WO2024113229A1 - Procédé de détection in situ de ppi, vecteur, réactif de diagnostic, kit et utilisation - Google Patents

Procédé de détection in situ de ppi, vecteur, réactif de diagnostic, kit et utilisation Download PDF

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WO2024113229A1
WO2024113229A1 PCT/CN2022/135467 CN2022135467W WO2024113229A1 WO 2024113229 A1 WO2024113229 A1 WO 2024113229A1 CN 2022135467 W CN2022135467 W CN 2022135467W WO 2024113229 A1 WO2024113229 A1 WO 2024113229A1
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smbit
lgbit
fusion protein
protein
antir
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PCT/CN2022/135467
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English (en)
Chinese (zh)
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彭琴
李倩倩
高毅勤
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深圳湾实验室
染色质(北京)科技有限公司
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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  • the present invention belongs to the technical field of biomolecules, and specifically relates to a PPI in-situ detection method and a carrier, a diagnostic reagent, a kit and an application thereof.
  • PPI Protein-protein interaction
  • MDM2 Protein-protein interaction
  • PPI inhibitors are a new field of drug development, providing new ideas for non-enzyme protein targets that are traditionally "undruggable”. PPI also provides important targets and directions for clinical diagnosis.
  • the main techniques for in vitro research on PPI include immunoprecipitation, yeast two-hybridization and GST fusion protein pull-down. These techniques require cell lysis or exogenous expression of yeast, and it is difficult to truly indicate protein interactions in mammalian cells or tissues.
  • Fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) developed based on fluorescent proteins can detect protein interactions in living cells, but both methods rely on the introduction and overexpression of heterologous genes in mammalian cells. And due to the limitation of cell autofluorescence, fluorescence-based detection technology is difficult to reduce the background, resulting in limited signal-to-noise ratio and sensitivity.
  • proximity ligation technology can realize the detection of PPI in situ in cells, but this method requires chemical coupling of different nucleotide chains on antibodies, and after the end of immunolabeling, complex color development steps such as rolling circle amplification and fluorescent probe incubation are required. This method has a high false positive rate, is time-consuming and has high reagent costs.
  • the PPI detection technology developed based on split luciferase utilizes its bioluminescence and enzyme catalysis characteristics to not only eliminate the background to the greatest extent, but also amplify the signal by orders of magnitude, significantly improving the signal-to-noise ratio.
  • Nanoluciferase (Nanoluciferase; referred to as: NanoLuc) is a new type of luciferase discovered in recent years.
  • the enzyme has a small molecular weight, compared with firefly luciferase (61kDa) and sea renilla luciferase (36kDa), the molecular weight is only 19KD, which is convenient for fusion expression; the luminescence intensity is two orders of magnitude higher than that of traditional luciferase, and the linear range is up to six orders of magnitude, which is conducive to improving the detection sensitivity; it is independent of ATP (adenosine triphosphate), and the reaction with the dedicated substrate furimazine (luminescent substrate of imidazopyrazinones) is stable and is not affected by factors such as ambient temperature.
  • ATP adenosine triphosphate
  • NanoBit system for short
  • NanoLuc luciferase Binary Technology
  • the NanoBit system splits the complete NanoLuc protein into two subunits, including a large fragment of 18kDa (LgBit for short) and a small fragment of 1.3kDa (SmBit for short).
  • LgBit and SmBit are fused and expressed separately on the PPI protein to be detected, and the interaction of the target proteins can be indicated by the complementary activated luciferase activity.
  • the NanoBit system mainly indicates the degree of protein interaction in cells by overexpressing the above fusion protein system in mammalian cells.
  • this method requires customized plasmids and overexpression of heterologous proteins that vary from protein to protein, making it difficult to achieve in situ detection and clinical diagnosis of PPIs in primary cells and pathological tissues.
  • the invention patent with publication number CN111198272A discloses an in vitro method for detecting protein-protein interactions based on the NanoBiT system.
  • this method requires customized plasmids and overexpression of heterologous proteins that vary according to the protein, making it difficult to achieve in situ detection of PPIs in primary cells and pathological tissues.
  • the patent does not disclose the application of nanoantibodies.
  • this patent combines the advantages of the NanoBit system and Nanobody (Nb) to develop a universal method for rapid detection of PPI in situ, high-throughput, and complex samples.
  • One of the objects of the present invention is to provide a diagnostic reagent for in situ detection of PPI.
  • the present invention adopts the following technical solutions:
  • a diagnostic reagent for in situ detection of PPI comprising a fusion protein 1 expressing LgBit and a nanobody secondary antibody and/or a fusion protein 2 expressing SmBit and a nanobody secondary antibody;
  • the nanobody in the fusion protein 1 is an anti-mouse IgG1 Fc nanobody, and the fusion protein 1 comprises LgBit with a nucleotide sequence as shown in SEQ ID NO.3;
  • the nanobody in the fusion protein 2 is an anti-rabbit IgG Fc nanobody, and the fusion protein 2 comprises SmBit-antiR Nb with a nucleotide sequence as shown in SEQ ID NO.8.
  • the second object of the present invention is to provide an in situ detection method for detecting protein interactions using the diagnostic reagent, which realizes the in situ detection of PPI by constructing and expressing a fusion protein of LgBit or SmBit and a nanobody secondary antibody.
  • the present invention adopts the following technical solutions:
  • the in situ detection method for detecting protein interactions using the diagnostic reagent is used to prepare a nano antibody modified with a label protein through genetic engineering and biological fermentation, thereby realizing in situ detection of protein interactions.
  • the label protein is LgBit and/or SmBit.
  • the method comprises the following steps:
  • S3 Construct the SmBit-antiR Nb vector using the SmBit and the anti-rabbit IgG Fc nanoantibody;
  • S5 incubating the fusion protein 1 and the fusion protein 2 in a fixed cell sample or pathological tissue, adding a luciferase substrate, and determining whether the target protein pair interacts with each other by detecting the chemiluminescence intensity.
  • nanobodies modified with tagged proteins can be obtained in large quantities simply by constructing plasmids and biofermentation, with low cost and good stability.
  • This patent is based on the principle of Split NanoLuc. It optimizes LgBit-antiM Nb and SmBit-antiR Nb through prokaryotic expression to stabilize active proteins as universal secondary antibodies for detecting protein interactions. Only three steps are needed to detect and diagnose protein interactions in situ: 1. Select appropriate primary antibodies to incubate cells or tissue samples for specific labeling of target protein pairs; 2. Use prokaryotic expressed secondary antibodies to incubate samples for universal detection of protein interactions; 3. In situ luminescence or imaging can also be used for large-scale high-throughput screening.
  • the method specifically comprises the following steps:
  • the luminescence of the substrate indicates that the target protein pair interacts with each other; the non-luminescence of the substrate indicates that the target protein pair does not interact with each other.
  • LgBit and SmBit complement each other to form a complete NanoLuc luciferase, which catalyzes the substrate to emit light.
  • the amount of protein interaction is quantitatively analyzed by microplate reader and/or microscope imaging.
  • the third object of the present invention is to provide a LgBit-antiM Nb expression vector for constructing the fusion protein 1.
  • the present invention adopts the following technical solutions:
  • the LgBit-antiM Nb expression vector used to construct the fusion protein 1 and the LgBit_For and LgBit_Rev primers for LgBit-antiM Nb amplification are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively.
  • the fourth object of the present invention is to provide a SmBit-antiR Nb expression vector for constructing the fusion protein 2.
  • the present invention adopts the following technical solutions:
  • the SmBit-antiR Nb expression vector used to construct the fusion protein 2 the primers for SmBit-antiR Nb amplification include SmBit_F1 with a nucleotide sequence as shown in SEQ ID NO.4, SmBit_F2 with a nucleotide sequence as shown in SEQ ID NO.5, SmBit_R1 with a nucleotide sequence as shown in SEQ ID NO.6, and SmBit_R2 with a nucleotide sequence as shown in SEQ ID NO.7.
  • a fifth object of the present invention is to provide a use of the diagnostic reagent in the preparation of a kit for in situ detection of protein interactions.
  • the diagnostic reagent is used in the preparation of a kit for detecting interactions between proteins with low expression levels.
  • the diagnostic reagent is used in the preparation of a kit for screening pathology-related PPI targets.
  • the diagnostic reagent is used in the preparation of a product for high-throughput screening of PPI inhibitors.
  • a sixth object of the present invention is to provide a kit for in situ detection of PPI.
  • the present invention adopts the following technical solutions:
  • kits for detecting protein interactions the kit containing the fusion protein 1 expressing LgBit and a nanobody secondary antibody and/or the fusion protein 2 expressing SmBit and a nanobody secondary antibody.
  • the method of the present invention covers the use of LgBit or SmBit coupled to a nanoantibody secondary antibody for in situ detection of protein interactions; detection means include but are not limited to microplate readers and microscope imaging; detection applications include but are not limited to in situ detection of PPI in cells or tissues, high-throughput screening of PPI inhibitors, etc.
  • the detection method provided by this patent can detect protein interactions in situ in primary cells and pathological sections without the need to introduce heterologous genes
  • This patent uses commercial primary antibodies, which does not require labeling or development of primary antibodies for target proteins, and is easy to use; at the same time, the present invention uses biological fermentation to obtain a large amount of LgBit-antiM Nb and SmBit-antiR Nb, which is low in cost;
  • the present invention is based on the principle of NanoLuc bioluminescence, with a low detection limit. Compared with time-resolved fluorescence resonance energy transfer (TR FRET), it can detect protein interactions with lower expression levels and achieve high-throughput in situ detection of protein interactions;
  • TR FRET time-resolved fluorescence resonance energy transfer
  • the detection method of this patent is stable and reliable, can be used for large-scale universal detection, and is suitable for various sample detections such as clinical pathology.
  • this detection method is easy to operate, can quickly achieve detection and diagnosis, and is suitable for clinical test kit development;
  • the method of the present invention solves the problem of extremely weak natural protein interaction signals in mammalian cells or tissues.
  • ultra-strong luminescence of NanoLuc and the signal amplification function of antibodies By utilizing the ultra-strong luminescence of NanoLuc and the signal amplification function of antibodies, in situ detection of protein interactions of cells in a 96-well plate is achieved.
  • FIG1 is a schematic diagram showing the principle of the in situ detection of protein interactions.
  • Figure 2 is the SDS PAGE image of LgBit-antiM Nb and SmBit-antiR Nb.
  • FIG. 3 is an immunofluorescence image of ⁇ -tubulin and ⁇ -tubulin.
  • Figure 4 is a graph showing the results of detecting the interaction between ⁇ -tubulin and ⁇ -tubulin in MCF-7 cells based on the present invention.
  • G1 represents LgBit-antiM Nb+SmBit-antiR Nb
  • G2 represents Primary antibody+LgBit-antiM Nb+SmBit-antiR Nb
  • G3 represents Primary antibody+LgBit-antiM Nb
  • the vertical axis Lum represents the luminescence intensity.
  • Figure 5 is a graph showing the results of detecting the interaction between ⁇ -tubulin and ⁇ -tubulin in U2OS cells based on the present invention.
  • G1 represents LgBit-antiM Nb+SmBit-antiR Nb
  • G2 represents Primary antibody+LgBit-antiM Nb+SmBit-antiR Nb
  • G3 represents Primary antibody+LgBit-antiM Nb
  • the vertical axis Lum represents the luminescence intensity.
  • Figure 6 shows the results of TR FRET detection of the interaction between ⁇ -tubulin and ⁇ -tubulin in U2OS cells; in the horizontal axis, A1 represents ⁇ / ⁇ tubulin antibody+antiR Eu+antiM Alexa647; A2 represents ⁇ / ⁇ tubulin+antiR Eu; A3 represents antiR Eu+antiM Alexa647; the vertical axis 665/615nm ratio represents the FRET ratio.
  • Anti-mouse IgG1 Fc Anti-crystallizable fragment of mouse immunoglobulin 1;
  • Anti-rabbit IgG Fc anti-rabbit immunoglobulin crystallizable fragment
  • IgG a type of immunoglobulin
  • LgBit_For large fragment upstream primer
  • LgBit_Rev large fragment downstream primer
  • SmBit_F1 small fragment upstream primer 1; SmBit_F2: small fragment upstream primer 2; SmBit_R1: small fragment downstream primer 1; SmBit_R2: small fragment downstream primer 2;
  • ⁇ -tubulin rabbit mAb ⁇ -tubulin rabbit monoclonal antibody
  • ⁇ -tubulin mouse mAb ⁇ -tubulin mouse monoclonal antibody
  • Gibson assembly homologous recombination method A molecular cloning method that allows the ligation of multiple DNA fragments in a single isothermal reaction and is a common method for inserting fragments into plasmids without the use of restriction enzymes;
  • DH5 ⁇ a mutagenic strain of Escherichia coli
  • BL21(DE3)pLysS competent cells The cells are competent cells obtained by special processing of E. coli BL21(DE3)pLysS strain, which can be used for heat shock transformation of DNA;
  • Sanger sequencing A method of obtaining the DNA base sequence by starting with a fixed point, randomly ending at a specific base, and fluorescently labeling each base to produce a series of four groups of nucleotides of different lengths ending with A, T, C, and G;
  • LB medium The full name is Luria-Bertani medium, which is the most widely used and most common bacterial basic medium, sometimes also called ordinary medium;
  • HEPES 4-hydroxyethylpiperazineethanesulfonic acid
  • Imidazole 1-formyl imidazole
  • Primary antibody primary antibody
  • LgBit-antiM Nb N-terminally fused with anti-mouse IgG1 Fc nanoantibody expressing LgBit;
  • SmBit-antiR Nb N-terminally fused with anti-rabbit IgG Fc nanoantibody expressing SmBit;
  • antiR Nb nanoantibody against rabbit IgG Fc
  • ⁇ / ⁇ tubulin antibody ⁇ or ⁇ tubulin primary antibody
  • antiR Eu anti-rabbit IgG antibody labeled with Eu nanoparticles
  • antiM Alexa647 anti-mouse IgG antibody labeled with Alexa647;
  • pFN33K LgBiT TK-neo Vector Commercial plasmid from Promega;
  • pTP1122 plasmid commercial plasmid from Addgene
  • pTP955 plasmid commercial plasmid from Addgene
  • HiFi DNA Assembly Master Mix A commercial reagent for DNA fragment ligation from New England Biolabs.
  • Alexa647 A commercial fluorescent small molecule with an excitation light of 647nm.
  • the principle of the in situ detection method of protein interaction is shown in FIG1 .
  • the method specifically comprises the following steps:
  • LgBit_For and LgBit_Rev primers for LgBiT amplification were shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and the amplified sequence is shown in SEQ ID NO.3.
  • the antiR Nb gene sequence with a partial SmBit sequence was amplified from the pTP955 plasmid (commercial plasmid) using primers SmBit_F1 and SmBit_R1.
  • the antiR Nb gene sequence with homology arms and the complete SmBit sequence was amplified using primers SmBit_F2 and SmBit_R2.
  • the pTP955 plasmid (commercial plasmid) was digested with Hind III and BamH1 to obtain the vector.
  • HiFi DNA Assembly Master Mix Instructions Connect SmBit-antiR Nb with the digested vector. Transform the ligation product into DH5 ⁇ competent colonies, plate and pick single clones, use Sanger sequencing to identify and select the correct plasmid to obtain the prokaryotic expression plasmid of SmBit-antiR Nb.
  • SmBit_F1 is shown in SEQ ID NO.4
  • SmBit_F2 is shown in SEQ ID NO.5
  • SmBit_R1 is shown in SEQ ID NO.6
  • SmBit_R2 is shown in SEQ ID NO.7.
  • the sequence after amplification is shown in SEQ ID NO.8.
  • the LgBit-antiM Nb constructed in Example 1 and the SmBit-antiR Nb plasmids constructed in Example 2 were introduced into the BL21 (DE3) pLyss competent medium, and single clones were screened. Pick a single clone into 5 mL of fresh LB medium and culture it overnight at 37 degrees. Dilute the cultured E. coli into 1 L of fresh LB medium and culture it at 37 degrees for 2-3 hours. When the OD600 (i.e., the absorbance of the solution at a wavelength of 600 nm) reaches 0.4, use 0.2 mM IPTG (isopropyl- ⁇ -D-thiogalactoside) to induce expression for 20 hours, and the expression temperature is 18 degrees.
  • IPTG isopropyl- ⁇ -D-thiogalactoside
  • the lysis buffer consists of 50 mM HEPES, 500 mM sodium chloride and 20 mM Imidazole, with a pH of 7.5. Centrifuge at 20000g for 30 minutes, take the supernatant and purify the target protein with Ni-NTA (nickel ion metal chelate affinity chromatography medium). Use 5mL elution buffer to elute the target protein, the elution buffer consists of 50mM HEPES, 500mM sodium chloride and 500mM Imidazole, pH 7.5.
  • ⁇ -tubulin and ⁇ -tubulin usually form heterodimers and further polymerize into microtubules, so they are a good model for testing PPI.
  • ⁇ -tubulin rabbit mAb Company: abcam, Catalog No.: ab52866, dilution: 1:250
  • ⁇ -tubulin mouse mAb Company: abclonal, Catalog No.: AC021, dilution: 1:100
  • anti-rabbit IgG Alexa 488 Company: abcam, Catalog No.: ab150077, dilution: 1:1000
  • anti-mouse IgG Alexa 594 Company: CST, Catalog No.: 8890S, dilution: 1:1000
  • Example 6 In situ detection of protein interactions using ⁇ -tubulin and ⁇ -tubulin as models
  • MCF-7 human breast cancer cells
  • 96-well cell culture plates were plated, with 30,000 cells per well. After overnight culture, adherent cells were washed with PBS. Cells were fixed with 4% paraformaldehyde for 10 minutes, membranes were broken with 0.25% Triton (membrane breaking solution), and blocked with 5% BSA (bovine serum albumin) for two hours. Then, cells were incubated with ⁇ -tubulin rabbit mAb (company: abcam, catalog number: ab52866, dilution factor: 1:250) and ⁇ -tubulin mouse mAb (company: abclonal, catalog number: AC021, dilution factor: 1:100) overnight or at 37°C for 1 hour.
  • ⁇ -tubulin rabbit mAb company: abcam, catalog number: ab52866, dilution factor: 1:250
  • BSA bovine serum albumin
  • U2OS human osteosarcoma cells
  • 96-well cell culture plates were plated, with 30,000 cells per well.
  • the interaction between ⁇ -tubulin and ⁇ -tubulin was detected by the method shown in Example 5.
  • the results are shown in Figure 5.
  • the control group that is, no primary antibody or SmBit-antiR Nb was added
  • the chemiluminescence intensity of the experimental group was 4-6 times higher.
  • 96-well plates were plated with the same cell density. After overnight culture, the cells were fixed, permeated, and blocked as shown above, and the cells were incubated with ⁇ -tubulin and ⁇ -tubulin antibodies overnight or at 37°C for 1 hour.

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Abstract

L'invention concerne un procédé de détection in situ de PPI, un vecteur, un réactif de diagnostic, un kit et l'utilisation. Un plasmide LgBit-antiM Nb est construit à l'aide de LgBit et d'un nanocorps Fe anti-IgG1 de souris, et une expression optimisée est réalisée pour obtenir une protéine de fusion 1; un plasmide SmBit-antiR Nb est construit à l'aide de SmBit et d'un nanocorps Fe anti-IgG de lapin, et une expression optimisée est réalisée pour obtenir une protéine de fusion 2; la protéine de fusion 1 et la protéine de fusion 2 sont incubées dans un échantillon de cellules fixées ou un tissu pathologique, un substrat de luciférase est ajouté et l'interaction d'une paire de protéines cibles est déterminée par la détection de l'intensité de la chimiluminescence. Le procédé combine les avantages d'un système NanoBit et des nanocorps, résout le problème des signaux d'interaction extrêmement faibles des protéines endogènes dans les cellules ou les tissu de mammifères, et permet la détection in situ à haut débit d'une interaction de protéines à faible quantité d'expression.
PCT/CN2022/135467 2022-11-30 2022-11-30 Procédé de détection in situ de ppi, vecteur, réactif de diagnostic, kit et utilisation WO2024113229A1 (fr)

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