure,
  • P1 is the first complementary fragment of an active protein
  • L1 is none, key or connector
  • NB1 is the first anchoring element connected to P1 through L1;
  • P2 is the second complementary fragment of an active protein
  • L2 is none, key or joint
  • NB2 is the second anchoring element connected to P2 through L2;
  • P3 is the third complementary fragment of an active protein
  • L3 is none, key or joint
  • NB3 is the third anchoring element connected to P3 through L3;
  • n is each independently 1-3.
  • the active protein is selected from the group consisting of enzymes, interacting protein complexes, and interacting proteins modified by small molecules that can generate signals.
  • the active protein is selected from the group consisting of luciferase, nuclease, endonuclease, protease and fluorescent protein.
  • the complementary fragment composite structure can catalyze the reaction of the substrate, thereby generating a detectable signal.
  • the detectable signal is selected from the group consisting of a fluorescent signal, an electrical signal or a chemical signal, or a combination thereof.
  • the substrate is selected from the group consisting of fluorescein, modified DNA fragments, modified polypeptide fragments, or a combination thereof.
  • each of the NBi is independently connected to the N-terminus, C-terminus, middle part, or a combination thereof of Pi through Li.
  • the predetermined detection object is selected from the group consisting of proteins, nucleic acids, and pathogenic microorganisms (pathogens).
  • the pathogen is selected from the group consisting of viruses, bacteria, chlamydia, fungi, and protists.
  • a protein assembly that can be assembled with each other is provided, the protein assembly includes a nanobody targeting FGL1 and a luciferase, and the two fragments of the nanobody targeting FGL1 and luciferase are mutually Assemble to form protein assemblies.
  • a protein assembly using a nanobody targeting FGL1 and two fragments of luciferase assembled with each other includes: two protein elements, wherein the first protein element has Ia As a result, the second protein component has an Ib structure,
  • P1 is the first complementary fragment of luciferase
  • L1 is a peptide linker
  • NB1 is the first anchoring element connected to P1 through L1;
  • P2 is the second complementary fragment of luciferase
  • L2 is a peptide linker
  • NB2 is a second anchoring element linked to P2 via L2.
  • each protein element binds to FGL1 through its respective nanobody, so that the two complementary fragments of luciferase can be reconstructed into luciferase, and the composite structure of the complementary fragments has or retains luciferase. active;
  • each protein element does not bind to FGL1, and P1 and P2 cannot be remodeled into luciferase (ie, cannot form active luciferase).
  • NB1 and NB2 are independently selected from Nanobodies targeting FGL1, and the complementarity determining regions (CDRs) of the VHH chains of the Nanobodies are one or more selected from the following group:
  • the VHH chain of the Nanobody further includes a framework region (FR), and the framework region is:
  • FR1 shown in SEQ ID NO:7 FR2 shown in SEQ ID NO:8, FR3 shown in SEQ ID NO:9, and FR4 shown in SEQ ID NO:10, or FR1 shown in SEQ ID NO:11 , FR2 shown in SEQ ID NO: 12, FR3 shown in SEQ ID NO: 13, and FR4 shown in SEQ ID NO: 14.
  • the VHH chain of the Nanobody has the amino acid sequence shown in any one of SEQ ID NOs: 15-16.
  • the peptide linkers described in L1 and L2 comprise 1-50 amino acids, preferably 10-20 amino acids.
  • nucleotide molecule encoding the mutually assembleable protein components of the first aspect.
  • the coding sequence encoding each protein element is located on one or more nucleotide sequences.
  • an expression vector in the third aspect of the present invention, contains the nucleotide molecule described in the second aspect.
  • the coding sequence encoding each protein element is located on one or more expression vectors.
  • the expression vector includes plasmid and viral vector.
  • a host cell comprises the expression vector of the third aspect; or the nucleotide molecule of the second aspect is integrated into the genome of the host cell .
  • the fifth aspect of the present invention there is provided a use of the mutually assembleable protein assemblies described in the first aspect, for the preparation of proteins, peptides, nucleic acids, viruses, bacteria, fungi, chlamydia, protist detection reagents or Reagent test kit.
  • a sixth aspect of the present invention provides a detection method, comprising:
  • the sample may contain a predetermined test object
  • the "observing whether a complementary fragment complex structure is formed in the detection system” comprises detecting a detectable signal generated by the complementary fragment complex structure.
  • the detection system there is a substrate that can be catalyzed by the complementary fragment complex structure.
  • the method is an in vitro or in vivo method.
  • the method is a non-diagnostic and non-therapeutic method.
  • the method is a diagnostic method.
  • Figure 1 shows an active protein that can be split into two complementary fragments
  • Figure 2 shows a schematic diagram of an example of the present invention, wherein two complementary fragments are linked to two different NB elements, respectively.
  • Figure 3 shows a schematic diagram of an example of the present invention in which two complementary fragments of luciferase are each linked to one or two different FGL1-targeting NB elements.
  • Figure 4 shows a schematic diagram of another example of the present invention, wherein two complementary fragments of a protein are linked to NB1 and NB2 elements, respectively; in addition, two complementary fragments of the protein are linked to NB3 and NB4 elements, respectively.
  • Figure 5 shows a schematic diagram of another example of the present invention, wherein two complementary fragments are attached to two different tandem NB elements, namely tandem NB1-NB2, tandem NB3 and NB4, respectively.
  • Figure 6 shows the results of gel electrophoresis of the fusion proteins 1D6-20X-LgBiT and 1E1-20X-SmBiT of the present invention.
  • Figure 7 shows the sensitivity test of the two fusion proteins of the present invention in the detection of hFGL1.
  • Figure 8 shows FGL1 concentrations in serum of esophageal cancer patients and normal subjects.
  • Figure 9 shows two single-domain antibodies acting on the RBD domain of the S protein of SARS-CoV-2, which are assembled into a system for detecting SARS-CoV-2 after being linked with the complementary fragment of luciferase.
  • Figure 10 is the data of virus detection using the system for detecting 2019-nCoV shown in Figure 9 (the RFU of the control group is much lower than the RFU of the experimental group).
  • single-domain antibodies including but not limited to nanobodies and synthetic single-domain antibodies, the definition of single-domain antibodies mentioned in the text is the same as here, and will not be repeated here.
  • a single domain antibody was found linked to the complementary fragment of biotinidase for detection of FGL1 molecules.
  • the present invention has been completed.
  • LAG3 (CD223), the lymphocyte activation gene 3, is an inhibitory receptor mainly expressed on the surface of T lymphocytes. Binding of LAG3 to its ligands transmits inhibitory signals to activated T cells, preventing immune-mediated tissue damage.
  • FGL1 emerged as a novel LAG3 ligand that binds to LAG3 to form a new immune checkpoint pathway independent of PD-1/PD-L1, leading to T cell exhaustion and subsequent dysfunction, and tumor cell evasion of immune surveillance.
  • FGL1 is upregulated in tumor tissues including lung, prostate, melanoma, colorectal, breast and brain tumors. Therefore, FGL1 has the potential to become another novel immune checkpoint target in clinical practice, especially in the targeted therapy of non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • the FGL1/LAG3 pathway plays an important role in immune evasion during cancer development, and antibodies to FGL1 may help overcome cancer immunotherapy resistance and become a promising new checkpoint target. Furthermore, plasma FGL1 secretion levels are a potential biomarker to identify patients who will not benefit from ICI therapy.
  • the new discovery of the immunologically high-affinity interaction of FGL1 and LAG3 marks a major breakthrough in the study of immune checkpoint blockade therapy. After targeting CTLA-4 and PD-1/PD-L1, this strategy has great potential as a third-generation immune checkpoint blocker.
  • a Nanobody targeting FGL1 is provided, and the complementarity determining region (CDR) of the VHH chain of the Nanobody is one or more selected from the following group:
  • Luciferase consists of two complementary large and small subunits, LgBiT of 18 kDa and SmBiT of 1.3 kDa, respectively.
  • the intrinsic affinity of the two complementary NanoBiT fragments in solution is too low to reconstitute luciferase.
  • SmBit or LgBiT fragments to link two nanobodies of FGL1 at appropriate distances, respectively, to help reconstitute luciferase and amplify bioluminescence.
  • single domain antibody sdAb, or VHH
  • nanobody a single domain antibody
  • VHH single domain antibody
  • VHH single domain antibody
  • Nanobody/single domain antibody (Nanobody), as a new type of small molecule antibody fragment, is cloned from the heavy chain variable region (VHH) of camelid natural heavy chain antibody.
  • VHH heavy chain variable region
  • Nanobody (Nb) has excellent biological properties, with a molecular weight of 12-15kDa, which is one tenth of that of a complete antibody. It has good tissue penetration, high specificity and good water solubility. Due to its special structural properties, it has the advantages of both traditional antibodies and small molecule drugs, and almost perfectly overcomes the shortcomings of traditional antibodies, such as long development cycle, low stability, and harsh storage conditions, and has gradually become a new generation of antibody therapy in the new generation. It shows broad application prospects in immunodiagnosis and therapy.
  • variable means that certain portions of the variable regions of an antibody differ in sequence that contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable regions are called the framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • the variable regions of native heavy and light chains each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs that form linking loops, and in some cases can form part of a ⁇ -sheet structure.
  • the CDRs in each chain are tightly packed together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.
  • variable region is used interchangeably with “complementarity determining region (CDR)”.
  • the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.
  • the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.
  • antibody of the present invention protein of the present invention
  • polypeptide of the present invention are used interchangeably and all refer to a polypeptide that specifically binds to FGL1, such as a protein or polypeptide having a heavy chain variable region. They may or may not contain the starting methionine.
  • the present invention also provides other protein or fusion expression products with the antibodies of the present invention.
  • the present invention includes any protein or protein conjugate and fusion expression product (ie, immunoconjugate and fusion expression product) having a variable region-containing heavy chain, as long as the variable region is associated with the heavy chain of an antibody of the invention
  • the variable regions are identical or at least 90% homologous, preferably at least 95% homologous.
  • variable regions which are separated into four framework regions (FRs), the amino acid sequence of the four FRs It is relatively conservative and does not directly participate in the binding reaction.
  • FRs framework regions
  • These CDRs form a circular structure, and the ⁇ -sheets formed by the FRs in between are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • Which amino acids make up the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.
  • fragment refers to polypeptides that retain substantially the same biological function or activity of an antibody of the invention.
  • a polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide with another compound (such as a compound that prolongs the half-life of a polypeptide, e.g.
  • polyethylene glycol polyethylene glycol
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or a secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or with 6His-tagged fusion protein.
  • the single-domain antibody or scFV or Fab fragment of the antibody acting on the detected biological substance is connected to the complementary fragment of the protease.
  • the enzyme is complementary. Fragments (complementary fragments of an enzyme refer to dividing the enzyme into different fragments, these different fragments will be combined to restore the activity of the enzyme) close together to form an enzyme with complementary activity, and the enzyme catalyzes the substrate to detect the amount of the substrate. Detect the presence or absence of biomolecules such as proteins, nucleic acids, viruses, and pathogenic microorganisms, and analyze their content. It is characterized in that the different single-domain antibodies or antibodies are linked with complementary fragments of various enzymes in various ways, and after the linkage, the antibodies bring these complementary fragments closer to form an active complementary enzyme.
  • FIG. 3 the schematic diagram of the detection method of the present invention is shown in FIG. 3 .
  • Two nanobodies targeting the fibrinogen-like protein FGL1 were linked to two complementary fragments of luciferase to form two fusion proteins.
  • the two fusion proteins were assembled into a system for detecting FGL1 for rapid detection of FGL1.
  • FGL1 is not present in the detection system, the two complementary fragments of luciferase cannot effectively combine to form luciferase.
  • the two nanobodies bind to FGL1, causing the two complementary fragments to come close to form a complete Luciferase, which emits light in the presence of a substrate.
  • Each protein element of the present invention has the structure of formula I,
  • Pi can be a biological enzyme.
  • the complementary fragments of biological enzymes are linked to different single-domain antibodies or scFV or Fab fragments of antibodies, and can be linked to the N-terminus or C-terminus of single-domain antibodies or scFV or Fab fragments of antibodies. It can be in any way, such as a linking sequence of amino acids, or a nucleic acid sequence or a polymeric molecular substance.
  • Complementary fragments of biological enzymes are linked with different single-domain antibodies or scFV or Fab fragments of antibodies, expressed by recombinant protein fusion or expressed separately and linked by chemical or biological methods.
  • the complementary fragments of biological enzymes are linked with different single-domain antibodies or scFV or Fab fragments of antibodies to form active enzymes.
  • the substrates are fluorescein, modified DNA fragments, and modified polypeptide fragments, and the product is a detectable fluorescent signal. , electrical or chemical signals.
  • each protein element of the present invention has the structure of formula I,
  • Pi can be a luciferase.
  • the complementary fragment of luciferase is linked to Nanobodies different from FGL1, and can be linked to the N-terminus or C-terminus of the Nanobody.
  • the complementary fragments of luciferase are linked with different FGL1 nanobodies to form active enzymes, the substrate is luciferin, and the product is a detectable luminescent signal.
  • the kind of active protein is not particularly limited.
  • Representative biological enzymes that can be linked to different single domain antibodies or scFV or Fab fragments of antibodies are enzymes that can be divided into complementary fragments, including (but not limited to): luciferases, nucleases, endonucleases Enzymes, proteases, and fluorescent proteins.
  • a detection system formed by linking complementary fragments of biological enzymes to different single-domain antibodies or scFV or Fab fragments of antibodies includes single use and several simultaneous use to improve detection sensitivity.
  • Complementary fragments of biological enzymes are linked to different monovalent or bivalent or multivalent tandem single domain antibodies or scFV or Fab fragments of antibodies to improve detection sensitivity.
  • the invention provides a new method for detecting biological substances, which is used for detecting proteins, peptides, nucleic acids, viruses, fungi, bacteria, chlamydia, etc., and is used for clinical diagnosis, animal husbandry and farming and agricultural planting.
  • the detection method of the present invention utilizes the protein assembly of the present invention, and the protein assembly is used for the detection of biological substances such as proteins, polypeptides, nucleic acids, etc., and the content of biomarker molecules of diseases is clinically detected for the diagnosis of diseases. It can also be used for routine physical examinations; it can be used to detect viruses, bacteria, fungi and other organisms to diagnose the source of infection of humans, animals and plants; it can also be used in laboratory research to detect the content and dynamic changes of biomolecules
  • the present invention provides a new method for detecting FGL1, which can be used for clinical diagnosis or treatment.
  • the detection method of the present invention utilizes the protein assembly of the present invention, applies the protein assembly to the detection of FGL1 molecules, and clinically detects the content of FGL1 molecules in the blood of diseased patients for disease diagnosis and treatment.
  • the protein assembly of the present invention can rapidly detect samples without the need to separate detection substances.
  • HEK 293S cells were used to express FGL1, and camels were immunized with FGL1 once a week. After seven weeks, the camel blood was drawn, lymphocytes were isolated, and RNA was extracted from them. Using this as a template for reverse transcription, a cDNA library was established. The heavy chain variable region VHH of the antibody was amplified therefrom and constructed into the pMECS vector.
  • the ELISA-positive bacterial liquid was sent to Suzhou Jinweizhi Co., Ltd. for Sanger sequencing. After sequence comparison, the base sequences corresponding to the nanobodies were found and classified according to sequence similarity.
  • the plasmid containing the nanobody sequence was transformed into E.coli WK6 competent cells, cultured in LB medium at 37°C, when the OD600 was between 0.6-0.8, 1mM IPTG was added to induce the expression of nanobody, and cultured at 28°C overnight, then Collect bacterial particles.
  • the bacterial particles were resuspended in buffer, and the bacteria were lysed by sonication to release intracellular proteins. Due to the 6x-His tag on the pMECS vector, Ni-NTA was used to purify the Nanobodies initially, and then further purification was carried out by size exclusion chromatography.
  • nanobodies 1D6 and 1H3 were obtained by recombinant production.
  • Nanobodies 1D6 and 1E1 against a pair of different epitopes of hFGL1 were connected to two fragments of NanoBiT, LgBiT and SmBiT, respectively, and the linker in the middle was a 20 amino acid peptide segment, and the final fusion protein was produced.
  • 1D6-20X-LgBiT and 1E1-20X-SmBiT were connected to two fragments of NanoBiT, LgBiT and SmBiT, respectively.
  • the plasmids containing the fusion protein sequence were transformed into E.coli WK6 competent cells, and cultured in LB medium at 37°C. When the OD 600 was between 0.6 and 0.8, 1 mM IPTG was added to induce the expression of nanobodies, and the cells were cultured at 28°C overnight. , and then collect the bacterial particles.
  • Ni-NTA was used to purify the nanobody initially, and then further purified by size exclusion chromatography.
  • a white opaque 96-well plate with Cat. No. Corning 3917 was used, and the buffer composition was: 100 mM MES, 1 mM CDTA, 0.5% NP40, 150 mM KCl, 1 mM DTT, 35 mM thiourea. 10nM 1D6-20X-LgBiT and 1E1-20X-SmBiT were added to each well, 1nM FGL1 was added to the experimental group, and an equal volume of buffer was added to the control group. After 1 h of incubation at 4°C, Furimazine was added to a final concentration of 10 ⁇ M and assayed immediately. The liquid volume per well was 120 ⁇ l.
  • Two single-domain antibodies acting on the RBD domain of the S protein of the new coronavirus are assembled with the complementary fragment of luciferase to form a new coronavirus detection system for rapid detection of the new coronavirus. As shown in Figures 9 and 10.

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  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé et une application permettant de tester le concept de substances biologiques telles que des protéines, des peptides, des virus et des micro-organismes ou de petites substances moléculaires biologiques par liaison d'un anticorps à domaine unique ou d'un fragment scFv ou Fab de l'anticorps à un fragment complémentaire d'une enzyme. En particulier, des molécules d'une protéase biologique sont séparées en au moins deux fragments différents, et le fragment complémentaire est lié à différents anticorps à domaine unique ou fragments scFv ou Fab des anticorps pour former un système de détection. Dans ledit système, l'anticorps à domaine unique agit sur une cible à tester, de sorte qu'une enzyme dotée d'une activité complémentaire soit assemblée, et une base est catalysée par l'activité de l'enzyme pour former un produit permettant d'analyser la teneur de la substance biologique testée, ce qui permet d'atteindre l'objectif du test de la cible.
PCT/CN2022/075886 2021-01-11 2022-02-10 Élément protéique assemblé et utilisation associée Ceased WO2022148491A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118255903A (zh) * 2024-05-07 2024-06-28 中国农业科学院兰州兽医研究所(中国动物卫生与流行病学中心兰州分中心) 一种检测非洲猪瘟病毒抗体的融合抗原、制备方法及检测试剂盒

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112876568A (zh) * 2021-01-11 2021-06-01 上海勇鉴生物科技有限公司 一种可组装蛋白原件及其用途

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112876568A (zh) * 2021-01-11 2021-06-01 上海勇鉴生物科技有限公司 一种可组装蛋白原件及其用途

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060240515A1 (en) * 2003-07-21 2006-10-26 Dimitrov Dimiter S Soluble fragments of the SARS-CoV spike glycoprotein
WO2016207313A1 (fr) * 2015-06-24 2016-12-29 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthodes et kits de détection d'interactions protéine-protéine
CN108226518A (zh) * 2017-11-27 2018-06-29 南京天纵易康生物科技股份有限公司 一种蛋白片段互补技术用于免疫诊断检测的方法
CN111592594B (zh) * 2020-03-13 2022-05-10 北京大学 一种抗新型冠状病毒的单克隆抗体及其应用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112876568A (zh) * 2021-01-11 2021-06-01 上海勇鉴生物科技有限公司 一种可组装蛋白原件及其用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MOUSTAQIL MEHDI, BHUMKAR AKSHAY, GONZALEZ LAURA, RAOUL LISA, HUNTER DOMINIC, CARRIVE PASCAL, SIERECKI EMMA, GAMBIN YANN: "A Split-Luciferase Reporter Recognizing GFP and mCherry Tags to Facilitate Studies of Protein–Protein Interactions", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 18, no. 12, 11 December 2017 (2017-12-11), pages 2681, XP055949893, DOI: 10.3390/ijms18122681 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118255903A (zh) * 2024-05-07 2024-06-28 中国农业科学院兰州兽医研究所(中国动物卫生与流行病学中心兰州分中心) 一种检测非洲猪瘟病毒抗体的融合抗原、制备方法及检测试剂盒

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