WO2018192365A1 - 一种检测系统 - Google Patents

一种检测系统 Download PDF

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WO2018192365A1
WO2018192365A1 PCT/CN2018/081798 CN2018081798W WO2018192365A1 WO 2018192365 A1 WO2018192365 A1 WO 2018192365A1 CN 2018081798 W CN2018081798 W CN 2018081798W WO 2018192365 A1 WO2018192365 A1 WO 2018192365A1
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cell
single domain
domain antibody
amino acid
protein
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PCT/CN2018/081798
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English (en)
French (fr)
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曹佳莉
袁权
张天英
赵菁华
张军
夏宁邵
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厦门大学
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • G01N33/56966—Animal cells
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
    • G01N2333/43595—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from coelenteratae, e.g. medusae

Definitions

  • the invention relates to the field of biotechnology.
  • the present invention relates to a detection system comprising a truncated body of a fluorescent protein and a single domain antibody against the fluorescent protein, the truncated body of the fluorescent protein being incapable of emitting fluorescence in a free state, but The single domain antibody is capable of emitting fluorescence upon binding.
  • the invention relates to various applications of the detection system.
  • Green Fluorescence protein (GFP) and other fluorescent proteins have been widely used for protein labeling, for example, in cells and even in animals. Targeting the protein of interest.
  • a protein tagging system based on complementary fragments of fluorescent proteins has also been reported (see Stéphanie Cabantous et al, Nature Biotechnology 23, 102-107 (2005)). Such systems can be used to detect the solubility of proteins, also known as shedding GFP systems.
  • the protein of interest is fused to a 16 amino acid fragment of GFP (amino acid 215-230, also known as GFP11 or G11), and simultaneously complements the complementary fragment of the GFP fragment (amino acids 1-214). ).
  • GFP11 or G11 16 amino acid fragment of GFP
  • These two GFP fragments are capable of spontaneously folding into intact GFP in a soluble state and fluoresce, thereby being useful for detecting and quantifying the solubility of proteins in vivo and in vitro.
  • shedding GFP system has also been applied to protein labeling, and it has been reported that multiple GFP11 repeats can enhance the fluorescence intensity of recombinant GFP (see Kamiyama D. et al., Nature Communications, 2016 Mar 18; 7: 11046). .
  • a single domain antibody is the heavy chain variable region of a camelid single chain antibody.
  • Camel single-chain antibodies contain only heavy chains and no light chains. Thus, the heavy chain variable region of a single chain antibody binds to the antigen.
  • These antibodies have the advantages of small molecular weight, good stability, high specificity, easy expression, good tissue permeability, etc., and have received extensive attention in the field of biotechnology research and diagnostic applications.
  • anti-GFP single domain antibodies can enhance or attenuate GFP fluorescence upon binding to GFP (see Kirchhofer A. et al, Nature Structural & Molecular Biology, 2010 Jan; 17(1): 133 -8).
  • the inventors have unexpectedly discovered that certain single domain antibodies against fluorescent proteins (eg, GFP) are capable of specifically binding to and fluoresce a truncated body of a fluorescent protein (eg, GFP) that is not fluorescent upon itself. .
  • the inventors of the present application designed and developed a new detection system based on the combined use of fluorescent protein-free non-luminescent fragments and single-domain antibodies against fluorescent proteins, and can be widely used in biotechnology research and diagnosis. field.
  • fluorescent protein refers to a protein that is capable of emitting light of a particular wavelength (fluorescence) under irradiation of a certain excitation light.
  • fluorescent proteins of various colors have been discovered, including, but not limited to, green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, red fluorescent protein and the like.
  • the structure of the fluorescent proteins of various colors and their luminescence mechanisms have been explained in detail (see, for example, Yang F et al. Nat Biotechnol. 1996 Oct; 14(10): 1246-51; Mark Wall et al. Nat. Struct. Biol. 7, 1133 - 1138, 2000; and Reid BG et al. Biochemistry. 1997 Jun 3; 36(22): 6786-91).
  • an exemplary amino acid sequence of green fluorescent protein is set forth in SEQ ID NO: 84; an exemplary amino acid sequence of blue fluorescent protein is set forth in SEQ ID NO: 85; an exemplary amino acid sequence of yellow fluorescent protein is SEQ. ID NO: 86 is shown.
  • fluorescent proteins of various colors have similar amino acid sequences and structures, and their main difference is that the domains involved in the excitation of fluorescence (for example, aa 65-67 of green fluorescent protein) are composed of different amino acid residues.
  • the technical effects confirmed by the present application based on green fluorescent protein can be extended to fluorescent proteins of other colors (for example, blue fluorescent protein and yellow fluorescent protein).
  • the expression "the C-terminus of a protein is truncated by 9-23 amino acid residues” means that 9-23 amino acid residues at the C-terminus of the protein are deleted.
  • the term "variant" when used in the context of a protein/polypeptide refers to a protein whose amino acid sequence is compared to the amino acid sequence of a reference protein/polypeptide (eg, a truncated body of the invention) Having one or more (eg, 1-15, 1-10, 1-5, or 1-3) amino acid differences (eg, additions, substitutions, or deletions of amino acid residues, such as conservative substitutions), or having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity, and which retains The necessary properties of the reference protein/polypeptide.
  • the essential property of the truncated body of the present invention may mean that it does not emit fluorescence in a free state, but is capable of emitting fluorescence upon binding to a single domain antibody.
  • the term "identity" is used to mean the matching of sequences between two polypeptides or between two nucleic acids.
  • a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or two
  • Each position in each of the polypeptides is occupied by lysine, and then each molecule is identical at that position.
  • the "percent identity" between the two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 of the 10 positions of the two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • the comparison is made when the two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0), using the PAM 120 weight residue table.
  • the gap length penalty of 12 and the gap penalty of 4 were used to determine the percent identity between the two amino acid sequences.
  • the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithms in the GAP program integrated into the GCG software package can be used, using the Blossum 62 matrix or The PAM250 matrix and the gap weight of 16, 14, 12, 10, 8, 6 or 4 and the length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the essential properties of a protein/polypeptide comprising an amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions of amino acid residues with similar side chains in place of amino acid residues, for example, physically or functionally similar to corresponding amino acid residues (eg, having similar size, shape, charge, chemical properties, including Substitution of residues by formation of a covalent bond or a hydrogen bond, etc.).
  • a family of amino acid residues having similar side chains has been defined in the art.
  • These families include basic side chains (eg, lysine, arginine, and histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine) , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg alanine, valine, leucine, isoluminescence) Acid, valine, phenylalanine, methionine), beta branch side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, Amino acids of phenylalanine, tryptophan, histidine).
  • basic side chains eg, lysine, arginine, and histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eg, glycine
  • single domain antibody means an antibody comprising an antibody heavy chain variable region but no light chain variable region.
  • An antibody also known as a heavy chain antibody
  • the antigen-binding region of the heavy chain antibody i.e., the heavy chain variable region
  • the antigen-binding region is isolated from the heavy chain antibody. It still has the function of binding antigen (see, for example, Hamers-Casterman C et al, Nature.
  • single domain antibody is intended to encompass such heavy chain antibodies comprising only the heavy chain but not the light chain, as well as antigen binding fragments thereof (eg, heavy chain variable regions).
  • a “single domain antibody” in the present application can comprise a heavy chain variable region comprising three CDRs and, optionally, can further comprise a hinge region, an Fc region, or a heavy chain constant region.
  • the single domain antibody comprises a heavy chain variable region comprising three CDRs.
  • the single domain antibody comprises a heavy chain variable region comprising three CDRs and a hinge region, an Fc region, or a heavy chain constant region.
  • vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector is referred to as an expression vector when the vector enables expression of the protein encoded by the inserted polynucleotide.
  • the vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be 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.
  • polypeptide and “protein” have the same meaning and are used interchangeably.
  • amino acids are generally represented by single letter and three letter abbreviations as are known in the art.
  • alanine can be represented by A or Ala.
  • the present application is based, at least in part, on the surprising discovery by the inventors that certain single domain antibodies against fluorescent proteins (eg, GFP) are capable of specifically binding to a truncated body of a fluorescent protein (eg, GFP) that is not fluorescent, and Fluorescence.
  • a fluorescent protein eg, GFP
  • the inventors of the present application designed and developed a new detection system based on the combined use of fluorescent protein-free non-luminescent fragments and single-domain antibodies against fluorescent proteins, and can be widely used in biotechnology research and diagnosis. field.
  • the invention provides a kit comprising two components, wherein the first component comprises:
  • (a2) a variant of a truncation body as defined in (a1), said variant having at least 85% identity with said truncation, or wherein said variant differs from said truncation by Addition, substitution or deletion of one or more amino acid residues; or
  • (a3) a nucleic acid molecule comprising a nucleotide sequence encoding a truncation as defined in (a1) or a variant as defined in (a2);
  • the second component comprises:
  • a single domain antibody against an anti-fluorescent protein preferably, it comprises CDR1, CDR2 and CDR3 selected from the group consisting of:
  • (b2) a nucleic acid molecule comprising a nucleotide sequence encoding a single domain antibody as defined in (b1);
  • the truncated body and the variant do not fluoresce in a free state, but are capable of emitting fluorescence upon binding to the single domain antibody.
  • the fluorescent protein is selected from the group consisting of a green fluorescent protein, a blue fluorescent protein, and a yellow fluorescent protein.
  • the green fluorescent protein has the amino acid sequence set forth in SEQ ID NO:84.
  • the blue fluorescent protein has the amino acid sequence set forth in SEQ ID NO:85.
  • the yellow fluorescent protein has the amino acid sequence set forth in SEQ ID NO:86.
  • the truncated body differs from the fluorescent protein in that the C-terminus of the fluorescent protein is truncated by 9-23 amino acid residues, eg, truncated 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acid residues.
  • the truncated body is a truncated form of green fluorescent protein and differs from green fluorescent protein in that the C-terminus of the green fluorescent protein is truncated by 9-23 amino acid residues, For example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acid residues are truncated.
  • the green fluorescent protein has the amino acid sequence set forth in SEQ ID NO:84.
  • the truncated form of the green fluorescent protein has the amino acid sequence set forth in SEQ ID NO:31.
  • the truncated body is a truncated form of blue fluorescent protein and differs from blue fluorescent protein in that the C-terminus of the blue fluorescent protein is truncated by 9-23 amino acids. Residues, for example, are truncated by 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acid residues.
  • the blue fluorescent protein has the amino acid sequence set forth in SEQ ID NO:85.
  • the truncated body is a truncated form of yellow fluorescent protein, and differs from yellow fluorescent protein in that the C-terminus of the yellow fluorescent protein is truncated by 9-23 amino acid residues, For example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acid residues are truncated.
  • the yellow fluorescent protein has the amino acid sequence set forth in SEQ ID NO:86.
  • the amino acid sequence of the variant has at least 85% identity to the amino acid sequence of the truncation, eg, at least 90%, at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.
  • the variant differs from the truncated body by the addition, substitution or deletion of one or more amino acid residues, such as no more than 15, no more than 14, no more than 13 No more than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3 Addition, substitution or deletion of no more than 2 or 1 amino acid residues.
  • the variant differs from the truncated body by a substitution (eg, a conservative substitution) of one or more amino acid residues, such as no more than 15, no more than 14, no more than 13, no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than A substitution of three, no more than two, or one amino acid residues (eg, a conservative substitution).
  • a substitution eg, a conservative substitution of one or more amino acid residues, such as no more than 15, no more than 14, no more than 13, no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than A substitution of three, no more than two, or one amino acid residues (eg, a conservative substitution).
  • the truncation or variant has an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-46.
  • the single domain antibody comprises a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-9 and 87-88. In certain preferred embodiments, the single domain antibody consists of the heavy chain variable region. In certain preferred embodiments, the single domain antibody comprises the heavy chain variable region, and optionally a hinge region, an Fc region, or a heavy chain constant region.
  • the nucleic acid molecule of (a3) comprises a nucleotide sequence encoding a truncation as defined in (a1) or a variant as defined in (a2), or by encoding as The truncated body defined in a1) or the nucleotide sequence of the variant as defined in (a2).
  • the nucleic acid molecule of (a3) is a vector comprising a nucleotide sequence encoding a truncation as defined in (a1) or a variant as defined in (a2) (eg, expression Carrier).
  • the nucleic acid molecule of (b2) comprises a nucleotide sequence encoding a single domain antibody as defined in (b1), or a core encoding a single domain antibody as defined in (b1)
  • the composition of the nucleotide sequence is a vector (eg, an expression vector) comprising a nucleotide sequence encoding a single domain antibody as defined in (b1).
  • the kit comprises a truncation as defined in (a1) or a variant as defined in (a2), and a single domain antibody as defined in (b1). In certain preferred embodiments, the kit comprises a truncation as defined in (a1) or a variant as defined in (a2), and the nucleic acid molecule of (b2).
  • the kit comprises the nucleic acid molecule of (a3), and the single domain antibody as defined in (b1). In certain preferred embodiments, the kit comprises the nucleic acid molecule of (a3), and (b2) the nucleic acid molecule.
  • the kit further comprises additional reagents.
  • additional reagents include, but are not limited to, reagents for molecular cloning or for constructing vectors, such as buffers for nucleic acid amplification, nucleic acid polymerases, endonucleases, ligases, for performing nucleic acids Purified reagents, reagents for performing nucleic acid transformation, transfection or transduction, and/or nucleic acid vectors (eg, plasmids or viral vectors).
  • reagents for molecular cloning or for constructing vectors such as buffers for nucleic acid amplification, nucleic acid polymerases, endonucleases, ligases, for performing nucleic acids Purified reagents, reagents for performing nucleic acid transformation, transfection or transduction, and/or nucleic acid vectors (eg, plasmids or viral vectors).
  • the invention provides a method of determining the location or distribution of a protein of interest comprising using a kit of the invention.
  • the invention provides a method of determining the location or distribution of a protein of interest, comprising:
  • Co-expressing (1) a truncated or mutant as defined above, and (2) a fusion protein comprising a single domain antibody as defined above and said protein of interest;
  • the method further comprises observing the cells using a fluorescence microscope.
  • the method further comprises observing the cells using a fluorescence microscope.
  • the method comprises the steps of:
  • the method comprises the steps of:
  • the method comprises the steps of:
  • the method comprises the steps of:
  • the method comprises the steps of:
  • the method comprises the steps of:
  • the vector can be introduced into the cell by any suitable means.
  • Such means include, but are not limited to, transformation (eg, protoplast transformation), transfection (eg, lipofection), electroporation, transduction (eg, phage transduction), and the like.
  • methods for stably expressing a protein of interest in a cell are known to those skilled in the art.
  • a protein of interest can be stably expressed in a cell by integrating an exogenous nucleotide sequence encoding a protein of interest into the genome of the cell.
  • Methods for integrating an exogenous nucleotide sequence into the genome of a target cell are also known to those skilled in the art (see, for example, Oberbek A et al, Biotechnol Bioeng. 2011 Mar; 108(3): 600-10).
  • the invention provides a method of determining whether cell fusion occurs, comprising using a kit of the invention.
  • the invention provides a method of determining whether cell fusion occurs, comprising:
  • step (2) after co-cultivating the first cell and the second cell, optionally, subjecting the first cell and the second cell to treatment, and then Fluorescence microscopy was used to see if fluorescence was present.
  • Fluorescence microscopy was used to see if fluorescence was present.
  • the first cell and the second cell are subjected to the treatment under the conditions in which the treatment is not performed, it takes a longer time to observe the fluorescence, or a weaker fluorescence is observed at the same time point. Then, it can be determined that the treatment prevents or inhibits cell fusion.
  • the treatment can be any desired operation, such as physical stimulation (eg, thermal stimulation, radiation, etc.), chemical stimulation (eg, contact with a candidate drug or agent), or biological stimulation (eg, contact with a pathogen (eg, a virus or bacteria)).
  • physical stimulation eg, thermal stimulation, radiation, etc.
  • chemical stimulation eg, contact with a candidate drug or agent
  • biological stimulation eg, contact with a pathogen (eg, a virus or bacteria)
  • the methods can be used to screen for stimuli, drugs, agents, or pathogens (eg, viruses or bacteria) that are capable of inducing or inhibiting cell fusion.
  • the invention provides a method of determining the ability of an agent or pathogen (eg, a virus or a bacterium) to induce or inhibit cell fusion, comprising the steps of:
  • step (3) if no fluorescence is observed in step (2) and fluorescence is observed in step (3), then the agent or pathogen can be determined to have the ability to induce cell fusion.
  • the invention provides a method of determining the ability of an agent or pathogen (eg, a virus or a bacterium) to induce or inhibit cell fusion, comprising the steps of:
  • the agent or pathogen is determined to have the ability to induce or promote cell fusion. Conversely, if more time is required to observe fluorescence in the experimental group culture than in the control culture, or if the experimental group culture exhibits weaker fluorescence at the same time point, then the reagent can be determined. Or the pathogen has the ability to block or inhibit cell fusion.
  • the first cell can express the truncation or mutant in a variety of suitable manners and cause the second cell to express the single domain antibody.
  • the first cell expresses the truncation or mutant by introducing a vector comprising a nucleotide sequence encoding the truncation or mutant into a first cell.
  • the first cell stably expresses the truncation or mutant by integrating a nucleotide sequence encoding the truncation or mutant into the genome of the first cell.
  • the second cell is expressed by the second cell by introducing a vector comprising a nucleotide sequence encoding the single domain antibody into the second cell.
  • the second cell is stably expressed by the second cell by integrating the nucleotide sequence encoding the single domain antibody into the genome of the second cell.
  • the vector can be introduced into the cell by any suitable means.
  • suitable means include, but are not limited to, transformation (eg, protoplast transformation), transfection (eg, lipofection), electroporation, transduction (eg, phage transduction), and the like.
  • transformation eg, protoplast transformation
  • transfection eg, lipofection
  • electroporation eg, phage transduction
  • transduction eg, phage transduction
  • methods for integrating an exogenous nucleotide sequence into the genome of a target cell are known to those skilled in the art (see, for example, Oberbek A et al, Biotechnol Bioeng. 2011 Mar; 108(3): 600-10). .
  • the invention provides a method of assessing the ability of an agent to promote or inhibit the passage of a polypeptide across a cell membrane, comprising using a kit of the invention.
  • the invention provides a method of assessing the ability of an agent to promote or inhibit the passage of a polypeptide across a cell membrane, comprising:
  • the cells of the experimental group exhibit stronger fluorescence, then it can be determined
  • the reagents have the ability to promote passage of the polypeptide across the cell membrane. Conversely, if it takes longer to observe fluorescence in the cells of the experimental group than at the control cells, or if the cells in the experimental group exhibit weaker fluorescence at the same time point, then it can be determined that the agent has a blocking polypeptide The ability to cross cell membranes.
  • the truncated or mutant can be expressed by the cells by any suitable means.
  • the vector is expressed by the cell, such that the vector comprising the nucleotide sequence encoding the truncation or mutant is introduced into the cell.
  • the cell stably expresses the truncation or mutant by integrating a nucleotide sequence encoding the truncation or mutant into the genome of the cell.
  • the invention provides a method of assessing the ability of an agent to promote or inhibit the passage of a polypeptide across a cell membrane, comprising:
  • the cells of the experimental group exhibit stronger fluorescence, then it can be determined
  • the reagents have the ability to promote passage of the polypeptide across the cell membrane. Conversely, if it takes longer to observe fluorescence in the cells of the experimental group than at the control cells, or if the cells in the experimental group exhibit weaker fluorescence at the same time point, then it can be determined that the agent has a blocking polypeptide The ability to cross cell membranes.
  • the cells can be expressed by the single domain antibody in a variety of suitable manners.
  • the single domain antibody is expressed by a cell by introducing a vector comprising a nucleotide sequence encoding the single domain antibody into a cell.
  • the single domain antibody is stably expressed by integrating a nucleotide sequence encoding the single domain antibody into the genome of the cell.
  • the vector can be introduced into the cell by any suitable means.
  • suitable means include, but are not limited to, transformation (eg, protoplast transformation), transfection (eg, lipofection), electroporation, transduction (eg, phage transduction), and the like.
  • transformation eg, protoplast transformation
  • transfection eg, lipofection
  • electroporation eg, phage transduction
  • transduction eg, phage transduction
  • methods for integrating an exogenous nucleotide sequence into the genome of a target cell are known to those skilled in the art (see, for example, Oberbek A et al, Biotechnol Bioeng. 2011 Mar; 108(3): 600-10). .
  • the single domain antibody GBP1 is capable of enhancing the fluorescence of GFP.
  • the single domain antibody GBP1 is capable of restoring luminescent ability to GFP truncates that have lost the ability to fluoresce.
  • the inventors have demonstrated for the first time that certain anti-GFP single domain antibodies (e.g., GBP1) are capable of restoring luminescent light to a non-luminescent, truncated body of a fluorescent protein (e.g., GFP). This property of such single domain antibodies (e.g., GBP1) is particularly advantageous.
  • various detection systems can be constructed using a combination of the single domain antibody (eg, GBP1) and a truncation of a fluorescent protein (eg, GFP), thereby facilitating various biological assays, such as Protein localization, detection of cell fusion, assessment of transmembrane ability, etc.
  • GBP1 single domain antibody
  • GFP fluorescent protein
  • the detection system of the present invention comprising a truncated body of a single domain antibody (e.g., GBP1) and a fluorescent protein (e.g., GFP) has the following advantages as compared to the previously reported exfoliated GFP system (sfGFP1-10 + G11):
  • G11 in the shedding GFP system is fused to the target protein.
  • G11 when G11 is linked to the N-terminus of the protein of interest, its ability to restore fluorescence to sfGFP1-10 may be affected or even lost.
  • the single domain antibody (for example, GBP1) in the detection system of the present invention does not have this problem, and can be fused to the N-terminus or C-terminus of the target protein by various linkage methods without affecting the function of the protein. .
  • G11 has a small molecular weight, and therefore, when it is freely expressed in cells, it is easily degraded.
  • single domain antibodies e.g., GBP1 in the detection system of the present invention do not have this problem and are relatively stable intracellularly.
  • the detection system of the present invention comprising a truncated body of a single domain antibody (for example, GBP1) and a fluorescent protein (for example, GFP) can be applied more widely, conveniently, and flexibly.
  • a single domain antibody for example, GBP1
  • a fluorescent protein for example, GFP
  • Figure 1 shows the results of fluorescence microscopy of co-transfected expression plasmids encoding single domain antibody and pTT22M-sfGFP1-10 Hela cells 48 h after transfection; for each cell of the experimental group, the upper panel shows red Observation of the light channel (used to indicate transfection efficiency), the figure below shows the observation of the green channel (used to show whether the cells emit green fluorescence); the "vector" group indicates that the empty vectors pTT5 and pTT22M-sfGFP1 were transfected -10 Hela cells.
  • Figure 2 shows the results of fluorescence microscopy of the expression plasmid encoding the C-terminal truncation variant of sfGFP and HeLa cells of PTT5 (Fig. 2A) or pTT5-GBP1 (Fig. 2B) at 48 h after transfection;
  • the "WT” group represents Hela cells co-transfected with an expression plasmid encoding the fluorescent protein sfGFP and pTT5 (Fig. 2A) or pTT5-GBP1 (Fig. 2B).
  • Figure 3 shows the results of fluorescence microscopy of HeLa cells co-transfected with pTT5-GBP1 and the expression plasmid encoding the sfGFP1-10 variant at 48 h after transfection; wherein the "Negative" group indicates co-transfection of pTT5-GBP1 and Hela cells encoding expression plasmids of irrelevant proteins.
  • Figure 4 shows the results of fluorescence microscopy of HeLa cells co-transfected with pTT5-GBP1 and pTT22M-BFP1-10 or pTT22M-YFP1-10 at 48 h after transfection; wherein “B/Y” indicates blue/yellow channel Observations; “R” indicates the observation of the red channel; “Merge” indicates the combination of the observations of the two channels.
  • Figure 5 shows the results of fluorescence microscopy of HeLa cells co-transfected with various expression plasmid combinations at 48 h after transfection; among the cells of each experimental group, the upper panel shows green fluorescence in Hela cells (by fusion) Distribution and location of GBP1+sfGFP1-10 in the protein; the middle panel shows the distribution and location of blue fluorescence (produced by BFP in the fusion protein) in Hela cells; the lower panel shows the upper and middle panels Merger.
  • Figure 6 shows the results of fluorescence microscopy of Hep2-GBP1 cell suspension, Hep2-Mbcd38 cell suspension, and cell suspension containing Hep2-GBP1 and Hep2-Mbcd38 after 48 hours of infection with RSV virus.
  • Figure 7 shows the results of fluorescence microscopy of U2OS cells expressing Mdc2-26 after incubation with GBP1 or GBP1+ transmembrane peptide pep1 for 6h, 8h, 10h or 12h.
  • Figure 8 shows the results of fluorescence microscopy of 293 cells co-transfected with various expression plasmid combinations at 48 h after transfection.
  • Figure 9 shows the results of fluorescence microscopy of Hela cells co-transfected with Mdc2-26 and GBP1 or GBPMT1 or GBPMTT at 48 h after transfection.
  • Variable region amino acid sequence of single domain antibody P-Nb1 28 Variable region amino acid sequence of single domain antibody S-Nb1 29 Variable region amino acid sequence of single domain antibody S-Nb5 30 Variable region amino acid sequence of single domain antibody S-Nb27 31 Amino acid sequence of sfGFP1-10 32 Amino acid sequence of Mdc2-26 33 Amino acid sequence of Mdc24 34 Amino acid sequence of Mbcd3 35 Amino acid sequence of Mbcd4 36 Amino acid sequence of Mbcd36 37 Amino acid sequence of Mbcd37 38 Amino acid sequence of Mbcd38 39 Amino acid sequence of Mbcd39 40 Amino acid sequence of Mbcd41 41 Amino acid sequence of Mbcd44 42 Amino acid sequence of Mbcd52 43 Amino acid sequence of test3-3 44 Amino acid sequence of test5-3 45 Amino acid sequence of BFP1-10 46 Amino acid sequence of YFP1-10 47-49
  • the molecular biology experimental methods and immunoassays used in the present invention are basically referred to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and The method described in FMAusubel et al., Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes according to the conditions recommended by the product manufacturer.
  • the invention is described by way of example, and is not intended to limit the scope of the invention.
  • Example 1 Construction of an expression plasmid encoding an anti-GFP single domain antibody
  • DNA fragments encoding these 30 single domain antibodies were synthesized by Shanghai Shenggong Bioengineering Co., Ltd., respectively.
  • the polymerase chain reaction (PCR) was carried out using the primers VHHF and VHHR using the 30 synthetic DNA fragments as templates.
  • the conditions of the PCR reaction were: 98 ° C, 10 min; 30 cycles (98 ° C, 30 s; 58 ° C, 30 s; 68 ° C, 30 s); 68 ° C, 5 min.
  • the sequences of the primers VHHF and VHHR are shown in Table 2.
  • a product having a size of about 400 bp was recovered.
  • the recovered PCR products were separately ligated into the commercially available pTT5 vector by the following steps: the pTT5 vector was digested with BamHI/HindIII, and then the recovered PCR product and the enzyme-cut were digested with NEB's Gibson Assembly reagent. The pTT5 vectors are ligated together. DH5 ⁇ competent cells were transformed with the obtained ligation product, and cultured in a 37 ° C incubator for 12 hours. Subsequently, monoclonal colonies were picked, plasmids were extracted, and sequenced to obtain an expression plasmid encoding an anti-GFP single domain antibody.
  • pTT5-GBP1 which encodes an anti-GFP single domain antibody GBP1 (SEQ ID NO: 1);
  • pTT5-NbsfGFP08 which encodes an anti-GFP single domain antibody NbsfGFP08 (SEQ ID NO: 2);
  • pTT5-S-Nb2 which encodes an anti-GFP single domain antibody S-Nb2 (SEQ ID NO: 3);
  • pTT5-S-Nb3 which encodes an anti-GFP single domain antibody S-Nb3 (SEQ ID NO: 4);
  • pTT5-S-Nb6 which encodes an anti-GFP single domain antibody S-Nb6 (SEQ ID NO: 5);
  • pTT5-S-Nb7 which encodes an anti-GFP single domain antibody S-Nb7 (SEQ ID NO: 6);
  • pTT5-S-Nb17 which encodes an anti-GFP single domain antibody S-Nb17 (SEQ ID NO: 7);
  • pTT5-S-Nb21 which encodes an anti-GFP single domain antibody S-Nb21 (SEQ ID NO: 8);
  • pTT5-S-Nb25 which encodes an anti-GFP single domain antibody S-Nb25 (SEQ ID NO: 9);
  • pTT5-GBP4 which encodes an anti-GFP single domain antibody GBP4 (SEQ ID NO: 10);
  • pTT5-GBPSR1 which encodes an anti-GFP single domain antibody GBPSR1 (SEQ ID NO: 11);
  • pTT5-GBPSR2 which encodes an anti-GFP single domain antibody GBPSR2 (SEQ ID NO: 12);
  • pTT5-LAG2 which encodes an anti-GFP single domain antibody LAG2 (SEQ ID NO: 13);
  • pTT5-LAG9 which encodes an anti-GFP single domain antibody LAG9 (SEQ ID NO: 14);
  • pTT5-LAG14 which encodes an anti-GFP single domain antibody LAG14 (SEQ ID NO: 15);
  • pTT5-GBP1 which encodes an anti-GFP single domain antibody LAG16 (SEQ ID NO: 16);
  • pTT5-LAG26 which encodes an anti-GFP single domain antibody LAG26 (SEQ ID NO: 17);
  • pTT5-LAG27 which encodes an anti-GFP single domain antibody LAG27 (SEQ ID NO: 18);
  • pTT5-LAG30 which encodes an anti-GFP single domain antibody LAG30 (SEQ ID NO: 19);
  • pTT5-LAG41 which encodes an anti-GFP single domain antibody LAG41 (SEQ ID NO: 20);
  • pTT5-NbsfGFP01 which encodes an anti-GFP single domain antibody NbsfGFP01 (SEQ ID NO: 21);
  • pTT5-NbsfGFP02 which encodes an anti-GFP single domain antibody NbsfGFP02 (SEQ ID NO: 22);
  • pTT5-NbsfGFP03 which encodes an anti-GFP single domain antibody NbsfGFP03 (SEQ ID NO: 23);
  • pTT5-NbsfGFP04 which encodes an anti-GFP single domain antibody NbsfGFP04 (SEQ ID NO: 24);
  • pTT5-NbsfGFP06 which encodes an anti-GFP single domain antibody NbsfGFP06 (SEQ ID NO: 25);
  • pTT5-NbsfGFP07 which encodes an anti-GFP single domain antibody NbsfGFP07 (SEQ ID NO: 26);
  • pTT5-P-Nb1 which encodes an anti-GFP single domain antibody P-Nb1 (SEQ ID NO: 27);
  • pTT5-S-Nb1 which encodes an anti-GFP single domain antibody S-Nb1 (SEQ ID NO: 28);
  • pTT5-S-Nb5 which encodes an anti-GFP single domain antibody S-Nb5 (SEQ ID NO: 29);
  • pTT5-S-Nb27 which encodes the anti-GFP single domain antibody S-Nb27 (SEQ ID NO: 30).
  • the PCR reaction was carried out using the synthetic sfGFP sequence (Stéphanie Cabantous et al, Nature Biotechnology 23, 102-107 (2005)) using the primers HdGFPF and BmGFP1-10R to obtain the coding sfGFP1-10 (SEQ ID NO: 31, which is sfGFP).
  • SEQ ID NO: 31 which is sfGFP.
  • a DNA fragment of the protein aa 1-214 i.e., the sfGFP protein having a C-terminally truncated 16 amino acid residue
  • the conditions of the PCR reaction were: 98 ° C, 10 min; 30 cycles (98 ° C, 30 s; 58 ° C, 30 s; 68 ° C, 30 s); 68 ° C, 5 min.
  • the sequences of the primers HdGFPF and BmGFP1-10R are shown in Table 3.
  • the PCR amplification product obtained above was ligated into the pTT22M vector (which is the engineered PTT22 vector in which the puromycin gene in the PTT22 vector was replaced with the gene encoding the red fluorescent protein mCherry) according to the method described in Example 1.
  • the expression plasmid pTT22M-sfGFP1-10 encoding sfGFP1-10 (SEQ ID NO: 31) was obtained.
  • the Hela cell suspension was plated into 96-well cell culture plates at a density of 10,000 cells per well at a culture volume of 100 ⁇ L per well. After 20 hours of culture, use according to the instructions of the kit.
  • LTX with Plus Reagent Invitrogen
  • the expression plasmid encoding the single domain antibody and pTT22M-sfGFP1-10 were co-transfected into HeLa cells.
  • the empty vector pTT5 and pTT22M-sfGFP1-10 were co-transfected into HeLa cells and used as a negative control.
  • Figure 1 shows the results of fluorescence microscopy of co-transfected expression plasmids encoding single domain antibody and pTT22M-sfGFP1-10 Hela cells 48 h after transfection; for each cell of the experimental group, the upper panel shows red Observation of the light channel (used to indicate transfection efficiency), the figure below shows the observation of the green channel (used to show whether the cells emit green fluorescence); the "vector" group indicates that the empty vectors pTT5 and pTT22M-sfGFP1 were transfected -10 Hela cells.
  • the experimental results in Figure 1 indicate that the single domain antibodies GBP1, NbsfGFP08, S-Nb2, S-Nb3, S-Nb6, S-Nb7, S-Nb17, S-Nb21 and S-Nb25 can specifically interact with sfGFP1-10. Act and make it glow green.
  • the results of Figure 1 also show that Hela cells co-expressing sfGFP1-10 and single domain antibody GBP1 have the strongest green fluorescence. Therefore, in some cases, the single domain antibody GBP1 is a preferred antibody capable of causing sfGFP1-10 to emit green fluorescence.
  • Example 3 it has been confirmed in Example 3 that sfGFP1-10 is capable of interacting with 9 single-domain antibodies and emitting fluorescence. In this example, it was evaluated whether other truncations of sfGFP have the same properties as sfGFP1-10.
  • CM5 its C-terminal truncation of 5 amino acid residues compared to sfGFP;
  • CM9 its C-terminal truncation of 9 amino acid residues compared to sfGFP;
  • CM10 its C-terminal truncation of 10 amino acid residues compared to sfGFP;
  • CM11 its C-terminal truncation of 11 amino acid residues compared to sfGFP;
  • CM16 (ie sfGFP1-10): its C-terminal truncation of 16 amino acid residues compared to sfGFP;
  • CM21 its C-terminal truncation of 21 amino acid residues compared to sfGFP;
  • CM22 its C-terminal truncation of 22 amino acid residues compared to sfGFP;
  • CM23 its C-terminal truncation of 23 amino acid residues compared to sfGFP;
  • CM24 its C-terminal truncation of 24 amino acid residues compared to sfGFP;
  • CM26 its C-terminal truncation of 26 amino acid residues compared to sfGFP;
  • CM28 its C-terminal truncation of 28 amino acid residues compared to sfGFP;
  • CM32 It has a C-terminal truncation of 32 amino acid residues compared to sfGFP.
  • Hela cell suspensions were plated into 96-well cell culture plates at a density of 10,000 cells per well at a culture volume of 100 ⁇ L per well. After 20 hours of culture, use according to the instructions of the kit.
  • LTX with Plus Reagent Invitrogen
  • a PTT5 vector and an expression plasmid encoding the sfGFP truncation for indicating whether the sfGFP truncation itself fluoresces
  • pTT5-GBP1 pTT5-GBP1 and an expression plasmid encoding the sfGFP truncation
  • the sfGFP truncation that is used to indicate whether GBP1 is capable of fluorescing itself does not fluoresce and is co-transfected into HeLa cells.
  • Figure 2 shows the results of fluorescence microscopy of the expression plasmid encoding the C-terminal truncation variant of sfGFP and HeLa cells of PTT5 (Fig. 2A) or pTT5-GBP1 (Fig. 2B) at 48 h after transfection;
  • the "WT" group represents Hela cells co-transfected with an expression plasmid encoding the fluorescent protein sfGFP and pTT5 (Fig. 2A) or pTT5-GBP1 (Fig. 2B).
  • Fig. 2A The experimental results of Fig. 2A show that the truncated body CM5 itself can display significant green fluorescence, and the truncated body CM9 can only display extremely weak green fluorescence, while other truncated bodies cannot display green fluorescence.
  • sfGFP protein truncation having a C-terminal truncation of 9-23 amino acid residues has the same properties as sfGFP1-10: that is, it does not fluoresce itself, but in the single domain antibody screened (eg Under the action of GBP1), it can emit fluorescence.
  • This experiment examined the degree of tolerance of sfGFP1-10 to mutations and obtained a preferred GFP fragment that can be used in combination with the single domain antibody GBP1.
  • sfGFP1-10 The sequence of sfGFP1-10 was randomly mutated to obtain a variant of sfGFP1-10. Subsequently, a variant of sfGFP1-10 and a single domain antibody GBP1 were co-expressed in HeLa cells according to the method described in Example 3, and the state and fluorescence of Hela cells were observed using a fluorescence microscope.
  • Hela cell suspensions were plated into 96-well cell culture plates at a density of 10,000 cells per well at a culture volume of 100 ⁇ L per well. After 20 hours of culture, use according to the instructions of the kit.
  • LTX with Plus Reagent Invitrogen
  • pTT5-GBP1 and an expression plasmid encoding the sfGFP1-10 variant were co-transfected into HeLa cells.
  • pTT22M-sfGFP1-10 and pTT5-GBP1 were co-transfected into HeLa cells for use as a positive control
  • pTT5-GBP1 and an expression plasmid encoding an irrelevant protein were co-transfected into HeLa cells and used as a negative control.
  • Figure 3 shows the results of fluorescence microscopy of HeLa cells co-transfected with pTT5-GBP1 and the expression plasmid encoding the sfGFP1-10 variant at 48 h after transfection; wherein the "Negative" group indicates co-transfection of pTT5-GBP1 and Hela cells encoding expression plasmids of irrelevant proteins.
  • green fluorescent protein and fluorescent proteins of other colors The main difference between green fluorescent protein and fluorescent proteins of other colors is that the domains involved in the excitation of fluorescence (especially aa 65-67) have different amino acid residues.
  • an expression plasmid encoding BFP1-10 or YFP1-10 was constructed based on the nucleic acid sequence encoding Mbcd38, and the interaction between GBP1 and BFP1-10 or YFP1-10 was verified.
  • the expression plasmid (pTT22M-Mbcd38) encoding Mbcd38 was used as a template, and PCR amplification was performed using primers HdGFPF and DrFPbR to obtain DNA fragment YFPa, and PCR amplification was performed using primers DrFPbF and BmGFP1-10R to obtain DNA. Fragment YFPb. Subsequently, PCR amplification was carried out using the DNA fragments YFPa and YFPb as a template using primers HdGFPF and BmGFP1-10R to obtain a DNA fragment encoding YFP1-10 (SEQ ID NO: 46).
  • PCR amplification was performed using primers HdGFPF and DrFPcR to obtain DNA fragment BFPa, and PCR amplification was performed using primers DrFPcF and BmGFP1-10R to obtain DNA fragments. BFPb.
  • PCR amplification was carried out using the DNA fragments BFPa and BFPb as a template using primers HdGFPF and BmGFP1-10R to obtain a DNA fragment encoding BFP1-10 (SEQ ID NO: 45).
  • the PCR amplification products obtained above were ligated into the pTT22M vector, respectively, according to the method described in Example 1, thereby obtaining an expression plasmid encoding BFP1-10 (SEQ ID NO: 45) (designated as pTT22M-BFP1-10) And an expression plasmid encoding YFP1-10 (SEQ ID NO: 46) (designated as pTT22M-YFP1-10).
  • Figure 4 shows the results of fluorescence microscopy of HeLa cells co-transfected with pTT5-GBP1 and pTT22M-BFP1-10 or pTT22M-YFP1-10 at 48 h after transfection; wherein "B/Y” indicates blue/yellow channel Observations; “R” indicates the observation of the red channel; “Merge” indicates the combination of the observations of the two channels.
  • GBP1 not only restores fluorescence to non-fluorescent GFP fragments, but also restores fluorescence to non-fluorescent BFP fragments and YFP fragments.
  • the principles and methods of the present invention are applicable to a variety of fluorescent proteins.
  • a fusion protein containing GBP1 and a protein of interest and sfGFP1-10 were co-expressed in cells, and then the distribution and location of the protein of interest in the cells were determined by the interaction between GBP1 and sfGFP1-10.
  • ACTB1, TUBB3, MAPRE3, H2B, LMNB1, PAXILLIN, EndoG can be found in GeneBank (GeneBank accession numbers are as follows: ACTB1, NM_001101; TUBB3, NM_006086; MAPRE3, XM_004028974; H2B, AK311849; LMNB1, BC012295; PAXILLIN, XM_015275216 ;EndoG, BC004922).
  • pTT5-GBP-ACTB1 which encodes the fusion protein GBP-ACTB1 comprising GBP1 and ACTB1, wherein GBP1 is linked to the N-terminus of ACTB1;
  • pTT5-BFP-ACTB1 which encodes a fusion protein BFP-ACTB1 comprising full-length BFP and ACTB1, wherein the BFP is linked to the N-terminus of ACTB1;
  • pTT5-TUBB3-GBP which encodes the fusion protein TUBB3-GBP containing GBP1 and TUBB3, wherein GBP1 is linked to the C-terminus of TUBB3;
  • pTT5-TUBB3-BFP which encodes the fusion protein TUBB3-BFP comprising full-length BFP and TUBB3, wherein the BFP is linked to the C-terminus of TUBB3;
  • pTT5-GBP-MAPRE3 which encodes a fusion protein GBP-MAPRE3 comprising GBP1 and MAPRE3, wherein GBP1 is linked to the N-terminus of MAPRE3;
  • pTT5-BFP-MAPRE3 which encodes a fusion protein BFP-MAPRE3 comprising full-length BFP and MAPRE3, wherein the BFP is linked to the N-terminus of MAPRE3;
  • pTT5-GBP-H2B which encodes a fusion protein GBP-H2B comprising GBP1 and H2B, wherein GBP1 is linked to the N-terminus of H2B;
  • pTT5-BFP-H2B which encodes a fusion protein BFP-H2B comprising full-length BFP and H2B, wherein the BFP is linked to the N-terminus of H2B;
  • pTT5-GBP-LMNB1 which encodes a fusion protein GBP-LMNB1 comprising GBP1 and LMNB1, wherein GBP1 is linked to the N-terminus of LMNB1;
  • pTT5-BFP-LMNB1 which encodes a fusion protein BFP-LMNB1 comprising full-length BFP and LMNB1, wherein the BFP is linked to the N-terminus of LMNB1;
  • pTT5-Paxillin-GBP which encodes the fusion protein Paxillin-GBP comprising GBP1 and Paxillin, wherein GBP1 is linked to the C-terminus of Paxillin;
  • pTT5-Paxillin-BFP which encodes a fusion protein Paxillin-BFP comprising full-length BFP and Paxillin, wherein BFP is linked to the C-terminus of Paxillin;
  • pTT5-EndoG-GBP which encodes the fusion protein EndoG-GBP containing GBP1 and EndoG, wherein GBP1 is linked to the C-terminus of EndoG;
  • pTT5-EndoG-BFP which encodes the fusion protein EndoG-BFP comprising full-length BFP and EndoG, wherein BFP is ligated to the C-terminus of EndoG.
  • Figure 5 shows the results of fluorescence microscopy of HeLa cells co-transfected with various expression plasmid combinations at 48 h after transfection; among the cells of each experimental group, the upper panel shows green fluorescence in Hela cells (by fusion) Distribution and location of GBP1+sfGFP1-10 in the protein; the middle panel shows the distribution and location of blue fluorescence (produced by BFP in the fusion protein) in Hela cells; the lower panel shows the upper and middle panels Merger.
  • the distribution of blue fluorescence and green fluorescence was consistent for each experimental group of Hela cells.
  • the GBP1/sfGFP1-10 combination of the present invention can also be used to accurately determine the intracellular distribution of various proteins of interest (eg, ACTB1, TUBB3, MAPRE3, H2B, LMNB1, PAXILLIN, EndoG). position.
  • the experimental results of Figure 5 also indicate that GBP1 can be linked to the protein of interest in a variety of ways. For example, GBP1 can be ligated to the N-terminus or C-terminus of the protein of interest without affecting its interaction with sfGFP1-10.
  • the use of the laryngeal carcinoma cell Hep2 as an example verified the use of GBP1/Mbcd38 in indicating cell fusion.
  • nucleotide sequences encoding Mbcd38 and BFP were stably integrated into the genome of laryngeal carcinoma cell line Hep2 using lentivirus infection methods well known in the art to construct stable expression of Mbcd38 and BFP.
  • the cell line Hep2-Mbcd38 The cell line Hep2-Mbcd38.
  • nucleotide sequences encoding the single domain antibodies GBP1 and iRFP near-infrared fluorescent protein
  • Hep2-GBP1 cell suspension, Hep2-Mbcd38 cell suspension, cell suspension containing Hep2-GBP1 and Hep2-Mbcd38 (1:1 ratio of the two cells), respectively, at a density of 30,000 cells per well Plate into 96-well cell culture plates. After 24 hours of culture, the cells in the culture plate were infected with RSV virus (respiratory syncytial virus; MOI 1), respectively. After 48 hours of infection, the state and fluorescence of the cells in each well were observed with a fluorescence microscope. The result is shown in Figure 6.
  • Figure 6 shows the results of fluorescence microscopy of Hep2-GBP1 cell suspension, Hep2-Mbcd38 cell suspension, and cell suspension containing Hep2-GBP1 and Hep2-Mbcd38 after 48 hours of infection with RSV virus.
  • transmembrane peptide pep1 (see Manceur A. et al., Analytical Biochemistry, 2007, 364(1): 51-59) was used as an example to verify that GBP1/Mdc2-26 is indicative of transmembrane peptide penetration. Application in membrane action.
  • Example 3 used LTX with Plus Reagent (Invitrogen), an expression plasmid encoding Mdc2-26 was transfected into U2OS cells to allow U2OS cells to express Mdc2-26.
  • FIG. 7 shows the results of fluorescence microscopy of U2OS cells expressing Mdc2-26 after incubation with GBP1 or GBP1+ transmembrane peptide pep1 for 6h, 8h, 10h or 12h.
  • the use of the present invention is compared to the conventional method of detecting the transmembrane action of a transmembrane peptide using FITC or EGFP (see Manceur A. et al., Analytical Biochemistry, 2007, 364(1): 51-59).
  • the GBP1/Mdc2-26 detection method has a lower background and does not require the removal of residual FITC or EGFP, making the operation simpler.
  • G11 amino acid 215-230 of GFP
  • G11 and sfGFP1-10 can be used as protein labeling systems.
  • six kinds of protein of interest (Agr2, HBc, NTCP, NP, TUBB3, hGBP1) were taken as examples to compare the performance and effects of GBP1/sfGFP1-10 and G11/sfGFP1-10.
  • the amino acid sequences of Agr2, HBc, NTCP, NP, TUBB3, hGBP1 can be found in GenBank (GenBank accession numbers are as follows: Agr2, KJ767789; HBc, AB818694; NTCP, BC074724; NP, EU330203; TUBB3, NM_006086; hGBP1, BC002666).
  • pTT5-Agr2-G11 which encodes the fusion protein Agr2-G11 comprising Agr2 and G11, wherein G11 is linked to the C-terminus of Agr2 via a flexible linker (GSSGGSSG; SEQ ID NO: 82);
  • pTT5-G11-Agr2 which encodes a fusion protein G11-Agr2 comprising Agr2 and G11, wherein G11 is linked to the N-terminus of Agr2 via a flexible linker (SEQ ID NO: 82);
  • pTT5-G11-2A-Agr2 which encodes a fusion protein G11-2A-Agr2 comprising Agr2 and G11, wherein G11 is linked to the N-terminus of Agr2 by a self-cleaving linker (GSSGGSSGGSGATNFSLLKQAG DVEENPGP; SEQ ID NO: 83);
  • pTT5-Agr2-GBP1 which encodes the fusion protein Agr2-GBP1 comprising Agr2 and GBP1, wherein GBP1 is linked to the C-terminus of Agr2 via a flexible linker (SEQ ID NO: 82);
  • pTT5-GBP1-Agr2 which encodes the fusion protein GBP1-Agr2 comprising Agr2 and GBP1, wherein GBP1 is linked to the N-terminus of Agr2 via a flexible linker (SEQ ID NO: 82);
  • pTT5-GBP1-2A-Agr2 which encodes the fusion protein GBP1-2A-Agr2 comprising Agr2 and GBP1, wherein GBP1 is linked to the N-terminus of Agr2 by a self-cleaving linker (SEQ ID NO: 83);
  • pTT5-HBc-G11 which encodes a fusion protein HBc-G11 comprising HBc and G11, wherein G11 is linked to the C-terminus of HBc via a flexible linker (SEQ ID NO: 82);
  • pTT5-G11-HBc which encodes a fusion protein G11-HBc comprising HBc and G11, wherein G11 is linked to the N-terminus of HBc via a flexible linker (SEQ ID NO: 82);
  • pTT5-G11-2A-HBc which encodes a fusion protein G11-2A-HBc comprising HBc and G11, wherein G11 is linked to the N-terminus of HBc via a self-cleaving linker (SEQ ID NO: 83);
  • pTT5-HBc-GBP1 which encodes the fusion protein HBc-GBP1 comprising HBc and GBP1, wherein GBP1 is linked to the C-terminus of HBc via a flexible linker (SEQ ID NO: 82);
  • pTT5-GBP1-HBc which encodes a fusion protein GBP1-HBc comprising HBc and GBP1, wherein GBP1 is linked to the N-terminus of HBc via a flexible linker (SEQ ID NO: 82);
  • pTT5-GBP1-2A-HBc which encodes the fusion protein GBP1-2A-HBc comprising HBc and GBP1, wherein GBP1 is linked to the N-terminus of HBc via a self-cleaving linker (SEQ ID NO: 83);
  • pTT5-NTCP-G11 which encodes a fusion protein NTCP-G11 comprising NTCP and G11, wherein G11 is linked to the C-terminus of NTCP via a flexible linker (SEQ ID NO: 82);
  • pTT5-G11-NTCP which encodes a fusion protein G11-NTCP comprising NTCP and G11, wherein G11 is linked to the N-terminus of NTCP via a flexible linker (SEQ ID NO: 82);
  • pTT5-G11-2A-NTCP which encodes a fusion protein G11-2A-NTCP comprising NTCP and G11, wherein G11 is linked to the N-terminus of NTCP via a self-cleaving linker (SEQ ID NO: 83);
  • pTT5-NTCP-GBP1 which encodes a fusion protein NTCP-GBP1 comprising NTCP and GBP1, wherein GBP1 is linked to the C-terminus of NTCP via a flexible linker (SEQ ID NO: 82);
  • pTT5-GBP1-NTCP which encodes a fusion protein GBP1-NTCP comprising NTCP and GBP1, wherein GBP1 is linked to the N-terminus of NTCP via a flexible linker (SEQ ID NO: 82);
  • pTT5-GBP1-2A-NTCP which encodes the fusion protein GBP1-2A-NTCP comprising NTCP and GBP1, wherein GBP1 is linked to the N-terminus of NTCP via a self-cleaving linker (SEQ ID NO: 83);
  • pTT5-NP-G11 which encodes a fusion protein NP-G11 comprising NP and G11, wherein G11 is linked to the C-terminus of NP via a flexible linker (SEQ ID NO: 82);
  • pTT5-G11-NP which encodes a fusion protein G11-NP comprising NP and G11, wherein G11 is linked to the N-terminus of NP via a flexible linker (SEQ ID NO: 82);
  • pTT5-G11-2A-NP which encodes a fusion protein G11-2A-NP comprising NP and G11, wherein G11 is linked to the N-terminus of NP via a self-cleaving linker (SEQ ID NO: 83);
  • pTT5-NP-GBP1 which encodes the fusion protein NP-GBP1 comprising NP and GBP1, wherein GBP1 is linked to the C-terminus of NP via a flexible linker (SEQ ID NO: 82);
  • pTT5-GBP1-NP which encodes a fusion protein GBP1-NP comprising NP and GBP1, wherein GBP1 is linked to the N-terminus of NP via a flexible linker (SEQ ID NO: 82);
  • pTT5-GBP1-2A-NP which encodes the fusion protein GBP1-2A-NP comprising NP and GBP1, wherein GBP1 is linked to the N-terminus of NP via a self-cleaving linker (SEQ ID NO: 83);
  • pTT5-hGBP1-G11 which encodes the fusion protein hGBP1-G11 comprising hGBP1 and G11, wherein G11 is linked to the C-terminus of hGBP1 via a flexible linker (SEQ ID NO: 82);
  • pTT5-G11-hGBP1 which encodes the fusion protein G11-hGBP1 comprising hGBP1 and G11, wherein G11 is linked to the N-terminus of hGBP1 via a flexible linker (SEQ ID NO: 82);
  • pTT5-G11-2A-hGBP1 which encodes the fusion protein G11-2A-hGBP1 comprising hGBP1 and G11, wherein G11 is linked to the N-terminus of hGBP1 by a self-cleaving linker (SEQ ID NO: 83);
  • pTT5-hGBP1-GBP1 which encodes the fusion protein hGBP1-GBP1 comprising hGBP1 and GBP1, wherein GBP1 is linked to the C-terminus of hGBP1 via a flexible linker (SEQ ID NO: 82);
  • pTT5-GBP1-hGBP1 which encodes the fusion protein GBP1-hGBP1 comprising hGBP1 and GBP1, wherein GBP1 is linked to the N-terminus of hGBP1 via a flexible linker (SEQ ID NO: 82);
  • pTT5-GBP1-2A-hGBP1 which encodes the fusion protein GBP1-2A-hGBP1 comprising hGBP1 and GBP1, wherein GBP1 is linked to the N-terminus of hGBP1 via a self-cleaving linker (SEQ ID NO: 83);
  • pTT5-TUBB3-G11 which encodes the fusion protein TUBB3-G11 comprising TUBB3 and G11, wherein G11 is linked to the C-terminus of TUBB3 via a flexible linker (SEQ ID NO: 82);
  • pTT5-G11-TUBB3 which encodes a fusion protein G11-TUBB3 comprising TUBB3 and G11, wherein G11 is linked to the N-terminus of TUBB3 via a flexible linker (SEQ ID NO: 82);
  • pTT5-G11-2A-TUBB3 which encodes a fusion protein G11-2A-TUBB3 comprising TUBB3 and G11, wherein G11 is linked to the N-terminus of TUBB3 via a self-cleaving linker (SEQ ID NO: 83);
  • pTT5-TUBB3-GBP1 which encodes the fusion protein TUBB3-GBP1 comprising TUBB3 and GBP1, wherein GBP1 is linked to the C-terminus of TUBB3 via a flexible linker (SEQ ID NO: 82);
  • pTT5-GBP1-TUBB3 which encodes the fusion protein GBP1-TUBB3 comprising TUBB3 and GBP1, wherein GBP1 is linked to the N-terminus of TUBB3 via a flexible linker (SEQ ID NO: 82);
  • pTT5-GBP1-2A-TUBB3 which encodes the fusion protein GBP1-2A-TUBB3 comprising TUBB3 and GBP1, wherein GBP1 is linked to the N-terminus of TUBB3 by a self-cleaving linker (SEQ ID NO: 83).
  • G11/sfGFP1-10 when G11/sfGFP1-10 is used to label proteins, G11 should be ligated to the C-terminus of the protein of interest; whereas the GBP1/sfGFP1-10 system of the present invention is not limited by the manner of attachment, and can be various The way to apply.
  • GBP1 can be freely expressed, or fused to the N-terminus or C-terminus of the protein of interest without substantially affecting the marker function of the GBP1/sfGFP1-10 system of the present invention.
  • the FR region of the GBP1 antibody was randomly mutated, and two mutants were obtained. These two mutants were named GBPMT1 and GBPMTT2, respectively, and their amino acid sequences are shown in SEQ ID NO: 87 and SEQ ID NO: 88, respectively.
  • the gene encoding GBPMT1 and the gene encoding GBPMT2 were synthesized and cloned into the PTT5 vector, respectively, according to the method described above.
  • the expression plasmids pTT22M-Mdc2-26 and the expression plasmid carrying the gene encoding GBPMT1 or GBPMTT2 were co-transfected into HeLa cells according to the method described in Example 3.
  • the expression plasmids pTT22M-Mdc2-26 and the expression plasmid carrying the gene encoding GBP1 were co-transfected into HeLa cells and used as a control. After 48 h of transfection, the fluorescence of Hela cells was observed with a fluorescence microscope. The result is shown in Figure 9.
  • Figure 9 shows that Hela cells co-transfected with Mdc2-26 and either GBP1 or GBPMMT1 or GBPMTT2 exhibited green fluorescence. This result indicates that GBP1 or GBPMTT1 or GBPMTT2 can restore Mdc2-26 to fluorescence.
  • a single domain antibody eg, GBP1
  • Mdc2-26 the function/property that restores fluorescent protein truncation
  • single domain antibodies eg, Mutation of the FR region of GBP1 does not affect its function/property.

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Abstract

一种荧光报告系统，其包含荧光蛋白的截短体和抗所述荧光蛋白的单域抗体，所述荧光蛋白的截短体在游离状态下不能发出荧光，但是在与所述单域抗体结合后能够发出荧光。

Description

一种检测系统 技术领域
本发明涉及生物技术领域。特别地，本发明涉及一种检测系统，其包含荧光蛋白的截短体和抗所述荧光蛋白的单域抗体，所述荧光蛋白的截短体在游离状态下不能发出荧光，但是在与所述单域抗体结合后能够发出荧光。此外，本发明还涉及所述检测系统的各种应用。
背景技术
绿色荧光蛋白(Green Fluorescence protein,GFP)及其他荧光蛋白(例如蓝色荧光蛋白(BFP)和黄色荧光蛋白(YFP))已经被广泛应用于蛋白的标记，例如用于在细胞内甚至在动物体内对目的蛋白进行定位。
之前已经描述过使用GFP片段的重组系统(参见Ozawa T.等人，Current Opinion in Chemical Biology,2001,5(5):578-83)。在此类系统中，将GFP蛋白拆分成不能自组装的两个片段，然后将这两个片段分别连接至两个不同的蛋白。若所述两个蛋白能够相互作用，则GFP的两个片段能够重组成完整的GFP，并发出荧光。因此，根据是否产生荧光，可以判断两个蛋白是否有相互作用。
还已经报道了一种基于荧光蛋白的互补片段的蛋白标签系统(参见Stéphanie Cabantous等人，Nature Biotechnology 23,102-107(2005))。此类系统可用于检测蛋白的可溶性，又被称为脱落GFP系统。在此类系统中，将目的蛋白与GFP的一个16个氨基酸的片段(氨基酸215-230，也被称为GFP11或G11)融合，并同时独立表达所述GFP片段的互补片段(氨基酸1-214)。这两个GFP片段在可溶状态下，能够自发折叠成完整的GFP，并发出荧光，由此可用于在体内和体外检测和定量蛋白的溶解性。此外，脱落GFP系统也被应用于蛋白的标记，并且已报道，多个GFP11的重复可以增强重组后的GFP的荧光强度(参见Kamiyama D.等人，Nature Communications,2016 Mar 18；7:11046)。
与GFP类似的其他荧光蛋白也能够拆分成可以重组和不能重组的两个片段进行应用(参见Kamiyama D.等人，Nature Communications,2016 Mar 18；7:11046)。
单域抗体是骆驼单链抗体的重链可变区。骆驼的单链抗体只包含重链，而不具有轻链。因此，单链抗体的重链可变区即可结合抗原。这类抗体具有分子量小，稳定性好，特异性高，易表达，组织渗透性好等优点，在生物技术研究与诊断应用领域已经得到广泛的关注。之前已经有多个团队报道，抗GFP的单域抗体在与GFP结合后，能够增强或减弱GFP的荧光(参见Kirchhofer A.等人，Nature Structural & Molecular Biology,2010 Jan；17(1):133-8)。
在本申请中，发明人意外地发现，某些抗荧光蛋白(例如GFP)的单域抗体能够与本身不能发出荧光的荧光蛋白(例如GFP)的截短体特异性结合，并使之发出荧光。基于此，本申请的发明人设计和开发了一种新的检测系统，其基于荧光蛋白的不发光片段和抗荧光蛋白的单域抗体的联合使用，并且可广泛用于生物技术研究领域与诊断领域。
发明内容
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“荧光蛋白”是指，在某一激发光照射下能够发射特定波 长的光(荧光)的蛋白。迄今为止，已发现了多种颜色的荧光蛋白，包括但不限于，绿色荧光蛋白，蓝色荧光蛋白，黄色荧光蛋白，红色荧光蛋白等。已对各种颜色的荧光蛋白的结构及其发光机理进行了详细的阐释(参见例如，Yang F等人Nat Biotechnol.1996 Oct；14(10):1246-51；Mark Wall等人Nat.Struct.Biol.7,1133–1138,2000；和Reid BG等人Biochemistry.1997Jun 3；36(22):6786-91)。在本申请中，绿色荧光蛋白的示例性氨基酸序列如SEQ ID NO:84所示；蓝色荧光蛋白的示例性氨基酸序列如SEQ ID NO:85所示；黄色荧光蛋白的示例性氨基酸序列如SEQ ID NO:86所示。
之前已报道，各种颜色的荧光蛋白具有类似的氨基酸序列和结构，并且它们的主要差异在于，参与激发荧光的结构域(例如，绿色荧光蛋白的aa 65-67)由不同的氨基酸残基构成(参见例如，ROGER HEIM等人Biochemistry Vol.91,pp.12501-12504,December 1994)。因此，本申请基于绿色荧光蛋白所证实的技术效果可被扩展至其他颜色的荧光蛋白(例如蓝色荧光蛋白和黄色荧光蛋白)。
如本文中所使用的，表述“蛋白的C端被截短9-23个氨基酸残基”是指，蛋白C端的9-23个氨基酸残基被缺失。
根据本发明，当在蛋白/多肽的背景中使用时，术语“变体”是指这样的蛋白，其氨基酸序列与参照蛋白/多肽(例如，本发明的截短体)的氨基酸序列相比，具有一个或多个(例如，1-15个、1-10个、1-5个或1-3个)氨基酸差异(例如氨基酸残基的添加、置换或缺失，例如保守置换)，或者具有至少85％，至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的同一性，并且其保留了参照蛋白/多肽的必要特性。在本申请中，本发明的截短体的必要特性可以指，其在游离状态下不发出荧光，但是在与单域抗体结合后，能够发出荧光。
根据本发明，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的必要特性的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨 酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
如本文中使用的，术语“单域抗体”意指，包含抗体重链可变区，但不包含轻链可变区的抗体。已在骆驼科动物和鲨鱼的血清中发现了一种抗体(也被称为重链抗体)，其仅包含重链而不包含轻链，并且具有特异性结合抗原的能力。此外，还已发现，重链抗体的抗原结合区(即，重链可变区)通过铰链区与Fc区连接，并且，该抗原结合区(即，重链可变区)自重链抗体上分离后仍具有结合抗原的功能(参见例如，Hamers-Casterman C等人,Nature.1993 Jun 3；363(6428):446-8)。因此，在本申请中，“单域抗体”意欲涵盖此类仅包含重链而不包含轻链的重链抗体，以及其抗原结合片段(例如，重链可变区)。例如，本申请中的“单域抗体”可以包含含有3个CDR的重链可变区，并且任选地，可以还包含铰链区、Fc区、或重链恒定区。在某些优选的实施方案中，所述单域抗体包含含有3个CDR的重链可变区。在某些优选的实施方案中，所述单域抗体包含，含有3个CDR的重链可变区以及铰链区、Fc区、或重链恒定区。
如本文中使用的，术语“载体”意指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌体；柯斯质粒等等。
在申请中，术语“多肽”和“蛋白质”具有相同的含义，可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
本申请至少部分基于本发明人的出人意料的发现：某些抗荧光蛋白(例如GFP)的单域抗体能够与本身不能发出荧光的荧光蛋白(例如GFP)的截短体特异性结合，并使之发出荧光。基于此，本申请的发明人设计和开发了一种新的检测系统，其基于荧光蛋白的不发光片段和抗荧光蛋白的单域抗体的联合使用，并且可广泛用于生物技术研究领域与诊断领域。
因此，在一个方面，本发明提供了一种试剂盒，其包含两种组分，其中，所述第一组分包含：
(a1)荧光蛋白的截短体，其与荧光蛋白的差异在于，荧光蛋白的C端被截短9-23个氨基酸残基；
(a2)如(a1)中定义的截短体的变体，所述变体与所述截短体具有至少85％的同一性，或者，所述变体与所述截短体的差异在于一个或多个氨基酸残基的添加、置换或缺失；或
(a3)核酸分子，其包含编码如(a1)中定义的截短体或如(a2)中定义的变体的核苷酸序列；
并且，所述第二组分包含：
(b1)抗荧光蛋白的单域抗体；优选地，其包含选自下列的CDR1、CDR2和CDR3：
(1)分别如SEQ ID NO:47-49所示的CDR1、CDR2和CDR3；
(2)分别如SEQ ID NO:50-52所示的CDR1、CDR2和CDR3；
(3)分别如SEQ ID NO:53-55所示的CDR1、CDR2和CDR3；
(4)分别如SEQ ID NO:56-58所示的CDR1、CDR2和CDR3；
(5)分别如SEQ ID NO:59-61所示的CDR1、CDR2和CDR3；
(6)分别如SEQ ID NO:62-64所示的CDR1、CDR2和CDR3；
(7)分别如SEQ ID NO:65-67所示的CDR1、CDR2和CDR3；
(8)分别如SEQ ID NO:68-70所示的CDR1、CDR2和CDR3；和
(9)分别如SEQ ID NO:71-73所示的CDR1、CDR2和CDR3；或
(b2)核酸分子，其包含编码如(b1)中定义的单域抗体的核苷酸序列；
其中，所述截短体和所述变体在游离状态下不发出荧光，但是在与所述单域抗体结合后，能够发出荧光。
在某些优选的实施方案中，所述荧光蛋白选自绿色荧光蛋白，蓝色荧光蛋白和黄色荧光蛋白。
在某些优选的实施方案中，所述绿色荧光蛋白具有如SEQ ID NO:84所示的氨基酸序列。在某些优选的实施方案中，所述蓝色荧光蛋白具有如SEQ ID NO:85所示的氨基酸序列。在某些优选的实施方案中，所述黄色荧光蛋白具有如SEQ ID NO:86所示的氨基酸序列。
在某些优选的实施方案中，所述截短体与荧光蛋白的差异在于，荧光蛋白的C端被截短9-23个氨基酸残基，例如被截短9、10、11、12、13、14、15、16、17、18、19、20、21、22、或23个氨基酸残基。
在某些优选的实施方案中，所述截短体为绿色荧光蛋白的截短体，并且其与绿色荧光蛋白的差异在于，绿色荧光蛋白的C端被截短9-23个氨基酸残基，例如被截短9、10、11、12、13、14、15、16、17、18、19、20、21、22、或23个氨基酸残基。在某些优选的实施方案中，所述绿色荧光蛋白具有如SEQ ID NO:84所示的氨基酸序列。在某些优选的实施方案中，所述绿色荧光蛋白的截短体具有如SEQ ID NO:31所示的氨基酸序列。
在某些优选的实施方案中，所述截短体为蓝色荧光蛋白的截短体，并且其与蓝色荧光蛋白的差异在于，蓝色荧光蛋白的C端被截短9-23个氨基酸残基，例如被截短9、10、11、12、13、14、15、16、17、18、19、20、21、22、或23个氨基酸残基。在某些优选的实施方案中，所述蓝色荧光蛋白具有如SEQ ID NO:85所示的氨基酸序列。
在某些优选的实施方案中，所述截短体为黄色荧光蛋白的截短体，并且其与黄色荧光蛋白的差异在于，黄色荧光蛋白的C端被截短9-23个氨基酸残基，例如被截短9、10、11、12、13、14、15、16、17、18、19、20、21、22、或23个氨基酸残基。在某些优选的实施方案中，所述黄色荧光蛋白具有如SEQ ID NO:86所示的氨基酸序列。
在某些优选的实施方案中，所述变体的氨基酸序列与所述截短体的氨基酸序列具有至少85％的同一性，例如至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的同一性。
在某些优选的实施方案中，所述变体与所述截短体的差异在于一个或多个氨基酸残基的添加、置换或缺失，例如不超过15个、不超过14个、不超过13个、不超过12个、不超过11个、不超过10个、不超过9个、不超过8个、不超过7个、不超过6个、不超过5个、不超过4个、不超过3个、不超过2个、或1个氨基酸残基的添加、置换或缺失。
在某些优选的实施方案中，所述变体与所述截短体的差异在于一个或多个氨基酸残基的置换(例如保守置换)，例如不超过15个、不超过14个、不超过13个、不超过12个、不超过11个、不超过10个、不超过9个、不超过8个、不超过7个、不超过6个、不超过5个、不超过4个、不超过3个、不超过2个、或1个氨基酸残基的置换(例如保守置换)。
在某些优选的实施方案中，所述截短体或所述变体具有选自下列的氨基酸序列：SEQ ID NO:31-46。
在某些优选的实施方案中，所述单域抗体包含重链可变区，所述重链可变区具有选自下列的氨基酸序列：SEQ ID NO:1-9和87-88。在某些优选的实施方案中，所述单域抗体由所述重链可变区组成。在某些优选的实施方案中，所述单域抗体包含所述重链可变区，以及任选的铰链区、Fc区、或重链恒定区。
在某些优选的实施方案中，(a3)所述的核酸分子包含编码如(a1)中定义的截短体或如(a2)中定义的变体的核苷酸序列，或者由编码如(a1)中定义的截短体或如(a2)中定义的变体的核苷酸序列组成。在某些优选的实施方案中，(a3)所述的核酸分子为包含编码如(a1)中定义的截短体或如(a2)中定义的变体的核苷酸序列的载体(例如表达载体)。
在某些优选的实施方案中，(b2)所述的核酸分子包含编码如(b1)中定义的单域抗体的核苷酸序列，或者由编码如(b1)中定义的单域抗体的核苷酸序列组成。在某些优选的实施方案中，(b2)所述的核酸分子为包含编码如(b1)中定义的单域抗体的核苷酸序列的载体(例如表达载体)。
在某些优选的实施方案中，所述试剂盒包含，如(a1)中定义的截短体或如(a2)中定义的变体，以及如(b1)中定义的单域抗体。在某些优选的实施方案中，所述试剂盒包含，如(a1)中定义的截短体或如(a2)中定义的变体，以及(b2)所述的核酸分子。
在某些优选的实施方案中，所述试剂盒包含，(a3)所述的核酸分子，以及如(b1)中定义的单域抗体。在某些优选的实施方案中，所述试剂盒包含，(a3)所述的核酸分子，以及(b2)所述的核酸分子。
在某些优选的实施方案中，所述试剂盒还包含额外的试剂。此类额外的试剂包括但不限于，用于进行分子克隆或用于构建载体的试剂,例如用于进行核酸扩增的缓冲液、核酸聚合酶、核酸内切酶、连接酶、用于进行核酸纯化的试剂、用于进行核酸转化、转染或转导的试剂，和/或核酸载体(例如质粒或病毒载体)。
在一个方面，本发明提供了一种确定目的蛋白的位置或分布的方法，其包括，使用本发明的试剂盒。
在一个方面，本发明提供了一种确定目的蛋白的位置或分布的方法，其包括：
共表达(1)如上文所定义的截短体或突变体，和(2)包含如上所定义的单域抗体和所述目的蛋白的融合蛋白；或者
共表达(3)如上文所定义的单域抗体，和(4)包含如上所定义的截短体或突变体和所述目的蛋白的融合蛋白。
在某些优选的实施方案中，在细胞内共表达(1)如上文所定义的截短体或突变体，和(2)包含如上所定义的单域抗体和所述目的蛋白的融合蛋白，从而确定所述目的蛋白在细胞中的位置或分布。在某些优选的实施方案中，所述单域抗体连接至所述目的蛋白的N端或C端，任选地通过接头。在某些优选的实施方案中，所述接头为柔性接头(例如，如SEQ ID NO:82所示的柔性接头)。在某些优选的实施方案中，所述方法还包括，使用荧光显微镜观察所述细胞。
在某些优选的实施方案中，在细胞内共表达(3)如上文所定义的单域抗体，和(4)包含如上所定义的截短体或突变体和所述目的蛋白的融合蛋白，从而确定所述目的蛋白在细胞中的位置或分布。在某些优选的实施方案中，所述截短体或突变体连接至所述目的蛋白的N端或C端，任选地通过接头。在某些优选的实施方案中，所述接头为柔性接头(例如，如SEQ ID NO:82所示的柔性接头)。在某些优选的实施方案中，所述方法还包括，使用荧光显微镜观察所述细胞。
在某些优选的实施方案中，所述方法包括下述步骤：
(1)提供包含编码如上文所定义的截短体或突变体的核苷酸序列的第一载体，以 及包含编码含有如上所定义的单域抗体和所述目的蛋白的融合蛋白的核苷酸序列的第二载体；
(2)将所述第一载体和第二载体共同导入细胞中，从而在所述细胞中共表达所述截短体或突变体，以及所述融合蛋白；和
(3)使用荧光显微镜观察所述细胞，并根据荧光的位置确定所述目的蛋白在所述细胞内的分布和位置，其中，所述荧光因所述截短体或突变体与所述融合蛋白包含的所述单域抗体之间的相互作用而产生。
在某些优选的实施方案中，所述方法包括下述步骤：
(1)提供包含编码如上文所定义的单域抗体的核苷酸序列的第一载体，以及包含编码含有如上所定义的截短体或突变体和所述目的蛋白的融合蛋白的核苷酸序列的第二载体；
(2)将所述第一载体和第二载体共同导入细胞中，从而在所述细胞中共表达所述单域抗体，以及所述融合蛋白；和
(3)使用荧光显微镜观察所述细胞，并根据荧光的位置确定所述目的蛋白在所述细胞内的分布和位置，其中，所述荧光因所述单域抗体与所述融合蛋白包含的所述截短体或突变体之间的相互作用而产生。
在某些优选的实施方案中，所述方法包括下述步骤：
(1)提供稳定表达如上文所定义的截短体或突变体的细胞，以及包含编码含有如上所定义的单域抗体和所述目的蛋白的融合蛋白的核苷酸序列的载体；
(2)将所述载体导入所述细胞中，从而在所述细胞中共表达所述截短体或突变体，以及所述融合蛋白；和
(3)使用荧光显微镜观察所述细胞，并根据荧光的位置确定所述目的蛋白在所述细胞内的分布和位置，其中，所述荧光因所述截短体或突变体与所述融合蛋白包含的所述单域抗体之间的相互作用而产生。
在某些优选的实施方案中，所述方法包括下述步骤：
(1)提供稳定表达含有如上所定义的单域抗体和所述目的蛋白的融合蛋白的细胞，以及包含编码如上文所定义的截短体或突变体的核苷酸序列的载体；
(2)将所述载体导入所述细胞中，从而在所述细胞中共表达所述截短体或突变体，以及所述融合蛋白；和
(3)使用荧光显微镜观察所述细胞，并根据荧光的位置确定所述目的蛋白在所述细胞内的分布和位置，其中，所述荧光因所述截短体或突变体与所述融合蛋白包含的所述单域抗体之间的相互作用而产生。
在某些优选的实施方案中，所述方法包括下述步骤：
(1)提供稳定表达如上文所定义的单域抗体的细胞，以及包含编码含有如上所定义的截短体或突变体和所述目的蛋白的融合蛋白的核苷酸序列的载体；
(2)将所述载体导入所述细胞中，从而在所述细胞中共表达所述单域抗体，以及所述融合蛋白；和
(3)使用荧光显微镜观察所述细胞，并根据荧光的位置确定所述目的蛋白在所述细胞内的分布和位置，其中，所述荧光因所述单域抗体与所述融合蛋白包含的所述截短体或突变体之间的相互作用而产生。
在某些优选的实施方案中，所述方法包括下述步骤：
(1)提供稳定表达含有如上所定义的截短体或突变体和所述目的蛋白的融合蛋白的细胞，以及包含编码如上文所定义的单域抗体的核苷酸序列的载体；
(2)将所述载体导入所述细胞中，从而在所述细胞中共表达所述单域抗体，以及所述融合蛋白；和
(3)使用荧光显微镜观察所述细胞，并根据荧光的位置确定所述目的蛋白在所述 细胞内的分布和位置，其中，所述荧光因所述单域抗体与所述融合蛋白包含的所述截短体或突变体之间的相互作用而产生。
可通过各种合适的方式将载体导入细胞中。此类方式包括但不限于转化(例如原生质体转化法)、转染(例如脂质体转染)、电穿孔、转导(例如噬菌体转导法)等。此外，在细胞中稳定表达目的蛋白的方法是本领域技术人员已知的。例如，可通过将编码目的蛋白的外源核苷酸序列整合入细胞的基因组中，从而在细胞中稳定表达目的蛋白。将外源核苷酸序列整合入目标细胞的基因组中的方法也是本领域技术人员已知的(参见例如，Oberbek A等人，Biotechnol Bioeng.2011Mar；108(3):600-10)。
在一个方面，本发明提供了一种确定是否发生细胞融合的方法，其包括，使用本发明的试剂盒。
在一个方面，本发明提供了一种确定是否发生细胞融合的方法，其包括：
(1)在第一细胞中表达如上文所定义的截短体或突变体，并且在第二细胞中表达如上所定义的单域抗体；
(2)将所述第一细胞和第二细胞共同培养，并使用荧光显微镜进行观察。
在此类方法中，如果在细胞内观察到因所述截短体或突变体与所述单域抗体之间的相互作用而产生的荧光，那么可确定第一细胞与第二细胞发生了细胞融合。反之，如果未在细胞内观察到所述荧光，那么可确定第一细胞与第二细胞未发生细胞融合。
在某些优选的实施方案中，在步骤(2)中，在将所述第一细胞和第二细胞共同培养之后，任选地，使所述第一细胞和第二细胞经历处理，然后再使用荧光显微镜观察是否出现了荧光。利用此类实施方案，可以判断所述处理是否诱导或抑制了细胞融合。例如，如果与未进行处理的情况相比，在所述第一细胞和第二细胞经历处理的条件下，在更短的时间内观察到荧光，或者在相同的时间点观察到更强的荧光，那么可确定所述处理诱导或促进了细胞融合。反之，如果与未进行处理的情况相比，在所述第一细胞和第二细胞经历处理的条件下，需要更长的时间才能观察到荧光，或者在相同的时间点观察到较弱的荧光，那么可确定所述处理阻止或抑制了细胞融合。
所述处理可以是任何期望的操作，例如物理刺激(例如热刺激，辐射等)，化学刺激(例如与候选药物或试剂接触)，或生物学刺激(例如与病原体(例如病毒或细菌)接触)。相应地，所述方法可以用于筛选能够诱导或抑制细胞融合的刺激方式、药物、试剂、或病原体(例如病毒或细菌)等。
因此，在某些优选的实施方案中，本发明提供了确定一种试剂或病原体(例如病毒或细菌)诱导或抑制细胞融合的能力的方法，其包括下述步骤：
(1)在第一细胞中表达如上文所定义的截短体或突变体，并且在第二细胞中表达如上所定义的单域抗体；
(2)将所述第一细胞和第二细胞共同培养，并使用荧光显微镜进行观察；
(3)将所述共同培养的第一细胞和第二细胞与所述试剂或病原体接触并继续培养，然后再使用荧光显微镜进行观察。
在此类实施方案中，如果在步骤(2)中未观察到荧光，而在步骤(3)中观察到荧光，那么可以确定所述试剂或病原体具有诱导细胞融合的能力。
在某些优选的实施方案中，本发明提供了确定一种试剂或病原体(例如病毒或细菌)诱导或抑制细胞融合的能力的方法，其包括下述步骤：
(1)在第一细胞中表达如上文所定义的截短体或突变体，并且在第二细胞中表达如上所定义的单域抗体；
(2)将所述第一细胞和第二细胞共同培养，并与所述试剂或病原体接触，用作实验组培养物；并且，将所述第一细胞和第二细胞共同培养，且不与所述试剂或病原体接触，用作对照组培养物；
(3)使用荧光显微镜观察所述实验组培养物和对照组培养物。
在此类实施方案中，与对照组培养物相比，如果在更短的时间内在实验组培养物中观察到荧光，或者在相同的时间点实验组培养物展示出更强的荧光，那么可以确定所述试剂或病原体具有诱导或促进细胞融合的能力。反之，与对照组培养物相比，如果需要更长的时间才能在实验组培养物中观察到荧光，或者在相同的时间点实验组培养物展示出较弱的荧光，那么可以确定所述试剂或病原体具有阻止或抑制细胞融合的能力。
可以通过各种合适的方式，使得第一细胞表达所述截短体或突变体，且使得第二细胞表达所述单域抗体。在某些优选的实施方案中，通过将包含编码所述截短体或突变体的核苷酸序列的载体导入第一细胞，使得第一细胞表达所述截短体或突变体。在某些优选的实施方案中，通过将编码所述截短体或突变体的核苷酸序列整合入第一细胞的基因组中，使得第一细胞稳定表达所述截短体或突变体。在某些优选的实施方案中，通过将包含编码所述单域抗体的核苷酸序列的载体导入第二细胞，使得第二细胞表达所述单域抗体。在某些优选的实施方案中，通过将编码所述单域抗体的核苷酸序列整合入第二细胞的基因组中，使得第二细胞稳定表达所述单域抗体。
可通过各种合适的方式将载体导入细胞中。此类方式包括但不限于转化(例如原生质体转化法)、转染(例如脂质体转染)、电穿孔、转导(例如噬菌体转导法)等。此外，将外源核苷酸序列整合入目标细胞的基因组中的方法是本领域技术人员已知的(参见例如，Oberbek A等人，Biotechnol Bioeng.2011 Mar；108(3):600-10)。
在一个方面，本发明提供了一种评估试剂促进或抑制多肽穿过细胞膜的能力的方法，其包括，使用本发明的试剂盒。
在一个方面，本发明提供了一种评估试剂促进或抑制多肽穿过细胞膜的能力的方法，其包括：
(1)在细胞中表达如上文所定义的截短体或突变体；
(2)将所述细胞与如上所定义的单域抗体和所述试剂接触，用作实验组细胞；并且，将所述细胞与如上所定义的单域抗体接触，用作对照组细胞；和
(3)使用荧光显微镜观察所述实验组细胞和对照组细胞。
在根据本发明的方法中，与对照组细胞相比，如果在更短的时间内在实验组细胞中观察到荧光，或者在相同的时间点实验组细胞展示出更强的荧光，那么可以确定所述试剂具有促进多肽穿过细胞膜的能力。反之，与对照组细胞相比，如果需要更长的时间才能在实验组细胞中观察到荧光，或者在相同的时间点实验组细胞展示出较弱的荧光，那么可以确定所述试剂具有阻止多肽穿过细胞膜的能力。
可以通过各种合适的方式，使得细胞表达所述截短体或突变体。在某些优选的实施方案中，通过将包含编码所述截短体或突变体的核苷酸序列的载体导入细胞，使得细胞表达所述截短体或突变体。在某些优选的实施方案中，通过将编码所述截短体或突变体的核苷酸序列整合入细胞的基因组中，使得细胞稳定表达所述截短体或突变体。
在一个方面，本发明提供了一种评估试剂促进或抑制多肽穿过细胞膜的能力的方法，其包括：
(1)在细胞中表达如上文所定义的单域抗体；
(2)将所述细胞与如上所定义的截短体或突变体和所述试剂接触，用作实验组细胞；并且，将所述细胞与如上所定义的截短体或突变体接触，用作对照组细胞；和
(3)使用荧光显微镜观察所述实验组细胞和对照组细胞。
在根据本发明的方法中，与对照组细胞相比，如果在更短的时间内在实验组细胞中观察到荧光，或者在相同的时间点实验组细胞展示出更强的荧光，那么可以确定所 述试剂具有促进多肽穿过细胞膜的能力。反之，与对照组细胞相比，如果需要更长的时间才能在实验组细胞中观察到荧光，或者在相同的时间点实验组细胞展示出较弱的荧光，那么可以确定所述试剂具有阻止多肽穿过细胞膜的能力。
可以通过各种合适的方式，使得细胞表达所述单域抗体。在某些优选的实施方案中，通过将包含编码所述单域抗体的核苷酸序列的载体导入细胞，使得细胞表达所述单域抗体。在某些优选的实施方案中，通过将编码所述单域抗体的核苷酸序列整合入细胞的基因组中，使得细胞稳定表达所述单域抗体。
可通过各种合适的方式将载体导入细胞中。此类方式包括但不限于转化(例如原生质体转化法)、转染(例如脂质体转染)、电穿孔、转导(例如噬菌体转导法)等。此外，将外源核苷酸序列整合入目标细胞的基因组中的方法是本领域技术人员已知的(参见例如，Oberbek A等人，Biotechnol Bioeng.2011 Mar；108(3):600-10)。
发明的有益效果
之前已报道，单域抗体GBP1能够增强GFP的荧光。然而，从未报道，单域抗体GBP1能够使已经丧失发出荧光能力的GFP截短体恢复发光能力。在本申请中，发明人首次证实，某些抗GFP单域抗体(例如GBP1)能够使荧光蛋白(例如GFP)的不能发光的截短体恢复发光的能力。此类单域抗体(例如GBP1)的这一性质是特别有利的。特别地，基于该性质，可利用所述单域抗体(例如GBP1)和荧光蛋白(例如GFP)的截短体的组合来构建各种检测系统，从而可方便地进行各种生物学检测，例如蛋白质的定位，细胞融合的检测，穿膜能力的评估等等。
此外，与之前报道的脱落GFP系统(sfGFP1-10+G11)相比，本发明的包含单域抗体(例如GBP1)和荧光蛋白(例如GFP)的截短体的检测系统还具有以下优势：
(1)脱落GFP系统中的G11与目的蛋白的融合方式受到了限制。例如，当将G11连接至目的蛋白的N端时，其使sfGFP1-10恢复荧光的能力可能会受到影响，甚至丧失。相比之下，本发明检测系统中的单域抗体(例如GBP1)则不存在这一问题，其可通过各种连接方式融合至目的蛋白的N端或C端，而不影响其功能的发挥。
(2)G11分子量很小，因此，当其在细胞内游离表达时，易于被降解。相比之下，本发明检测系统中的单域抗体(例如GBP1)则不存在这一问题，其在细胞内是相对稳定的。
因此，本发明的包含单域抗体(例如GBP1)和荧光蛋白(例如GFP)的截短体的检测系统可更加广泛、方便、灵活地进行应用。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示，共转染了编码单域抗体的表达质粒和pTT22M-sfGFP1-10的Hela细胞在转染后48h的荧光显微镜观察结果；其中，对于每一个实验组的细胞，上图显示了红光通道的观察结果(用于指示转染效率)，下图显示了绿光通道的观察结果(用于显示细胞是否发出绿色荧光)；“vector”组表示转染了空载体pTT5和pTT22M-sfGFP1-10的Hela细胞。
图2显示，共转染了编码sfGFP的C端截短变体的表达质粒以及PTT5(图2A)或pTT5-GBP1(图2B)的Hela细胞在转染后48h的荧光显微镜观察结果；其中，“WT”组表示共转染了编码荧光蛋白sfGFP的表达质粒以及pTT5(图2A)或pTT5-GBP1(图2B)的Hela细胞。
图3显示，共转染了pTT5-GBP1和编码sfGFP1-10变体的表达质粒的Hela细胞在转染后48h的荧光显微镜观察结果；其中，“Negative”组表示共转染了pTT5-GBP1和编码无关蛋白的表达质粒的Hela细胞。
图4显示，共转染了pTT5-GBP1与pTT22M-BFP1-10或pTT22M-YFP1-10的Hela细胞在转染后48h的荧光显微镜观察结果；其中，“B/Y”表示蓝光/黄光通道的观察结果；“R”表示红光通道的观察结果；“Merge”表示两种通道的观察结果的合并。
图5显示，共转染了各种表达质粒组合的Hela细胞在转染后48h的荧光显微镜观察结果；其中，对于每一个实验组的细胞，上图显示了Hela细胞中的绿色荧光(由融合蛋白中的GBP1+sfGFP1-10产生)的分布和位置；中图显示了Hela细胞中的蓝色荧光(由融合蛋白中的BFP产生)的分布和位置；下图显示了，上图和中图的合并。
图6显示了，Hep2-GBP1细胞悬液、Hep2-Mbcd38细胞悬液以及含有Hep2-GBP1和Hep2-Mbcd38的细胞悬液在感染RSV病毒48h后的荧光显微镜观察结果。
图7显示，表达Mdc2-26的U2OS细胞在与GBP1或者GBP1+穿膜肽pep1一起温育6h、8h、10h或12h后的荧光显微镜观察结果。
图8显示，共转染了各种表达质粒组合的293细胞在转染后48h的荧光显微镜观察结果。
图9显示，共转染了Mdc2-26以及GBP1或GBPMT1或GBPMT2的Hela细胞在转染后48h的荧光显微镜观察结果。
序列信息
本申请所涉及的序列的信息概述于表1中。
表1：序列信息
SEQ ID NO: 序列描述
1 单域抗体GBP1的可变区氨基酸序列
2 单域抗体NbsfGFP08的可变区氨基酸序列
3 单域抗体S-Nb2的可变区氨基酸序列
4 单域抗体S-Nb3的可变区氨基酸序列
5 单域抗体S-Nb6的可变区氨基酸序列
6 单域抗体S-Nb7的可变区氨基酸序列
7 单域抗体S-Nb17的可变区氨基酸序列
8 单域抗体S-Nb21的可变区氨基酸序列
9 单域抗体S-Nb25的可变区氨基酸序列
10 单域抗体GBP4的可变区氨基酸序列
11 单域抗体GBPSR1的可变区氨基酸序列
12 单域抗体GBPSR2的可变区氨基酸序列
13 单域抗体LAG2的可变区氨基酸序列
14 单域抗体LAG9的可变区氨基酸序列
15 单域抗体LAG14的可变区氨基酸序列
16 单域抗体LAG16的可变区氨基酸序列
17 单域抗体LAG26的可变区氨基酸序列
18 单域抗体LAG27的可变区氨基酸序列
19 单域抗体LAG30的可变区氨基酸序列
20 单域抗体LAG41的可变区氨基酸序列
21 单域抗体NbsfGFP01的可变区氨基酸序列
22 单域抗体NbsfGFP02的可变区氨基酸序列
23 单域抗体NbsfGFP03的可变区氨基酸序列
24 单域抗体NbsfGFP04的可变区氨基酸序列
25 单域抗体NbsfGFP06的可变区氨基酸序列
26 单域抗体NbsfGFP07的可变区氨基酸序列
27 单域抗体P-Nb1的可变区氨基酸序列
28 单域抗体S-Nb1的可变区氨基酸序列
29 单域抗体S-Nb5的可变区氨基酸序列
30 单域抗体S-Nb27的可变区氨基酸序列
31 sfGFP1-10的氨基酸序列
32 Mdc2-26的氨基酸序列
33 Mdc24的氨基酸序列
34 Mbcd3的氨基酸序列
35 Mbcd4的氨基酸序列
36 Mbcd36的氨基酸序列
37 Mbcd37的氨基酸序列
38 Mbcd38的氨基酸序列
39 Mbcd39的氨基酸序列
40 Mbcd41的氨基酸序列
41 Mbcd44的氨基酸序列
42 Mbcd52的氨基酸序列
43 test3-3的氨基酸序列
44 test5-3的氨基酸序列
45 BFP1-10的氨基酸序列
46 YFP1-10的氨基酸序列
47-49 单域抗体GBP1的CDR1-CDR3的氨基酸序列
50-52 单域抗体NbsfGFP08的CDR1-CDR3的氨基酸序列
53-55 单域抗体S-Nb2的CDR1-CDR3的氨基酸序列
56-58 单域抗体S-Nb3的CDR1-CDR3的氨基酸序列
59-61 单域抗体S-Nb6的CDR1-CDR3的氨基酸序列
62-64 单域抗体S-Nb7的CDR1-CDR3的氨基酸序列
65-67 单域抗体S-Nb17的CDR1-CDR3的氨基酸序列
68-70 单域抗体S-Nb21的CDR1-CDR3的氨基酸序列
71-73 单域抗体S-Nb25的CDR1-CDR3的氨基酸序列
74-81 引物
82 柔性接头的氨基酸序列
83 自切割接头的氨基酸序列
84 绿色荧光蛋白的氨基酸序列
85 蓝色荧光蛋白的氨基酸序列
86 黄色荧光蛋白的氨基酸序列
87 GBPMT1的氨基酸序列
88 GBPMT2的氨基酸序列
Figure PCTCN2018081798-appb-000001
Figure PCTCN2018081798-appb-000002
Figure PCTCN2018081798-appb-000003
Figure PCTCN2018081798-appb-000004
Figure PCTCN2018081798-appb-000005
Figure PCTCN2018081798-appb-000006
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley & Sons,Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1.编码抗GFP单域抗体的表达质粒的构建
根据之前的文献报道(参见Kirchhofer A.等人，Nature Structural & Molecular Biology,2010 Jan；17(1):133-8；Fleetwood,F.等人,Cellular & Molecular Life Sciences,2013.70(6):p.1081-93；Ryckaert S.等人,Journal of biotechnology,2010 Jan 15；145(2):93-8；Aya Twair等人，Molecular Biology Reports,October 2014,Volume 41,Issue 10,pp 6887-6898)，获得了30种不同的抗GFP单域抗体的序列(SEQ ID NO:1-30)。随后，由上海生工生物工程股份有限公司分别合成了编码这30种单域抗体的DNA片段。分别以这30种合成的DNA片段为模板，利用引物VHHF和VHHR进行聚合酶链式反应(PCR)。PCR反应的条件为：98℃，10min；30个循环的(98℃，30s；58℃，30s；68℃，30s)；68℃，5min。引物VHHF和VHHR的序列如表2所示。
表2：引物的序列
SEQ ID NO: 引物名称 引物序列(5'-3')
74 VHHF gctagcaagcttgccaccatggcc
75 VHHR gtcgaggtcgggggatcctta
PCR反应后，回收大小为约400bp的产物。通过下述步骤，将回收的PCR产物分别连接入可商购获得的pTT5载体：将pTT5载体用BamHⅠ/HindⅢ进行酶切，然后用NEB公司的Gibson Assembly试剂将回收的PCR产物和经酶切的pTT5载体连接在一起。用所获得的连接产物转化DH5α感受态细胞，并在37℃培养箱中培养 12小时。随后，挑取单克隆菌落，提取质粒，并进行测序，从而获得编码抗GFP单域抗体的表达质粒。
共获得了如下30种表达质粒：
pTT5-GBP1，其编码抗GFP单域抗体GBP1(SEQ ID NO:1)；
pTT5-NbsfGFP08，其编码抗GFP单域抗体NbsfGFP08(SEQ ID NO:2)；
pTT5-S-Nb2，其编码抗GFP单域抗体S-Nb2(SEQ ID NO:3)；
pTT5-S-Nb3，其编码抗GFP单域抗体S-Nb3(SEQ ID NO:4)；
pTT5-S-Nb6，其编码抗GFP单域抗体S-Nb6(SEQ ID NO:5)；
pTT5-S-Nb7，其编码抗GFP单域抗体S-Nb7(SEQ ID NO:6)；
pTT5-S-Nb17，其编码抗GFP单域抗体S-Nb17(SEQ ID NO:7)；
pTT5-S-Nb21，其编码抗GFP单域抗体S-Nb21(SEQ ID NO:8)；
pTT5-S-Nb25，其编码抗GFP单域抗体S-Nb25(SEQ ID NO:9)；
pTT5-GBP4，其编码抗GFP单域抗体GBP4(SEQ ID NO:10)；
pTT5-GBPSR1，其编码抗GFP单域抗体GBPSR1(SEQ ID NO:11)；
pTT5-GBPSR2，其编码抗GFP单域抗体GBPSR2(SEQ ID NO:12)；
pTT5-LAG2，其编码抗GFP单域抗体LAG2(SEQ ID NO:13)；
pTT5-LAG9，其编码抗GFP单域抗体LAG9(SEQ ID NO:14)；
pTT5-LAG14，其编码抗GFP单域抗体LAG14(SEQ ID NO:15)；
pTT5-GBP1，其编码抗GFP单域抗体LAG16(SEQ ID NO:16)；
pTT5-LAG26，其编码抗GFP单域抗体LAG26(SEQ ID NO:17)；
pTT5-LAG27，其编码抗GFP单域抗体LAG27(SEQ ID NO:18)；
pTT5-LAG30，其编码抗GFP单域抗体LAG30(SEQ ID NO:19)；
pTT5-LAG41，其编码抗GFP单域抗体LAG41(SEQ ID NO:20)；
pTT5-NbsfGFP01，其编码抗GFP单域抗体NbsfGFP01(SEQ ID NO:21)；
pTT5-NbsfGFP02，其编码抗GFP单域抗体NbsfGFP02(SEQ ID NO:22)；
pTT5-NbsfGFP03，其编码抗GFP单域抗体NbsfGFP03(SEQ ID NO:23)；
pTT5-NbsfGFP04，其编码抗GFP单域抗体NbsfGFP04(SEQ ID NO:24)；
pTT5-NbsfGFP06，其编码抗GFP单域抗体NbsfGFP06(SEQ ID NO:25)；
pTT5-NbsfGFP07，其编码抗GFP单域抗体NbsfGFP07(SEQ ID NO:26)；
pTT5-P-Nb1，其编码抗GFP单域抗体P-Nb1(SEQ ID NO:27)；
pTT5-S-Nb1，其编码抗GFP单域抗体S-Nb1(SEQ ID NO:28)；
pTT5-S-Nb5，其编码抗GFP单域抗体S-Nb5(SEQ ID NO:29)；
pTT5-S-Nb27，其编码抗GFP单域抗体S-Nb27(SEQ ID NO:30)。
实施例2.编码sfGFP1-10的表达质粒的构建
以合成的sfGFP序列(Stéphanie Cabantous等人，Nature Biotechnology 23,102-107(2005))为模板，利用引物HdGFPF和BmGFP1-10R进行PCR反应，以获得编码sfGFP1-10(SEQ ID NO:31，其为sfGFP蛋白的aa 1-214(即C端截短了16个氨基酸残基的sfGFP蛋白))的DNA片段。PCR反应的条件为：98℃，10min；30个循环的(98℃，30s；58℃，30s；68℃，30s)；68℃，5min。引物HdGFPF和BmGFP1-10R的序列如表3所示。
表3：引物的序列
SEQ ID NO: 引物名称 引物序列(5'-3')
76 HdGFPF gagggcccgtttctgctagcaagcttatggtttcgaaaggcgaggag
77 BmGFP1-10R gccagaggtcgaggtcgggggatccttatttctcgtttgggtctt
按照实施例1描述的方法，将如上获得的PCR扩增产物连接入pTT22M载体(其 为经改造的PTT22载体，其中PTT22载体中的嘌呤霉素基因被替换成编码红色荧光蛋白mCherry的基因)中，从而获得编码sfGFP1-10(SEQ ID NO:31)的表达质粒pTT22M-sfGFP1-10。
实施例3.能够使sfGFP1-10恢复荧光的单域抗体的鉴定
以每孔10000个细胞的密度，将Hela细胞悬液铺板到96孔细胞培养板中，培养体积为每孔100μL。培养20h后，依照试剂盒的说明书，使用
Figure PCTCN2018081798-appb-000007
LTX with Plus Reagent(Invitrogen公司)，将编码单域抗体的表达质粒和pTT22M-sfGFP1-10共同转染至Hela细胞中。另外，还将空载体pTT5和pTT22M-sfGFP1-10共同转染至Hela细胞中，用作阴性对照。
转染48h后，用荧光显微镜观察各个孔中的细胞的状态和荧光。结果如图1所示。图1显示，共转染了编码单域抗体的表达质粒和pTT22M-sfGFP1-10的Hela细胞在转染后48h的荧光显微镜观察结果；其中，对于每一个实验组的细胞，上图显示了红光通道的观察结果(用于指示转染效率)，下图显示了绿光通道的观察结果(用于显示细胞是否发出绿色荧光)；“vector”组表示转染了空载体pTT5和pTT22M-sfGFP1-10的Hela细胞。
图1的结果显示，转染后，所有实验组的细胞都能够发出红色荧光，这表明pTT22M-sfGFP1-10(其携带编码红色荧光蛋白mCherry的基因)已被成功转染入Hela细胞，并表达出红色荧光蛋白mCherry。进一步，图1的结果显示，单独表达sfGFP1-10的Hela细胞不能发出绿色荧光(“vector”组)；并且，共表达sfGFP1-10和单域抗体GBP4、GBPSR1、GBPSR2、LAG2、LAG9、LAG14、LAG16、LAG26、LAG27、LAG30、LAG41、NbsfGFP01、NbsfGFP02、NbsfGFP03、NbsfGFP04、NbsfGFP06、NbsfGFP07、P-Nb1、S-Nb1、S-Nb5或S-Nb27的Hela细胞也不能够发出绿色荧光；但是，共表达sfGFP1-10和单域抗体GBP1、NbsfGFP08、S-Nb2、S-Nb3、S-Nb6、S-Nb7、S-Nb17、S-Nb21或S-Nb25的Hela细胞则能够发出绿色荧光。
图1的实验结果表明，单域抗体GBP1、NbsfGFP08、S-Nb2、S-Nb3、S-Nb6、S-Nb7、S-Nb17、S-Nb21和S-Nb25能够与sfGFP1-10发生特异性相互作用，并使之发出绿色荧光。另外，图1的结果还显示，共表达sfGFP1-10和单域抗体GBP1的Hela细胞的绿色荧光最强。因此，在某些情况下，单域抗体GBP1是能够使sfGFP1-10发出绿色荧光的优选抗体。
另外，还通过本领域熟知的Kabat方法(Kabat EA,Wu TT,Perry HM,Gottesman KS,Coeller K.Sequences of proteins of immunological interest,U.S Department of Health and Human Services,PHS,NIH,Bethesda,1991)，确定了单域抗体GBP1、NbsfGFP08、S-Nb2、S-Nb3、S-Nb6、S-Nb7、S-Nb17、S-Nb21和S-Nb25的互补决定区(CDR)序列。结果如表4所示。
表4：9株单域抗体的CDR序列
Figure PCTCN2018081798-appb-000008
Figure PCTCN2018081798-appb-000009
实施例4.sfGFP的其他截短体的验证
如上所述，已在实施例3中证实，sfGFP1-10能够与9株单域抗体相互作用，并发出荧光。在本实施例中，评估了sfGFP的其他截短体是否具有与sfGFP1-10相同的性质。
简言之，基本上按照实施例2中描述的方案，制备了编码下述sfGFP截短体的表达质粒：
CM5：其与sfGFP相比，C端截短了5个氨基酸残基；
CM9：其与sfGFP相比，C端截短了9个氨基酸残基；
CM10：其与sfGFP相比，C端截短了10个氨基酸残基；
CM11：其与sfGFP相比，C端截短了11个氨基酸残基；
CM16(即sfGFP1-10)：其与sfGFP相比，C端截短了16个氨基酸残基；
CM21：其与sfGFP相比，C端截短了21个氨基酸残基；
CM22：其与sfGFP相比，C端截短了22个氨基酸残基；
CM23：其与sfGFP相比，C端截短了23个氨基酸残基；
CM24：其与sfGFP相比，C端截短了24个氨基酸残基；
CM26：其与sfGFP相比，C端截短了26个氨基酸残基；
CM28：其与sfGFP相比，C端截短了28个氨基酸残基；
CM32：其与sfGFP相比，C端截短了32个氨基酸残基。
随后，按照实施例3中描述的方法，在Hela细胞中表达sfGFP的各种截短体，或者共表达sfGFP的各种截短体和单域抗体GBP1，并使用荧光显微镜观察Hela细胞的状态和荧光。
简言之，以每孔10000个细胞的密度，将Hela细胞悬液铺板到96孔细胞培养板中，培养体积为每孔100μL。培养20h后，依照试剂盒的说明书，使用
Figure PCTCN2018081798-appb-000010
LTX with Plus Reagent(Invitrogen公司)，将PTT5载体和编码sfGFP截短体的表达质粒(用于指示sfGFP截短体本身是否发出荧光)，或者将pTT5-GBP1和编码sfGFP截短体的表达质粒(用于指示GBP1是否能够使本身不发出荧光的sfGFP截短体恢复荧光)，共同转染至Hela细胞中。
转染48h后，用荧光显微镜观察各个孔中的细胞的荧光。结果如图2所示。图2显示，共转染了编码sfGFP的C端截短变体的表达质粒以及PTT5(图2A)或pTT5-GBP1(图2B)的Hela细胞在转染后48h的荧光显微镜观察结果；其中，“WT”组表示共转染了编码荧光蛋白sfGFP的表达质粒以及pTT5(图2A)或pTT5-GBP1(图2B)的Hela细胞。
图2A的实验结果显示，截短体CM5本身能够显示明显的绿色荧光，截短体CM9 仅能够显示极其微弱的绿色荧光，而其他截短体均不能显示绿色荧光。这些结果表明，当sfGFP蛋白的C端截短9个或更多个氨基酸残基时，所产生的截短体基本上丧失了发出绿色荧光的能力。
进一步，图2B的实验结果显示，共表达GBP1和CM9，CM10,CM11,CM16,CM21,CM22或CM23的Hela细胞能够发出绿色荧光；但共表达GBP1和CM24，CM26,CM28或CM32的Hela细胞不能发出绿色荧光。这些结果表明，GBP1能够与CM9,CM10,CM11,CM16,CM21,CM22或CM23相互作用，并使其恢复发出绿色荧光的能力。
上述实验结果表明，C端截短了9-23个氨基酸残基的sfGFP蛋白截短体具有与sfGFP1-10相同的性质：即，其本身不能发出荧光，但是在所筛选的单域抗体(例如GBP1)的作用下，能够发出荧光。
实施例5.sfGFP1-10的突变
本实验考察了sfGFP1-10对突变的耐受程度，并获得了能够与单域抗体GBP1组合使用的优选GFP片段。
对sfGFP1-10的序列进行随机突变，以获得sfGFP1-10的变体。随后，按照实施例3中描述的方法，在Hela细胞中共表达sfGFP1-10的变体和单域抗体GBP1，并使用荧光显微镜观察Hela细胞的状态和荧光。
简言之，以每孔10000个细胞的密度，将Hela细胞悬液铺板到96孔细胞培养板中，培养体积为每孔100μL。培养20h后，依照试剂盒的说明书，使用
Figure PCTCN2018081798-appb-000011
LTX with Plus Reagent(Invitrogen公司)，将pTT5-GBP1和编码sfGFP1-10变体的表达质粒共同转染至Hela细胞中。另外，还将pTT22M-sfGFP1-10和pTT5-GBP1共同转染至Hela细胞中，用作阳性对照；将pTT5-GBP1和编码无关蛋白的表达质粒共同转染至Hela细胞中，用作阴性对照。
转染48h后，用荧光显微镜观察各个孔中的细胞的荧光。结果如图3所示。图3显示，共转染了pTT5-GBP1和编码sfGFP1-10变体的表达质粒的Hela细胞在转染后48h的荧光显微镜观察结果；其中，“Negative”组表示共转染了pTT5-GBP1和编码无关蛋白的表达质粒的Hela细胞。
图3的结果显示，共表达单域抗体GBP1和sfGFP1-10或其变体(Mdc2-26、Mdc24、Mbcd3、Mbcd4、Mbcd36、Mbcd37、Mbcd38、Mbcd39、Mbcd41、Mbcd44、Mbcd52、Test3-3或Test5-3)的Hela细胞能够发出绿色荧光；但是，共表达单域抗体GBP1和无关蛋白的Hela细胞不能够发出荧光。
Mdc2-26、Mdc24、Mbcd3、Mbcd4、Mbcd36、Mbcd37、Mbcd38、Mbcd39、Mbcd41、Mbcd44、Mbcd52、Test3-3和Test5-3的氨基酸序列分别如SEQ ID NO:32-44所示，它们与sfGFP1-10的比较如表5所示。
表5：sfGFP1-10变体与sfGFP1-10的比较
名称 突变残基的数目 同一性(％)
sfGFP1-10 0 100
Mdc2-26 5 97.67
Mdc24 1 99.53
test3-3 4 98.14
test5-3 3 98.60
Mbcd3 10 95.35
Mbcd39 9 95.81
Mbcd41 8 96.28
Mbcd52 6 97.21
Mbcd36 10 95.35
Mbcd4 12 94.42
Mbcd37 12 94.42
Mbcd38 14 93.49
Mbcd44 13 93.95
图3的实验结果表明，sfGFP1-10能够耐受一定程度的突变，而不影响其与GBP1相互作用并发出荧光的能力。因此，可通过各种已知的方法(例如定点诱变法和随机突变法)，对sfGFP1-10的序列进行各种突变和改造，并通过如上所述的方法，筛选获得能够与GBP1相互作用并发出荧光的各种变体。本申请意欲涵盖所有的此类变体。
此外，图3的实验结果还显示，共表达某些sfGFP1-10变体与GBP1的Hela细胞的荧光强度显著高于共表达sfGFP1-10与GBP1的Hela细胞的荧光强度。例如，共表达Mbcd38与GBP1的Hela细胞具有最高的荧光强度。此外，还发现，当将Mdc2-26与GBP1组合使用时，能获得最佳的信噪比：即，单独表达Mdc2-26的Hela细胞的荧光背景极低，并且共表达Mdc2-26与GBP1的Hela细胞的荧光强度增加最为显著。此类sfGFP1-10变体在某些情况下可能是特别有利的。
实施例6.编码BFP1-10或YFP1-10的表达质粒的构建
绿色荧光蛋白与其他颜色的荧光蛋白的主要差异在于，参与激发荧光的结构域(特别是aa 65-67)具有不同的氨基酸残基。在本实施例中，基于编码Mbcd38的核酸序列，构建了编码BFP1-10或YFP1-10的表达质粒，并验证了GBP1与BFP1-10或YFP1-10之间的相互作用。
简言之，以编码Mbcd38的表达质粒(pTT22M-Mbcd38)为模板，利用引物HdGFPF与DrFPbR进行PCR扩增，以获得DNA片段YFPa，且利用引物DrFPbF与BmGFP1-10R进行PCR扩增，以获得DNA片段YFPb。随后，以DNA片段YFPa和YFPb为模板，利用引物HdGFPF与BmGFP1-10R进行PCR扩增，以获得编码YFP1-10(SEQ ID NO:46)的DNA片段。
类似地，以编码Mbcd38的表达质粒(pTT22M-Mbcd38)为模板，利用引物HdGFPF与DrFPcR进行PCR扩增，以获得DNA片段BFPa，且利用引物DrFPcF与BmGFP1-10R进行PCR扩增，以获得DNA片段BFPb。随后，以DNA片段BFPa和BFPb为模板，利用引物HdGFPF与BmGFP1-10R进行PCR扩增，以获得编码BFP1-10(SEQ ID NO:45)的DNA片段。
上述PCR反应所使用的引物的序列如表6所示。
表6：引物的序列
SEQ ID NO: 引物名称 引物序列(5'-3')
76 HdGFPF gagggcccgtttctgctagcaagcttatggtttcgaaaggcgaggag
77 BmGFP1-10R gccagaggtcgaggtcgggggatccttatttctcgtttgggtctt
78 DrFPbF ggctacggcctgcagtgcttcgccagatatccggaccacatg
79 DrFPbR ggcgaagcactgcaggccgtagcccagtgttgtcactagtgttggcca
80 DrFPcF agccacggcgtgcagtgcttcgccagatatccggaccacatg
81 DrFPcR ggcgaagcactgcacgccgtggctcagtgttgtcactagtgttggcca
按照实施例1描述的方法，将如上获得的PCR扩增产物分别连接入pTT22M载体中，从而获得编码BFP1-10(SEQ ID NO:45)的表达质粒(将其命名为pTT22M-BFP1-10)和编码YFP1-10(SEQ ID NO:46)的表达质粒(将其命名为pTT22M-YFP1-10)。
随后，按照实施例3描述的方法，验证GBP1与BFP1-10或YFP1-10之间的相互作用。简言之，以每孔10000个细胞的密度，将Hela细胞悬液铺板到96孔细胞培养板中，培养体积为每孔100μL。培养20h后，依照试剂盒的说明书，使用
Figure PCTCN2018081798-appb-000012
LTX with Plus Reagent(Invitrogen公司)，将编码单域抗体GBP1的表达质粒 (pTT5-GBP1)与pTT22M-BFP1-10或pTT22M-YFP1-10共同转染至Hela细胞中。另外，还将空载体pTT5与pTT22M-BFP1-10或pTT22M-YFP1-10共同转染至Hela细胞中，用作阴性对照。
转染48h后，用荧光显微镜观察各个孔中的细胞的状态和荧光。结果如图4所示。图4显示，共转染了pTT5-GBP1与pTT22M-BFP1-10或pTT22M-YFP1-10的Hela细胞在转染后48h的荧光显微镜观察结果；其中，“B/Y”表示蓝光/黄光通道的观察结果；“R”表示红光通道的观察结果；“Merge”表示两种通道的观察结果的合并。
图4的结果显示，单独表达BFP1-10或YFP1-10的Hela细胞不能发出荧光(“BFP1-10”组和“YFP1-10”组)；而共表达BFP1-10和单域抗体GBP1的Hela细胞能够发出蓝色荧光，共表达YFP1-10和单域抗体GBP1的Hela细胞能够发出黄色荧光。
这些结果表明，GBP1不仅能够使不发荧光的GFP片段恢复荧光，而且能够使不发荧光的BFP片段和YFP片段恢复荧光。因此，本发明的原理和方法可适用于各种荧光蛋白。
实施例7.GBP1/sfGFP1-10在蛋白定位中的应用
在本实施例中，以7种目的蛋白(ACTB1,TUBB3,MAPRE3,H2B,LMNB1,PAXILLIN,EndoG)为例，验证了GBP1/sfGFP1-10在蛋白定位中的应用。简言之，在细胞中共表达含有GBP1和目的蛋白的融合蛋白以及sfGFP1-10，随后通过GBP1和sfGFP1-10之间的相互作用，确定目的蛋白在细胞内的分布和位置。ACTB1,TUBB3,MAPRE3,H2B,LMNB1,PAXILLIN,EndoG的氨基酸序列均可参见GeneBank(GeneBank登录号分别如下：ACTB1,NM_001101；TUBB3,NM_006086；MAPRE3,XM_004028974；H2B,AK311849；LMNB1,BC012295；PAXILLIN,XM_015275216；EndoG,BC004922)。
按照一般的分子克隆方案，构建了下述表达质粒：
pTT5-GBP-ACTB1，其编码包含GBP1和ACTB1的融合蛋白GBP-ACTB1，其中GBP1连接至ACTB1的N端；
pTT5-BFP-ACTB1，其编码包含全长BFP和ACTB1的融合蛋白BFP-ACTB1，其中BFP连接至ACTB1的N端；
pTT5-TUBB3-GBP，其编码包含GBP1和TUBB3的融合蛋白TUBB3-GBP，其中GBP1连接至TUBB3的C端；
pTT5-TUBB3-BFP，其编码包含全长BFP和TUBB3的融合蛋白TUBB3-BFP，其中BFP连接至TUBB3的C端；
pTT5-GBP-MAPRE3，其编码包含GBP1和MAPRE3的融合蛋白GBP-MAPRE3，其中GBP1连接至MAPRE3的N端；
pTT5-BFP-MAPRE3，其编码包含全长BFP和MAPRE3的融合蛋白BFP-MAPRE3，其中BFP连接至MAPRE3的N端；
pTT5-GBP-H2B，其编码包含GBP1和H2B的融合蛋白GBP-H2B，其中GBP1连接至H2B的N端；
pTT5-BFP-H2B，其编码包含全长BFP和H2B的融合蛋白BFP-H2B，其中BFP连接至H2B的N端；
pTT5-GBP-LMNB1，其编码包含GBP1和LMNB1的融合蛋白GBP-LMNB1，其中GBP1连接至LMNB1的N端；
pTT5-BFP-LMNB1，其编码包含全长BFP和LMNB1的融合蛋白BFP-LMNB1，其中BFP连接至LMNB1的N端；
pTT5-Paxillin-GBP，其编码包含GBP1和Paxillin的融合蛋白Paxillin-GBP，其 中GBP1连接至Paxillin的C端；
pTT5-Paxillin-BFP，其编码包含全长BFP和Paxillin的融合蛋白Paxillin–BFP，其中BFP连接至Paxillin的C端；
pTT5-EndoG–GBP，其编码包含GBP1和EndoG的融合蛋白EndoG–GBP，其中GBP1连接至EndoG的C端；
pTT5-EndoG-BFP，其编码包含全长BFP和EndoG的融合蛋白EndoG-BFP，其中BFP连接至EndoG的C端。
随后，按照实施例3描述的方法，在Hela细胞中分别共转染下述表达质粒的组合：
(1)pTT5-GBP-ACTB1+pTT5-BFP-ACTB1+pTT22M-sfGFP1-10；
(2)pTT5-TUBB3-GBP+pTT5-TUBB3-BFP+pTT22M-sfGFP1-10；
(3)pTT5-GBP-MAPRE3+pTT5-BFP-MAPRE3+pTT22M-sfGFP1-10；
(4)pTT5-GBP-H2B+pTT5-BFP-H2B+pTT22M-sfGFP1-10；
(5)pTT5-GBP-LMNB1+pTT5-BFP-LMNB1+pTT22M-sfGFP1-10；
(6)pTT5-Paxillin-GBP+pTT5-Paxillin-BFP+pTT22M-sfGFP1-10；或
(7)pTT5-EndoG-GBP+pTT5-EndoG-BFP+pTT22M-sfGFP1-10。
转染48h后，用荧光显微镜观察Hela细胞的荧光。结果如图5所示。图5显示，共转染了各种表达质粒组合的Hela细胞在转染后48h的荧光显微镜观察结果；其中，对于每一个实验组的细胞，上图显示了Hela细胞中的绿色荧光(由融合蛋白中的GBP1+sfGFP1-10产生)的分布和位置；中图显示了Hela细胞中的蓝色荧光(由融合蛋白中的BFP产生)的分布和位置；下图显示了，上图和中图的合并。
从图5的实验结果可以看出，对于每一个实验组的Hela细胞，蓝色荧光与绿色荧光的分布是一致的。这表明，与全长BFP一样，本发明的GBP1/sfGFP1-10组合也能够用于准确确定各种目的蛋白(例如ACTB1,TUBB3,MAPRE3,H2B,LMNB1,PAXILLIN,EndoG)在细胞内的分布和位置。另外，图5的实验结果还表明，GBP1可以以各种方式与目的蛋白相连接。例如，可以将GBP1连接至目的蛋白的N端或C端，而不影响其与sfGFP1-10之间的相互作用。
实施例8.GBP1/Mbcd38在指示细胞融合中的应用
在本实施例中，以喉癌细胞Hep2为例，验证了GBP1/Mbcd38在指示细胞融合中的应用。
简言之，使用本领域熟知的慢病毒感染法，将编码Mbcd38和BFP(蓝色荧光蛋白)的核苷酸序列稳定地整合入喉癌细胞Hep2的基因组中，从而构建获得稳定表达Mbcd38和BFP的细胞株Hep2-Mbcd38。另外，将编码单域抗体GBP1和iRFP(近红外荧光蛋白)的核苷酸序列稳定地整合入喉癌细胞Hep2的基因组中，从而构建获得稳定表达GBP1和iRFP的细胞株Hep2-GBP1。
随后，以每孔30000个细胞的密度，将Hep2-GBP1细胞悬液、Hep2-Mbcd38细胞悬液、含有Hep2-GBP1和Hep2-Mbcd38的细胞悬液(两种细胞的比例为1:1)分别铺板到96孔细胞培养板中。培养24h后，用RSV病毒(呼吸道合胞病毒；MOI＝1)分别感染培养板中的细胞。感染48h后，用荧光显微镜观察各个孔中的细胞的状态和荧光。结果如图6所示。图6显示了，Hep2-GBP1细胞悬液、Hep2-Mbcd38细胞悬液以及含有Hep2-GBP1和Hep2-Mbcd38的细胞悬液在感染RSV病毒48h后的荧光显微镜观察结果。
图6的结果显示，在感染RSV病毒后，在含有单独的Hep2-Mbcd38的培养物中，能够观察到蓝色荧光(由BFP蛋白产生)，而无法观察到近红外荧光或绿色荧光；在含有单独的Hep2-GBP1的培养物中，能够观察到近红外荧光(由iRFP蛋白产生)， 而无法观察到蓝色荧光或绿色荧光；在含有Hep2-GBP1和Hep2-Mbcd38的培养物中，能够观察到蓝色荧光(由BFP蛋白产生)，近红外荧光(由iRFP蛋白产生)，以及绿色荧光(由GBP1+Mbcd38产生)。这些结果表明：(1)Hep2-Mbcd38已稳定整合了编码Mbcd38和BFP的核苷酸序列，能够表达Mbcd38和BFP，从而能够发出蓝色荧光；(2)Hep2-GBP1已稳定整合了编码GBP1和iRFP的核苷酸序列，能够表达GBP1和iRFP，从而能够发出近红外荧光；(3)在感染RSV病毒后，混合培养的Hep2-GBP1和Hep2-Mbcd38发生了细胞融合，由此这两种细胞各自表达的GBP1和Mbcd38发生了相互作用，产生了绿色荧光。因此，这些实验结果证实，本发明的GBP1/Mbcd38组合可用于指示细胞融合，例如由RSV感染引起的细胞融合。
实施例9.GBP1/Mdc2-26在指示穿膜肽的穿膜作用中的应用
在本实施例中，以穿膜肽pep1(参见Manceur A.等人，Analytical Biochemistry,2007,364(1):51-59)为例，验证了GBP1/Mdc2-26在指示穿膜肽的穿膜作用中的应用。
如实施例3所述，使用
Figure PCTCN2018081798-appb-000013
LTX with Plus Reagent(Invitrogen公司)，将编码Mdc2-26的表达质粒转染入U2OS细胞中，以使得U2OS细胞表达Mdc2-26。
转染后36h，去除U2OS细胞培养物的培养液，并添加新鲜培养基，所述新鲜培养基含有80μg GBP1蛋白或者80μg GBP1蛋白与10μg穿膜肽pep1的混合物。随后，用荧光显微镜观察U2OS细胞。结果如图7所示。图7显示，表达Mdc2-26的U2OS细胞在与GBP1或者GBP1+穿膜肽pep1一起温育6h、8h、10h或12h后的荧光显微镜观察结果。
图7的实验结果显示，与不使用pep1的情况相比，在使用pep1的情况下，在U2OS细胞培养物中观察到显著更强的荧光。这些结果表明，pep1能够促进GBP1蛋白进入U2OS细胞中，从而U2OS细胞中具有更多的GBP1蛋白，能够与Mdc2-26产生更强的相互作用，发出更强的绿色荧光。因此，这些结果进一步表明，本发明的GBP1/Mdc2-26能够用于指示穿膜肽(例如pep1)的穿膜作用。
此外，相比于使用FITC或者EGFP来检测穿膜肽的穿膜作用的常规方法(参见Manceur A.等人，Analytical Biochemistry,2007,364(1):51-59)而言，本发明的使用GBP1/Mdc2-26的检测方法的本底更低，并且不需要把残余的FITC或者EGFP清洗掉，操作更为简单。
实施例10.GBP1/sfGFP1-10与G11/sfGFP1-10的比较
之前已报道，G11(GFP的氨基酸215-230)能够与sfGFP1-10相互作用，并恢复sfGFP1-10的荧光。因此，G11与sfGFP1-10可用作蛋白标签系统。在本实施例中，以6种目的蛋白(Agr2,HBc,NTCP,NP,TUBB3,hGBP1)为例，比较了GBP1/sfGFP1-10与G11/sfGFP1-10的性能和效果。Agr2,HBc,NTCP,NP,TUBB3,hGBP1的氨基酸序列均可参见GenBank(GenBank登录号分别如下：Agr2,KJ767789；HBc,AB818694；NTCP,BC074724；NP,EU330203；TUBB3,NM_006086；hGBP1,BC002666)。
简言之，按照一般的分子克隆方案，构建了下述表达质粒：
pTT5-Agr2-G11，其编码包含Agr2和G11的融合蛋白Agr2-G11，其中G11通过柔性接头(GSSGGSSG；SEQ ID NO:82)连接至Agr2的C端；
pTT5-G11-Agr2，其编码包含Agr2和G11的融合蛋白G11-Agr2，其中G11通过柔性接头(SEQ ID NO:82)连接至Agr2的N端；
pTT5-G11-2A-Agr2，其编码包含Agr2和G11的融合蛋白G11-2A-Agr2，其中G11通过自切割接头(GSSGGSSGGSGATNFSLLKQAG DVEENPGP；SEQ ID NO:83)连接至Agr2的N端；
pTT5-Agr2-GBP1，其编码包含Agr2和GBP1的融合蛋白Agr2-GBP1，其中GBP1 通过柔性接头(SEQ ID NO:82)连接至Agr2的C端；
pTT5-GBP1-Agr2，其编码包含Agr2和GBP1的融合蛋白GBP1-Agr2，其中GBP1通过柔性接头(SEQ ID NO:82)连接至Agr2的N端；
pTT5-GBP1-2A-Agr2，其编码包含Agr2和GBP1的融合蛋白GBP1-2A-Agr2，其中GBP1通过自切割接头(SEQ ID NO:83)连接至Agr2的N端；
pTT5-HBc-G11，其编码包含HBc和G11的融合蛋白HBc-G11，其中G11通过柔性接头(SEQ ID NO:82)连接至HBc的C端；
pTT5-G11-HBc，其编码包含HBc和G11的融合蛋白G11-HBc，其中G11通过柔性接头(SEQ ID NO:82)连接至HBc的N端；
pTT5-G11-2A-HBc，其编码包含HBc和G11的融合蛋白G11-2A-HBc，其中G11通过自切割接头(SEQ ID NO:83)连接至HBc的N端；
pTT5-HBc-GBP1，其编码包含HBc和GBP1的融合蛋白HBc-GBP1，其中GBP1通过柔性接头(SEQ ID NO:82)连接至HBc的C端；
pTT5-GBP1-HBc，其编码包含HBc和GBP1的融合蛋白GBP1-HBc，其中GBP1通过柔性接头(SEQ ID NO:82)连接至HBc的N端；
pTT5-GBP1-2A-HBc，其编码包含HBc和GBP1的融合蛋白GBP1-2A-HBc，其中GBP1通过自切割接头(SEQ ID NO:83)连接至HBc的N端；
pTT5-NTCP-G11，其编码包含NTCP和G11的融合蛋白NTCP-G11，其中G11通过柔性接头(SEQ ID NO:82)连接至NTCP的C端；
pTT5-G11-NTCP，其编码包含NTCP和G11的融合蛋白G11-NTCP，其中G11通过柔性接头(SEQ ID NO:82)连接至NTCP的N端；
pTT5-G11-2A-NTCP，其编码包含NTCP和G11的融合蛋白G11-2A-NTCP，其中G11通过自切割接头(SEQ ID NO:83)连接至NTCP的N端；
pTT5-NTCP-GBP1，其编码包含NTCP和GBP1的融合蛋白NTCP-GBP1，其中GBP1通过柔性接头(SEQ ID NO:82)连接至NTCP的C端；
pTT5-GBP1-NTCP，其编码包含NTCP和GBP1的融合蛋白GBP1-NTCP，其中GBP1通过柔性接头(SEQ ID NO:82)连接至NTCP的N端；
pTT5-GBP1-2A-NTCP，其编码包含NTCP和GBP1的融合蛋白GBP1-2A-NTCP，其中GBP1通过自切割接头(SEQ ID NO:83)连接至NTCP的N端；
pTT5-NP-G11，其编码包含NP和G11的融合蛋白NP-G11，其中G11通过柔性接头(SEQ ID NO:82)连接至NP的C端；
pTT5-G11-NP，其编码包含NP和G11的融合蛋白G11-NP，其中G11通过柔性接头(SEQ ID NO:82)连接至NP的N端；
pTT5-G11-2A-NP，其编码包含NP和G11的融合蛋白G11-2A-NP，其中G11通过自切割接头(SEQ ID NO:83)连接至NP的N端；
pTT5-NP-GBP1，其编码包含NP和GBP1的融合蛋白NP-GBP1，其中GBP1通过柔性接头(SEQ ID NO:82)连接至NP的C端；
pTT5-GBP1-NP，其编码包含NP和GBP1的融合蛋白GBP1-NP，其中GBP1通过柔性接头(SEQ ID NO:82)连接至NP的N端；
pTT5-GBP1-2A-NP，其编码包含NP和GBP1的融合蛋白GBP1-2A-NP，其中GBP1通过自切割接头(SEQ ID NO:83)连接至NP的N端；
pTT5-hGBP1-G11，其编码包含hGBP1和G11的融合蛋白hGBP1-G11，其中G11通过柔性接头(SEQ ID NO:82)连接至hGBP1的C端；
pTT5-G11-hGBP1，其编码包含hGBP1和G11的融合蛋白G11-hGBP1，其中G11通过柔性接头(SEQ ID NO:82)连接至hGBP1的N端；
pTT5-G11-2A-hGBP1，其编码包含hGBP1和G11的融合蛋白G11-2A-hGBP1， 其中G11通过自切割接头(SEQ ID NO:83)连接至hGBP1的N端；
pTT5-hGBP1-GBP1，其编码包含hGBP1和GBP1的融合蛋白hGBP1-GBP1，其中GBP1通过柔性接头(SEQ ID NO:82)连接至hGBP1的C端；
pTT5-GBP1-hGBP1，其编码包含hGBP1和GBP1的融合蛋白GBP1-hGBP1，其中GBP1通过柔性接头(SEQ ID NO:82)连接至hGBP1的N端；
pTT5-GBP1-2A-hGBP1，其编码包含hGBP1和GBP1的融合蛋白GBP1-2A-hGBP1，其中GBP1通过自切割接头(SEQ ID NO:83)连接至hGBP1的N端；
pTT5-TUBB3-G11，其编码包含TUBB3和G11的融合蛋白TUBB3-G11，其中G11通过柔性接头(SEQ ID NO:82)连接至TUBB3的C端；
pTT5-G11-TUBB3，其编码包含TUBB3和G11的融合蛋白G11-TUBB3，其中G11通过柔性接头(SEQ ID NO:82)连接至TUBB3的N端；
pTT5-G11-2A-TUBB3，其编码包含TUBB3和G11的融合蛋白G11-2A-TUBB3，其中G11通过自切割接头(SEQ ID NO:83)连接至TUBB3的N端；
pTT5-TUBB3-GBP1，其编码包含TUBB3和GBP1的融合蛋白TUBB3-GBP1，其中GBP1通过柔性接头(SEQ ID NO:82)连接至TUBB3的C端；
pTT5-GBP1-TUBB3，其编码包含TUBB3和GBP1的融合蛋白GBP1-TUBB3，其中GBP1通过柔性接头(SEQ ID NO:82)连接至TUBB3的N端；
pTT5-GBP1-2A-TUBB3，其编码包含TUBB3和GBP1的融合蛋白GBP1-2A-TUBB3，其中GBP1通过自切割接头(SEQ ID NO:83)连接至TUBB3的N端。
随后，按照实施例3描述的方法，在293细胞中共转染表达质粒pTT22M-sfGFP1-10以及如上制备的36种表达质粒中的任一种。转染48h后，用荧光显微镜观察293细胞的荧光。结果如图8所示。图8显示，共转染了各种表达质粒组合的293细胞在转染后48h的荧光显微镜观察结果。
图8的实验结果显示，当G11连接至目的蛋白的C端时，G11与sfGFP1-10的共表达能够产生较强的绿色荧光；但是，当G11通过柔性接头连接至Agr2,HBc,NTCP或通过自切割接头连接至任一目的蛋白的N端时，G11与sfGFP1-10的共表达只能产生很弱的绿色荧光。相比之下，对于所有6种蛋白以及所有3种连接方式，GBP1与sfGFP1-10的共表达均能够产生强绿色荧光。GBP1与sfGFP1-10的之间的相互作用不受目的蛋白的种类和连接方式的影响。
这些实验结果表明，当使用G11/sfGFP1-10来标记蛋白时，应当将G11连接至目的蛋白的C端；而本发明的GBP1/sfGFP1-10系统则不受连接方式的限制，可以以各种方式进行应用。例如，可以将GBP1游离表达，或融合至目的蛋白的N端或者C端，而基本上不影响本发明的GBP1/sfGFP1-10系统的标记功能。
实施例11.GBP1抗体FR区的突变
在本实施例中，对GBP1抗体的FR区进行了随机突变，获得了2个突变体。这2个突变体分别被命名为GBPMT1和GBPMT2，并且其氨基酸序列分别如SEQ ID NO:87和SEQ ID NO:88所示。
合成编码GBPMT1的基因和编码GBPMT2的基因，并按照上文描述的方法，将它们分别克隆入PTT5载体。
随后，按照实施例3描述的方法，将表达质粒pTT22M-Mdc2-26以及携带编码GBPMT1或GBPMT2的基因的表达质粒共转染入Hela细胞中。另外，将表达质粒pTT22M-Mdc2-26和携带编码GBP1的基因的表达质粒共转染入Hela细胞中，用作对照。转染48h后，用荧光显微镜观察Hela细胞的荧光。结果如图9所示。
图9显示，共转染了Mdc2-26以及GBP1或GBPMT1或GBPMT2的Hela细胞都能展示出绿色荧光。这个结果说明，GBP1或GBPMT1或GBPMT2均能够使Mdc2-26恢复荧光。这进一步说明：单域抗体(例如GBP1)的功能/性质(即，使荧光蛋白截短体(例如Mdc2-26)恢复荧光的功能/性质)主要由其CDR1-3决定；单域抗体(例如GBP1)的FR区的突变不影响其功能/性质。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公开的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (21)

  1. 一种试剂盒，其包含两种组分，其中，所述第一组分包含：
    (a1)荧光蛋白的截短体，其与荧光蛋白的差异在于，荧光蛋白的C端被截短9-23个氨基酸残基；
    (a2)如(a1)中定义的截短体的变体，所述变体与所述截短体具有至少85％的同一性，或者，所述变体与所述截短体的差异在于一个或多个氨基酸残基的添加、置换或缺失；或
    (a3)核酸分子，其包含编码如(a1)中定义的截短体或如(a2)中定义的变体的核苷酸序列；
    并且，所述第二组分包含：
    (b1)抗荧光蛋白的单域抗体；优选地，其包含选自下列的CDR1、CDR2和CDR3：
    (1)分别如SEQ ID NO:47-49所示的CDR1、CDR2和CDR3；
    (2)分别如SEQ ID NO:50-52所示的CDR1、CDR2和CDR3；
    (3)分别如SEQ ID NO:53-55所示的CDR1、CDR2和CDR3；
    (4)分别如SEQ ID NO:56-58所示的CDR1、CDR2和CDR3；
    (5)分别如SEQ ID NO:59-61所示的CDR1、CDR2和CDR3；
    (6)分别如SEQ ID NO:62-64所示的CDR1、CDR2和CDR3；
    (7)分别如SEQ ID NO:65-67所示的CDR1、CDR2和CDR3；
    (8)分别如SEQ ID NO:68-70所示的CDR1、CDR2和CDR3；和
    (9)分别如SEQ ID NO:71-73所示的CDR1、CDR2和CDR3；或
    (b2)核酸分子，其包含编码如(b1)中定义的单域抗体的核苷酸序列；
    其中，所述截短体和所述变体在游离状态下不发出荧光，但是在与所述单域抗体结合后，能够发出荧光。
  2. 权利要求1的试剂盒，其中，所述荧光蛋白选自绿色荧光蛋白，蓝色荧光蛋白和黄色荧光蛋白；
    优选地，所述绿色荧光蛋白具有如SEQ ID NO:84所示的氨基酸序列；和/或，所述蓝色荧光蛋白具有如SEQ ID NO:85所示的氨基酸序列；和/或，所述黄色荧光蛋白具有如SEQ ID NO:86所示的氨基酸序列。
  3. 权利要求1-2任一项的试剂盒，其中，所述截短体与荧光蛋白的差异在于，荧光蛋白的C端被截短9-23个氨基酸残基，例如被截短9、10、11、12、13、14、15、16、17、18、19、20、21、22、或23个氨基酸残基；
    例如，所述截短体为绿色荧光蛋白的截短体，并且其与绿色荧光蛋白的差异在于，绿色荧光蛋白的C端被截短9-23个氨基酸残基，例如被截短9、10、11、12、13、14、15、16、17、18、19、20、21、22、或23个氨基酸残基(例如，所述截短体具有如SEQ ID NO:31所示的氨基酸序列)；或者，所述截短体为蓝色荧光蛋白的截短体，并且其与蓝色荧光蛋白的差异在于，蓝色荧光蛋白的C端被截短9-23个氨基酸残基，例如被截短9、10、11、12、13、14、15、16、17、18、19、20、21、22、或23个氨基酸残基；或者，所述截短体为黄色荧光蛋白的截短体，并且其与黄色荧光蛋白的差异在于，黄色荧光蛋白的C端被截短9-23个氨基酸残基，例如被截短9、10、11、12、13、14、15、16、17、18、19、20、21、22、或23个氨基酸残基。
  4. 权利要求1-3任一项的试剂盒，其中，所述变体与所述截短体的差异在于一个或 多个氨基酸残基的添加、置换或缺失，例如不超过15个、不超过14个、不超过13个、不超过12个、不超过11个、不超过10个、不超过9个、不超过8个、不超过7个、不超过6个、不超过5个、不超过4个、不超过3个、不超过2个、或1个氨基酸残基的添加、置换或缺失；
    优选地，所述变体与所述截短体的差异在于一个或多个氨基酸残基的置换(例如保守置换)，例如不超过15个、不超过14个、不超过13个、不超过12个、不超过11个、不超过10个、不超过9个、不超过8个、不超过7个、不超过6个、不超过5个、不超过4个、不超过3个、不超过2个、或1个氨基酸残基的置换(例如保守置换)。
  5. 权利要求1-4任一项的试剂盒，其中，所述截短体或所述变体具有选自下列的氨基酸序列：SEQ ID NO:31-46。
  6. 权利要求1-5任一项的试剂盒，其中，所述单域抗体包含重链可变区，所述重链可变区具有选自下列的氨基酸序列：SEQ ID NO:1-9和87-88；
    优选地，所述单域抗体由所述重链可变区组成，或者包含所述重链可变区，以及任选的铰链区、Fc区、或重链恒定区。
  7. 权利要求1-6任一项的试剂盒，其中，(a3)所述的核酸分子包含编码如(a1)中定义的截短体或如(a2)中定义的变体的核苷酸序列，或者由编码如(a1)中定义的截短体或如(a2)中定义的变体的核苷酸序列组成；
    例如，(a3)所述的核酸分子为包含编码如(a1)中定义的截短体或如(a2)中定义的变体的核苷酸序列的载体(例如表达载体)。
  8. 权利要求1-7任一项的试剂盒，其中，(b2)所述的核酸分子包含编码如(b1)中定义的单域抗体的核苷酸序列，或者由编码如(b1)中定义的单域抗体的核苷酸序列组成；
    例如，(b2)所述的核酸分子为包含编码如(b1)中定义的单域抗体的核苷酸序列的载体(例如表达载体)。
  9. 权利要求1-8任一项的试剂盒，其中，所述试剂盒包含：
    如(a1)中定义的截短体或如(a2)中定义的变体，以及如(b1)中定义的单域抗体；或者
    如(a1)中定义的截短体或如(a2)中定义的变体，以及(b2)所述的核酸分子；或者
    如(a3)所述的核酸分子，以及如(b1)中定义的单域抗体；或者
    如(a3)所述的核酸分子，以及(b2)所述的核酸分子。
  10. 权利要求1-9任一项的试剂盒，其中，所述试剂盒还包含额外的试剂，例如用于进行分子克隆或用于构建载体的试剂,例如用于进行核酸扩增的缓冲液、核酸聚合酶、核酸内切酶、连接酶、用于进行核酸纯化的试剂、用于进行核酸转化、转染或转导的试剂，和/或核酸载体。
  11. 一种确定目的蛋白的位置或分布的方法，其包括，使用权利要求1-10任一项的试剂盒。
  12. 一种确定目的蛋白的位置或分布的方法，其包括：
    共表达(1)如权利要求1所定义的截短体或突变体，和(2)包含如权利要求1所定义的单域抗体和所述目的蛋白的融合蛋白；或者
    共表达(3)如权利要求1所定义的单域抗体，和(4)包含如权利要求1所定义的 截短体或突变体和所述目的蛋白的融合蛋白。
  13. 权利要求12的方法，其中，所述方法包括，在细胞内共表达(1)如权利要求1所定义的截短体或突变体和(2)包含如权利要求1所定义的单域抗体和所述目的蛋白的融合蛋白；
    优选地，所述单域抗体连接至所述目的蛋白的N端或C端，任选地通过接头；
    优选地，所述方法还包括，使用荧光显微镜观察所述细胞。
  14. 权利要求12的方法，其中，所述方法包括，在细胞内共表达(3)如权利要求1所定义的单域抗体，和(4)包含如权利要求1所定义的截短体或突变体和所述目的蛋白的融合蛋白；
    优选地，所述截短体或突变体连接至所述目的蛋白的N端或C端，任选地通过接头；
    优选地，所述方法还包括，使用荧光显微镜观察所述细胞。
  15. 一种确定是否发生细胞融合的方法，其包括，使用权利要求1-10任一项的试剂盒。
  16. 一种确定是否发生细胞融合的方法，其包括：
    (1)在第一细胞中表达如权利要求1所定义的截短体或突变体，并且在第二细胞中表达如权利要求1所定义的单域抗体；
    (2)将所述第一细胞和第二细胞共同培养，并使用荧光显微镜进行观察。
  17. 确定一种试剂或病原体(例如病毒或细菌)诱导或抑制细胞融合的能力的方法，其包括下述步骤：
    (1)在第一细胞中表达如权利要求1所定义的截短体或突变体，并且在第二细胞中表达如权利要求1所定义的单域抗体；
    (2)将所述第一细胞和第二细胞共同培养，并使用荧光显微镜进行观察；
    (3)将所述共同培养的第一细胞和第二细胞与所述试剂或病原体接触并继续培养，然后再使用荧光显微镜进行观察。
  18. 确定一种试剂或病原体(例如病毒或细菌)诱导或抑制细胞融合的能力的方法，其包括下述步骤：
    (1)在第一细胞中表达如权利要求1所定义的截短体或突变体，并且在第二细胞中表达如权利要求1所定义的单域抗体；
    (2)将所述第一细胞和第二细胞共同培养，并与所述试剂或病原体接触，用作实验组培养物；并且，将所述第一细胞和第二细胞共同培养，且不与所述试剂或病原体接触，用作对照组培养物；
    (3)使用荧光显微镜观察所述实验组培养物和对照组培养物。
  19. 一种评估试剂促进或抑制多肽穿过细胞膜的能力的方法，其包括，使用权利要求1-10任一项的试剂盒。
  20. 一种评估试剂促进或抑制多肽穿过细胞膜的能力的方法，其中，所述方法包括下述步骤：
    (1)在细胞中表达如权利要求1所定义的截短体或突变体；
    (2)将所述细胞与如权利要求1所定义的单域抗体和所述试剂接触，用作实验组细 胞；并且，将所述细胞与所述单域抗体接触，用作对照组细胞；和
    (3)使用荧光显微镜观察所述实验组细胞和对照组细胞。
  21. 一种评估试剂促进或抑制多肽穿过细胞膜的能力的方法，其中，所述方法包括下述步骤：
    (1)在细胞中表达如权利要求1所定义的单域抗体；
    (2)将所述细胞与如权利要求1所定义的截短体或突变体和所述试剂接触，用作实验组细胞；并且，将所述细胞与如权利要求1所定义的截短体或突变体接触，用作对照组细胞；和
    (3)使用荧光显微镜观察所述实验组细胞和对照组细胞。
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