WO2017130610A1 - Fusion protein and method for detecting antigen using same - Google Patents

Abstract

In order to establish an antigen detection means capable of simply and quickly detecting an antigen and having excellent detection sensitivity, etc., this fusion protein contains an enzyme variant which is activated by multimeric formation, a linker peptide linked to the enzyme variant, and the V_H domain or V_L domain of an antibody which binds to the linker peptide, and is characterized in that the enzyme variant has an introduced mutation which reduces linking affinity between monomers.

Description

Fusion protein and antigen detection method using the same

The present invention relates to a fusion protein comprising an enzyme variant and an antibody variable region, and an antigen detection method and an antigen detection kit using the same.

Currently, immunoassays are becoming an increasingly important measurement technique in clinical diagnosis. In adopting individual immunoassays, not only improvements in sensitivity and specificity, but also rapid and simple measurement are important factors. In the current mainstream immunoassay, the sandwich method is used for the detection of protein biomarkers, and the competitive method is used for the detection of small molecules. However, both of them are enzyme immunoassays that mainly measure the enzyme activity used for the label after several times of reaction and washing, and there is a problem that the measurement takes time and several hours. In comparison, a homogeneous immunoassay method in which a sample and a measurement reagent are mixed and reacted to detect them allows simple and rapid measurement. Homogeneous immunoassay methods such as fluorescence polarization, EMIT, CEDIA, OS-FIA, and Quenchbody are known, and practical sensitivity comparable to that of conventional competitive methods can be obtained for small molecule detection. There are many things that have been made. However, in the method using fluorescence, the sensitivity is limited by the detection sensitivity of the fluorescent label, and there is a problem that signal amplification for improving sensitivity is difficult. On the other hand, in the method using an enzyme that can improve sensitivity by signal amplification, In many cases, the stability of the problem became a problem. For example, the CEDIA method and OS-ECIA method (Non-patent Documents 1 and 2) using activity complementation between β-galactosidase deletion mutants have the advantage that the substrate is easy to use, but the enzyme stability, specific activity, and further activity The small amount of change was a big problem in practical use.

T. Yokozeki, H. Ueda, R. Arai, W. Mahoney and T. Nagamune. Anal. Chem. 74, 2500-2504 (2002). H. Ueda et al., J. Immunol. Methods 279, 209-218 (2003)

As described above, the homogeneous immunoassay has a merit that antigen can be detected easily and rapidly, but there are also problems with the stability and activity of the reagent. The object of the present invention is to solve the problems of the conventional homogeneous immunoassay.

As a result of intensive studies to solve the above problems, the present inventor has discovered that β-glucuronidase (GUS) having reduced tetramer-forming ability instead of the β-galactosidase deletion mutant used in the OS-ECIA method. By using the mutant, it was found that the instability and low activity of the reagent, which were disadvantages of the OS-ECIA method, can be remarkably improved (Example 13), and the present invention has been completed.

The present invention takes advantage of the property of β-glucuronidase, which exhibits enzyme activity only by forming a tetramer. On the other hand, β-galactosidase is an enzyme that exhibits activity by forming a tetramer, but such properties are not used at all in the OS-ECIA method. Therefore, the present invention and the OS-ECIA method are based on completely different ideas.
The present invention has been completed based on the above findings.

That is, the present invention provides the following (1) to (8).

(1) A fusion protein comprising a mutant of an enzyme activated by formation of a multimer, a linker peptide that binds to the mutant of the enzyme, and a V _H region or a V _L region of an antibody that binds to the linker peptide. The fusion protein, wherein the mutant of the enzyme is a mutant into which a mutation that reduces the binding affinity between monomers is introduced.

(2) The fusion protein according to (1), wherein the mutant of the enzyme activated by formation of a multimer is a mutant of β-glucuronidase.

(3) The fusion according to (2), wherein the mutant of β-glucuronidase is a mutant in which the 516th methionine and the 517th tyrosine in the amino acid sequence of Escherichia coli β-glucuronidase are substituted with other amino acids. protein.

(4) The mutant of β-glucuronidase is a mutant in which the 516th methionine in the amino acid sequence of Escherichia coli β-glucuronidase is replaced with lysine and the 517th tyrosine is replaced with glutamic acid. The fusion protein described.

(5) The fusion protein according to any one of (2) to (4), wherein the fusion protein contains one or two β-glucuronidase mutants.

(6) A nucleic acid encoding the fusion protein according to any one of (1) to (5).

(7) A method for detecting an antigen in a sample, the sample comprises a V _L region of a fusion protein and antibody according to any one of including the V _H region of an antibody (1) to (5) (1 A method for detecting an antigen, comprising the steps of contacting the fusion protein according to any one of (1) to (5) and detecting the formation of multimers by a change in enzyme activity.

(8) The fusion protein according to any one of (1) to (5) including the V _H region of the antibody and the fusion protein according to any one of (1) to (5) including the _VL region of the antibody. An antigen detection kit.

This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2016-011475, which is the basis of the priority of the present application.

The antigen detection method of the present invention can detect an antigen simply and rapidly, and is excellent in terms of detection sensitivity.

The figure which shows construction | assembly (A) of a GUS variant, and the primer (B) used for it. Electrophoresis photograph showing PCR product and amplified GUS mutant gene. lane M: 100 bp DNA ladder, lane 1: GUS gene, lane 2: mutated GUS gene fragment (5 'side), lane 3: mutated GUS gene fragment (3' side), lane 4: mutated GUS gene. Electrophoresis photograph showing restriction enzyme-treated vector and GUS mutant gene. lane M: DNA marker, lane 1: GUS mutant gene (after restriction enzyme treatment), lane 2: GUS mutant gene (before restriction enzyme treatment), lane 3: V _H (NP) vector (after restriction enzyme treatment), lane 4: V _H (NP) vector (before restriction enzyme treatment), lane 5: V _L (NP) vector (after restriction enzyme treatment), lane 6: V _L (NP) vector (before restriction enzyme treatment). Construction of GUS mutant expression vector having GS linker (A), electrophoresis photograph showing amplified (G ₄ S) ₃ gene (B), and electrophoresis photograph showing GUS mutant expression vector having GS linker (C). (B) lane M: DNA marker, lane 1-4: amplified (G ₄ S) ₃ gene. (C) Lane 1: pET32-V _H (NP) -GUS vector before restriction enzyme treatment, lane 2: pET32-V _H (NP) -GUS vector treated with NotI, lane 3: pET32 treated with HindIII -V _H (NP) -GUS vector. Electricity of lysates of Escherichia coli expressing V _H (NP)-(G ₄ S) ₃ -GUS mutant (A) and V _L (NP)-(G ₄ S) ₃ -GUS mutant (B) Electrophoresis photograph. lane M: protein molecular weight marker, lane 1: whole cell protein before inducing expression, lane 2: whole cell protein after inducing expression. The arrow indicates the target protein. _{V H (NP) - (G} 4 S) 3 -GUS variants and _{V L (NP) - (G} 4 S) 3 electrophoretogram of purification process of-GUS variant. (A) Purification by TALON-immobilized metal affinity chromatography, lane M: protein molecular weight marker, lane 1-5: V _H (NP)-(G ₄ S) ₃ -GUS mutant, lane 6-10: V _L ( NP)-(G ₄ S) ₃ -GUS mutant, lane 1,6: insoluble fraction, lane 2,7: soluble fraction, lane 3,8: flow-through, lane 4, 5, 9, 10: elution Fraction. _{(B) V L (NP)} - (G 4 S) 3 Purification by anion exchange chromatography -GUS mutants, lane 11: TALON purification eluted fraction, lane 12-15: elution fractions. The figure which shows the time-dependent change of GUS activity (absorbance) with and without an antigen. The figure which shows an antigen concentration and GUS activity (fluorescence) response. Construction of pET32-VH (BGP)-(G4S) 3-GUSm and pET32-VL (BGP)-(G4S) 3-GUSm (A), electrophoresis showing amplified VH (BGP) gene and VL (BGP) gene A photograph (B) and an electrophoresis photograph of the vector after restriction enzyme treatment (C). (B) Left figure: PCR product of VH (BGP) gene, right figure: PCR product of VL (BGP) gene. (C) Lane 1: vector after NcoI treatment, lane 2: vector after HindIII and NcoI treatment. Construction of pRsetSA-VL (BGP)-(G4S) 3-GUSm (A), electrophoresis photograph of pRsetSA plasmid treated with restriction enzyme (B), and electrophoresis photograph of PCR product (C). (B) lane 1: before restriction enzyme treatment, lane 2: treatment with BamHI, lane 3: treatment with BamHI and HindIII. (C) lane 1: PCR product, lane 2: PCR product after restriction enzyme treatment. Expression and purification of V _H (BGP)-(G ₄ S) ₃ -GUSm and V _L (BGP)-(G ₄ S) ₃ -GUSm. lane M: protein molecular weight marker, lane 1: total intracellular protein before induction of expression, lane 2: total intracellular protein after expression induction, lane 3: insoluble fraction, lane 4: soluble fraction, lane 5: flow Through, lane 6: eluted protein. The figure which shows the time-dependent change of GUS activity (absorbance) with and without an antigen. The figure which shows an antigen concentration and GUS activity response. Left: fluorescence intensity, right: absorbance. The figure which shows the kinetic curve of the fusion protein containing a GUS variant. The figure which shows the kinetic curve of the fusion protein containing wild type GUS.

Hereinafter, the present invention will be described in detail.

(1) Fusion protein and nucleic acid The fusion protein of the present invention comprises a mutant of an enzyme activated by formation of a multimer, a linker peptide that binds to the mutant of the enzyme, and a _VH region of an antibody that binds to the linker peptide. Alternatively, it is a fusion protein containing a _VL region, wherein the mutant of the enzyme is a mutant into which a mutation that reduces the binding affinity between monomers is introduced.

“An enzyme activated by the formation of a multimer” means an enzyme that shows an activity or an improvement in activity only when several monomers are bonded. The number of monomers constituting the multimer is not particularly limited, and may be an enzyme activated by any of dimer, trimer, tetramer, pentamer, hexamer, and the like. The enzyme activated by the formation of multimers is preferably an enzyme whose activity can be easily detected or measured. For example, an enzyme that can detect or measure a product or substrate by absorbance, fluorescence intensity, or luminescence intensity. Specific examples of enzymes activated by the formation of multimers include those generally used as reporter enzymes, such as β-glucuronidase (activated by tetramer), β-galactosidase (activated by tetramer). Alkaline phosphatase (activated by dimer), malate dehydrogenase (activated by dimer), and the like.

Examples of the mutation that reduces the binding affinity between monomers include a mutation introduced at a binding site between monomers. Many of the enzymes that are activated by the formation of multimers have been clarified in their amino acid sequences and binding sites between monomers. I understand how affinity can be reduced. The degree of the affinity decrease may be such that it is difficult to form a multimer due to a decrease in the binding affinity between the monomers, thereby recognizing the difference in activity from the wild-type enzyme. Mutants that have introduced mutations that reduce the binding affinity between monomers include those that have reduced the affinity of all bonds between monomers, as well as the affinity of some bonds between monomers. Also included are those that only declined. Therefore, a mutant that forms a dimer but does not easily form a tetramer, such as a mutant of β-glucuronidase described later, is introduced with a mutation that reduces the binding affinity between the monomers. Included in the mutant.

Specific examples of mutations that reduce the binding affinity between monomers include mutants in which the 516th methionine and the 517th tyrosine in the amino acid sequence of Escherichia coli β-glucuronidase are substituted with other amino acids. The other amino acid substituted with the 516th methionine includes lysine, and the other amino acid substituted with the 517th tyrosine includes glutamic acid. In addition, since the 516th methionine and the 517th tyrosine indicate the position in the amino acid sequence of β-glucuronidase derived from Escherichia coli, the methionine and tyrosine are not present in the aforementioned positions in the amino acid sequence of β-glucuronidase derived from other organisms. There is also. In such a case, the amino acid sequence is aligned with the amino acid sequence of β-glucuronidase derived from E. coli, and methionine and tyrosine corresponding to 516th methionine and 517th tyrosine are substituted with other amino acids. Like that. The amino acid sequence of wild-type β-glucuronidase derived from E. coli is shown in SEQ ID NO: 1, and the amino acid sequence of β-glucuronidase into which the above mutation has been introduced is shown in SEQ ID NO: 2.

Any linker may be used as long as the enzyme, V _H region, and _VL region can function normally. If the distance between the enzyme and the V _H region or _VL region is not sufficient, the enzyme may not exhibit activity, so the linker needs to have a certain length. The length of the linker varies depending on the type of enzyme and antibody used, but is usually 10 to 60 mm, preferably 30 to 40 mm. The number of amino acids in the linker is not limited as long as it is the above-mentioned length, but is usually 5 to 50, preferably 15 to 20. The amino acid sequence of the linker may be the same as the amino acid sequence of a general linker used in the production of a fusion protein. Specifically, Gly-Gly-Gly-Gly-Ser (G ₄ S) repetitive sequence (repetition number is usually 2-5), Glu-Ala-Ala-Ala-Lys (EAAAK) repetitive sequence, Asp- Asp-Ala-Lys-Lys (DDAKK) repetitive sequence and the like can be mentioned.

The antibody V _H region or V _L region can be selected from any antibody V _H region or V _L region according to the antigen to be detected, and is limited to a specific antibody V _H region or V _L region. Not. Specifically, the V _H region or _VL region of an antibody that specifically binds to the antigen to be detected, which will be described later, can be used.

The fusion protein of the present invention may consist of only three of the above-mentioned enzyme mutant activated by the formation of a multimer, linker peptide, _VH region or _VL region, but other peptides and proteins, etc. May be included. Examples of such peptides include tag sequences for purification such as His-Tag, tag sequences that solubilize expressed proteins such as thioredoxin and amyloid precursor protein-derived solubilized tag sequences, and the like.

In the fusion protein of the present invention, an enzyme mutant activated by multimer formation, a linker peptide, a V _H region or a _VL region are arranged in this order. The V _H region or _VL region may be the C terminus at the N terminus, and conversely, the mutant of the enzyme activated by formation of a multimer may be the C terminus and the V _H region or _VL region may be the N terminus.

One mutant of the enzyme that is activated by the formation of a multimer may be included in the fusion protein, but may be included in two or more. When two variants of the enzyme are included, they are arranged adjacent to each other via a linker. The linker used at this time may be the same as the linker disposed between the mutant of the enzyme and the _VH region or _VL region.

The present invention includes not only the fusion protein described above but also a nucleic acid encoding the fusion protein. Here, “nucleic acid” mainly refers to deoxyribonucleic acid, but also includes ribonucleic acid and modifications of these nucleic acids.

(2) Antigen detection method The antigen detection method of the present invention is a method for detecting an antigen in a sample, the sample comprising the fusion protein of the present invention containing the antibody V _H region and the antibody V _L region. Including the step of contacting with the fusion protein of the present invention, and the step of detecting the formation of multimers.

This method is based on the principle of the open sandwich method developed by the present inventors (H. Ueda et al., Nat. Biotechnol. 14 (12), 1714-1718 (1996)), that is, V _H region and V _L It takes advantage of the principle that domain interactions are enhanced by the presence of antigen. In the conventional antigen detection method using the open sandwich method (for example, Non-Patent Documents 1 and 2), an N-terminal deletion mutant and a C-terminal deletion mutant are used. In the present invention, multimer formation is performed. Antigen detection is performed using a mutant of the enzyme activated by. The detection principle in the case where the above-described mutant of β-glucuronidase is used as a mutant of an enzyme activated by the formation of a multimer is described below. β-glucuronidase becomes active by forming a tetramer. The above-mentioned mutant of β-glucuronidase forms a dimer because a mutation that reduces the binding affinity between some monomers is introduced, but does not form a tetramer under normal conditions. . If the sample does not exist antigen, since the interaction of V _H and V _L domains is a weak remains almost tetramer dimers in intact variants of β-glucuronidase which connects the V _H region and the V _L region It will not be. On the other hand, when the antigen is present in the sample, the interaction between the V _H region and the _VL region is enhanced, and this interaction also causes a β-glucuronidase mutant dimer linked to the V _H region and the _VL region. It binds to form a tetramer and becomes active. Therefore, by measuring β-glucuronidase activity, it can be determined whether or not the antigen is present in the sample.

Any antigen can be detected, but the method of the present invention is suitable for detecting low molecular weight compounds (for example, compounds having a molecular weight of 1000 or less). Is preferred. Further, since the method of the present invention can be used for disease diagnosis, food toxicity test, environmental analysis, and the like, it is preferable to use substances related to these as detection targets. Specifically, neonicotinoid pesticides such as imidacloprid, environmental pollutants such as polychlorinated biphenyls and bisphenol A, toxic substances such as mycotoxins, osteocalcin (effective for diagnosis of osteoporosis), corticoids, estradiol, aldosterone, lysozyme (chicken) And biological substances such as egg white lysozyme) and drugs such as digoxin.

The sample may be any sample that may contain the antigen to be detected. For example, human samples (blood, saliva, urine, etc.), contaminated water, Examples include soil, food, and food ingredients.

Although the method for contacting the sample and the fusion protein is not particularly limited, it is usually performed by allowing the sample and the fusion protein to coexist in the solution. Moreover, you may coexist with the cell which expresses not a fusion protein itself but a fusion protein. Conditions such as temperature, time, pH of the solution, and the amount of the fusion protein to be used in this contact step may be those generally used for the enzyme contained in the fusion protein. For example, when the fusion protein contains a variant of β-glucuronidase, the temperature in this contact step is preferably about 20 to 37 ° C., the contact time is preferably about 10 to 60 minutes, and the pH of the solution is 6.8 to About 7.5 is preferable, and the concentration of the fusion protein in the solution is preferably about 10 to 100 nM.

The formation of multimers can be detected by a change (increase or expression) in the activity of the enzyme mutant contained in the fusion protein. The activity of the enzyme variant contained in the fusion protein can be measured by an activity measurement method generally used for the enzyme. For example, when the enzyme mutant contained in the fusion protein is a mutant of β-glucuronidase, the activity can be measured by adding a chromogenic substrate or a fluorescent substrate and quantifying a substance produced from the substrate. Examples of chromogenic substrates for β-glucuronidase include X-Gluc, 4-nitrophenyl α-glucopyranoside, 4-nitrophenyl β-D-glucuronide, and fluorescent substrates include 4-methylumbelliferyl-β-D- Examples include glucuronide. Quantification of substances produced from these substrates can be performed by measuring absorbance, fluorescence intensity, etc. at a specific wavelength. For example, if the substrate is 4-nitrophenyl β-D-glucuronide, the product can be quantified by measuring the absorbance near 405 nm, and if the substrate is 4-methylumbellylphenyl-β-D-glucuronide, 340 The product can be quantified by exciting with nm fluorescence and measuring the fluorescence intensity around 480 nm.

(3) Antigen detection kit The antigen detection kit of the present invention comprises the fusion protein of the present invention containing the V _H region of an antibody and the fusion protein of the present invention containing the _VL region of an antibody. This kit can detect an antigen in a sample according to the principle of antigen detection described above.

This kit may contain other than the fusion protein of the present invention containing the V _H region of the antibody and the fusion protein of the present invention containing the _VL region of the antibody. For example, since a substrate is required for measuring enzyme activity, it may be included. Further, it may contain a reagent or equipment for quantifying a substance produced from the substrate, or a fusion protein or a substance for stabilizing the substrate.

Hereinafter, the present invention will be described in more detail with reference to examples.

[Example 1] Construction of GUS mutant expression vector Mutations were introduced into the GUS gene by the method shown in Fig. 1A. The primer sequence (5′-3 ′) used is shown in FIG. 1B. Specifically, first, the 5 ′ side of the GUS gene was amplified by polymerase chain reaction (PCR) using primer a (SEQ ID NO: 3) and primer d (SEQ ID NO: 6). Further, the 3 ′ side of the GUS gene was amplified by PCR using primer c (SEQ ID NO: 5) and primer b (SEQ ID NO: 4). Primers c and d contain the codon sequence of the amino acid mutation to be introduced. Thereafter, Splice Overlap Extension PCR was performed using both PCR products as a template and primers a and b to construct a GUS mutant gene (hereinafter, this GUS mutant may be referred to as “GUSm”). . In PCR, KOD-Plus-Neo polymerase was used to first denature the DNA by incubating at 94 ° C for 2 minutes, and then 30 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 68 ° C for 1 minute were performed. Gel electrophoresis was performed to confirm the amplified DNA sample. The result is shown in FIG. Lane 1 is amplified using primers a and b using the GUS gene before mutagenesis as a template, Lane 2 is the 5 'fragment of the mutagenized GUS gene, Lane 3 is the 3' fragment of the mutagenized GUS gene, and Lane 4 is a mutant GUS gene amplification product. For insertion into an expression vector, restriction sites NotI and XhoI were cleaved at the 5 ′ and 3 ′ sides of the GUS gene.

[Example 2] Construction of GUS mutant expression vector The GUS gene and mutant gene prepared in Example 1 were cloned into an expression vector. First, the amplified GUS gene DNA was digested with restriction enzymes NotI and XhoI. On the other hand, E. coli expression vectors pET-VH (NP) -Rluc and pET-VL (NP)-(G3S) 3 encoding high affinity anti-NP (4-hydroxy-3-nitrophenyl acetic acid: molecular weight 198) antibody variable region -EYFP (Non-patent Document 1) was treated with NotI and XhoI, respectively, and confirmed by agarose gel electrophoresis. The results are shown in FIG. Lane M is a molecular weight marker, and lanes 1 and 2 show GUS mutant genes after and before restriction enzyme treatment. Lanes 3 and 4 are pET-VH (NP) -Rluc vectors after and before restriction enzyme treatment, and lanes 5 and 6 are pET32-VL (NP)-(G4S) 3-EYFP vectors after and before restriction enzyme treatment. . After agarose gel electrophoresis, the separated DNA fragment was excised, purified, and the restriction enzyme-treated gene and plasmid were ligated using T4 ligase, and the expression vectors pET32-VH (NP) -GUSm and pET32-VL ( NP)-(G4S) 3-GUSm was prepared.

[Example 3] Insertion of (G ₄ S) ₃ linker between VH (NP) gene and mutant GUS gene (G ₄ S) ₃ linker sequence in pET32-VH (NP) -GUSm constructed in Example 2 Was inserted (FIG. 4A), the following genetic manipulation was performed. Specifically, first, pET-VL (NP)-(G3S) 3-EYFP was templated using primer e (TCC AAGCTT GCGGCCGGTGGATCCGGT) (SEQ ID NO: 7) and primer f (CGTAACATA GCGGCCGC GCTACCGCCACCGCCGG) (SEQ ID NO: 8). The GS linker gene was amplified. For PCR, KOD-Plus-Neo polymerase was used to incubate at 94 ° C. for 2 minutes to denature the DNA. Thereafter, a reaction of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 1 minute was performed for 30 cycles, and agarose gel electrophoresis was performed to confirm the amplified DNA sample. The result is shown in FIG. 4B. The amplified GS linker gene fragment was treated with HindIII / NotI and ligated with a vector (FIG. 4C) treated with the same enzyme set with T4 ligase to construct pET32-VH (NP)-(G4S) 3-GUSm. .

Example 4 Expression of V _H (NP)-(G ₄ S) ₃ -GUSm and V _L (NP)-(G ₄ S) ₃ -GUSm pET32-VH (NP)-(G4S) 3-GUSm and pET32-VL (NP)-(G4S) 3-GUSm was used to transform E. coli SHuffle T7 lysY. After culturing the plasmid-carrying E. coli in 500 ml of LBA medium (10 g / L tryptone, 5 g / L yeast, 5 g / L NaCl, 100 μg / mL ampicillin) at 30 ° C. until OD ₆₀₀ is 0.6 Then, 0.5 mM IPTG was added, and the cells were further cultured at 16 ° C. for 16 hours. After collecting the cells by centrifugation, Escherichia coli was crushed by an ultrasonic crusher to prepare a cell lysate. E. coli before and after expression induction was sampled and analyzed by SDS-PAGE. The results are shown in FIG. Lane M is a protein molecular weight marker, lane 1 is the total intracellular protein before induction of expression, and lane 2 is the total intracellular protein after induction of expression. The arrow indicates the target protein.

Example 5 Purification of V _H (NP)-(G ₄ S) ₃ -GUSm and V _L (NP)-(G ₄ S) ₃ -GUSm 40 ml Extraction buffer (50 mM sodium phosphate, 300 mM Escherichia coli suspended in sodium chloride, pH 7.0) was crushed with a sonicator, centrifuged at 1000 g for 20 minutes, and the supernatant was collected and purified by immobilized metal affinity chromatography. Specifically, an appropriate amount of TALON (Takara Bio, Clontech) agarose gel was added to the supernatant and stirred for 2 hours. Thereafter, the column was transferred to a column and washed three times with 10 ml of extraction buffer, and the protein bound to the gel was eluted using 2.5 ml of extraction buffer containing 150 mM imidazole. Samples were taken at each stage of the purification process and analyzed by SDS-PAGE. The result is shown in FIG. 6A. Lane M shows molecular weight markers, and lanes 1-5 show V _H (NP)-(G ₄ S) ₃ -GUS mutants. Lane 1 is the insoluble fraction, lane 2 is the soluble fraction, lane 3 is the flow-through, and lanes 4 and 5 are the eluted protein. Lanes 6-10 show V _L (NP)-(G ₄ S) ₃ -GUS mutants. Lane 6 shows the insoluble fraction, lane 7 is the soluble fraction, lane 8 is the flow-through, and lanes 9 and 10 are the eluted protein. Thus, V _H (NP)-(G ₄ S) ₃ -GUSm gave a band of almost single molecular weight (~ 100 kd), but V _L (NP)-(G ₄ S) ₃ -GUSm In the case of Nd, a band of contaminants that seemed to contain N-terminal thioredoxin tag (14 kd), V _L (12 kd) and GUS degradation products was observed in the vicinity of 35 kd. For this reason, V _L (NP)-(G ₄ S) ₃ -GUSm was subsequently purified using an anion exchange resin, and finally a protein consisting of a single band was obtained. The result is shown in FIG. 6B. Lane 11 is a concentrated TALON purified elution fraction, and lanes 12-15 show a fraction eluted with NaCl.

[Example 6] Change in GUS activity (absorbance) with and without antigen over time V _H (NP)-(G ₄ S) ₃ -GUSm and V _L (NP)-(G ₄ S) ₃ -GUSm were added to 137 mM NaCl. The buffer was replaced with 10 mM phosphate buffer (pH 7.4) containing 2.7 mM KCl to prepare a final concentration of 100 nM. Antigen NP was added to a final concentration of 5 μM and incubated at 25 ° C. for 10 minutes. Then, the substrate 4-nitrophenyl β-D-glucuronide (4-NPG, Tokyo Chemical Industry Co., Ltd.) was added, and 2 Absorbance at 405 nm was measured at minute intervals (n = 3). At this time, the absorbance at 655 nm in each well was reduced as a background. The result is shown in FIG. When NP is added to a sample containing only V _H (NP)-(G ₄ S) ₃ -GUSm, or V _L (NP)-(G ₄ S) ₃ -GUSm, and each 50 nM V _H ( When NP)-(G ₄ S) ₃ -GUSm and _VL (NP)-(G ₄ S) ₃ -GUSm were added but no NP was added, the absorbance increased slightly after 28 minutes. When NP is added to 50 nM each of V _H (NP)-(G ₄ S) ₃ -GUSm and V _L (NP)-(G ₄ S) ₃ -GUSm, the absorbance or GUS activity is greatly increased did.

[Example 7] Antigen concentration and GUS activity (fluorescence) response A solution containing 50 nM each of V _H (NP)-(G ₄ S) ₃ -GUSm and V _L (NP)-(G ₄ S) ₃ -GUSm To the final concentration of 5, 10, 50, 100, 500, 1000, 5000, 10000 nM and add NP or NIP (5-iodo-NP, which binds to the antibody about 10 times stronger than NP), and 25 Incubated for 10 minutes at ° C. After that, the fluorescent substrate 4-methylumbellylphenyl-β-D-glucuronide (MUG, Wako Pure Chemical Industries, Ltd.) was added and incubated in a black half-well microplate at 25 ° C for 15 minutes, then excited at 340 nm and excited at 480 nm. The fluorescence intensity of was measured. Then, NP and NIP calibration curves based on the fluorescence intensity at each concentration were prepared. As shown in FIG. 8, as the NP and NIP concentrations increased, the fluorescence intensity gradually increased. When 10 μM NIP was added, the fluorescence intensity increased 5 times or more compared to when no antigen was added. In contrast, the responsiveness to NP was low, and the fluorescence was increased only 2.5-fold with 10 μM NP. This seems to reflect the difference in affinity of both antigens for this antibody (NP-5x10 ⁵ / M, NIP-5x10 ⁶ / M).

[Example 8] Construction of GUS mutant expression vector pET32-VH (NP)-(G4S) 3-GUSm and pET32-VL (NP)-(G4S) 3-GUSm constructed in Examples 2 and 3 (FIG. 9A) In order to insert the VH (BGP) and VL (BGP) genes into), the following operation was performed. First, primers a and b (VH (KTM) NcoBack: 5'-ATATGCCATGGATCAAGTAAAGCTGCAGCAGTC-3 '(SEQ ID NO: 9), VH_HindFor: 5'-CCCAAGCTTGCTCGAGAGACGGTGACCGT-3' (SEQ ID NO: 10)) and primers c, d (Vk (KTM) NcoSalBack: 5'-CATGCCATGGGGTCGACGGACATTGAGCTCACCCAG-3 '(SEQ ID NO: 11), Vk_HindFor: 5'-CCCAAGCTTCCGTTTTATTTCCAGCTT-3' (SEQ ID NO: 12)) (BGP) and VL (BGP) genes were amplified. For PCR, KOD-Plus-Neo polymerase was used to incubate at 94 ° C. for 2 minutes to denature the DNA. Thereafter, a reaction of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 1 minute was performed for 30 cycles, and agarose gel electrophoresis was performed to confirm the amplified DNA sample. The result is shown in FIG. 9B. The amplified VH (BGP) and VL (BGP) gene fragments were treated with NcoI / HindIII, ligated with the vector treated with the same enzyme set (FIG. 9C) with T4 ligase, and pET32-VH (BGP)-(G4S) 3-GUSm and pET32-VL (BGP)-(G4S) 3-GUSm were constructed.

[Example 9] Insertion of VL (BGP)-(G4S) 3-GUSm gene into pRsetSA plasmid amyloid precursor protein-derived solubilized tag sequence APP hyper at the N-terminal of VL (BGP)-(G4S) 3-GUSm In order to add an acidic region, the following operation was performed (FIG. 10A). Primer e (VLBGPback_to_pRSETSA (Bam): 5'- GGAGGAGGTGGGATCCATGGGGTCGACGGACATTG-3 '(SEQ ID NO: 13)) and primer f (GUSmutXhoFor_to_pRSETSA (Hd): 5'- CAGCCGGATCAAGCTCTCGAGTAGTCATTGTTTGC-3' (Example 14) VL (BGP)-(G4S) 3-GUSm was amplified using pET32-VL (BGP)-(G4S) 3-GUSm constructed in step 1 as a template. For PCR, KOD-Plus-Neo polymerase was used to incubate at 94 ° C. for 2 minutes to denature the DNA. Thereafter, a reaction of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 2 minutes was performed for 30 cycles, and agarose gel electrophoresis was performed to confirm the amplified DNA sample. The result is shown in FIG. 10C. Amplified VL (BGP)-(G4S) 3-GUSm was treated with BamHI and HindIII, ligated with pRsetSA plasmid (Fig. 10B, Dr. Tetsuya Kitaguchi, Waseda University) treated with the same enzyme set, and pRsetSA -VL (BGP)-(G4S) 3-GUSm was constructed.

[Example 10] Expression and purification of V _H (BGP)-(G ₄ S) ₃ -GUSm and V _L (BGP)-(G ₄ S) ₃ -GUSm pET32-VH (BGP)-(G4S) 3- E. coli SHuffle T7 lysY was transformed with GUSm and pRsetSA-VL (BGP)-(G4S) 3-GUSm. After culturing the plasmid-carrying E. coli in 500 ml of LBA medium (10 g / L tryptone, 5 g / L yeast, 5 g / L NaCl, 100 μg / mL ampicillin) at 30 ° C. until OD ₆₀₀ is 0.6 Then, 0.5 mM IPTG was added, and the cells were further cultured at 16 ° C. for 16 hours. After collecting the cells by centrifugation, Escherichia coli was crushed by an ultrasonic crusher to prepare a cell lysate. E. coli before and after expression induction was sampled and analyzed by SDS-PAGE. The results are shown in FIG. Lane M is a protein molecular weight marker, lane 1 is the total intracellular protein before induction of expression, and lane 2 is the total intracellular protein after induction of expression. Escherichia coli suspended in 40 ml of Extraction buffer (50 mM sodium phosphate, 300 mM sodium chloride, pH 7.0) was disrupted with a sonicator, then centrifuged at 1000 g for 20 minutes, the supernatant was collected, and the immobilized metal affinity was collected. Purification was performed by chromatography. Specifically, an appropriate amount of TALON (Takara Bio, Clontech) agarose gel was added to the supernatant and stirred for 2 hours. Thereafter, the column was transferred to a column and washed three times with 10 ml of extraction buffer, and the protein bound to the gel was eluted using 2.5 ml of extraction buffer containing 150 mM imidazole. Samples were taken at each stage of the purification process and analyzed by SDS-PAGE. The result is shown in FIG. Lane 3 is the insoluble fraction, lane 4 is the soluble fraction, lane 5 is the flow-through, and lane 6 is the eluted protein. Thus, V _H (BGP)-(G ₄ S) ₃ -GUSm and V _L (BGP)-(G ₄ S) ₃ -GUSm gave bands of almost single molecular weight (˜100 kd). The arrow indicates the target protein.

[Example 11] Change in GUS activity (absorbance) with and without antigen over time V _H (BGP)-(G ₄ S) ₃ -GUSm and V _L (BGP)-(G ₄ S) ₃ -GUSm The buffer was exchanged with an acid buffer (pH 7.4) to prepare a final concentration of 0.1 μM. Antigen BGP-C7 (7-residue peptide at the C-terminal of osteocalcin) was added to a final concentration of 10 μM and incubated at 25 ° C. for 10 minutes, and then the substrate 4-nitrophenyl β-D-glucuronide (4-NPG, Tokyo Chemical Industry Co., Ltd.) was added and the absorbance at 405 nm was measured at intervals of 2 minutes (n = 3). At this time, the absorbance at 655 nm in each well was reduced as a background. The result is shown in FIG. When V _H (BGP)-(G ₄ S) ₃ -GUSm and V _L (BGP)-(G ₄ S) ₃ -GUSm are added but BGP-C7 is not added, the absorbance after 28 minutes increases slightly. In contrast, when BGP-C7 was added to V _H (BGP)-(G ₄ S) ₃ -GUSm and V _L (BGP)-(G ₄ S) ₃ -GUSm, the absorbance or GUS activity was Increased significantly.

Example 12 Antigen Concentration and GUS Activity Response In a solution containing V _H (BGP)-(G ₄ S) ₃ -GUSm and V _L (BGP)-(G ₄ S) ₃ -GUSm, the final concentration is 0, BGP-C7 was added to 1, 10, 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ nM and incubated at 25 ° C. for 10 minutes. Subsequently, the fluorescent substrate 4-methylumbellylphenyl-β-D-glucuronide (MUG, Wako Pure Chemical Industries) was added, incubated in a black half-well microplate for 15 minutes at 25 ° C., then excited at 340 nm and 480 nm The fluorescence intensity at was measured. A calibration curve for BGP-C7 based on the fluorescence intensity at each concentration was prepared. In addition, the substrate 4-NPG was added, and the absorbance at 405 nm was measured (n = 3). At this time, the absorbance at 655 nm in each well was reduced as a background, and a calibration curve for BGP-C7 based on the absorbance at each concentration was prepared. As shown in FIG. 13, the fluorescence intensity and absorbance gradually increased as the BGP-C7 concentration increased.

[Example 13] Comparison of activity between GUS mutant and wild-type GUS Fusion protein (V _H (NP)-(G ₄ S) ₃ -GUSm and V _L (NP) containing the GUS mutant prepared in Example 5 -(G ₄ S) ₃ -GUSm) and the enzyme activity of a fusion protein containing wild-type GUS (a fusion protein in which the GUS mutant in the fusion protein containing the GUS mutant prepared in Example 5 is replaced with wild-type GUS) It measured by the following method.

Both fusion proteins were mixed with 1 μM NP in PBST buffer, and 1 mg / mL enzyme substrate (4-nitrophenyl β-D-glucuronide) was added thereto. After incubating for 10 minutes until the reaction rate became stable, the absorbance at 405 nm was measured every 15 seconds for 15 minutes using a spectrophotometer (Beckmann DU530). FIG. 14 shows the time course of absorbance when a fusion protein containing a GUS mutant is used, and FIG. 15 shows the time course of absorbance when a fusion protein containing wild-type GUS is used. As shown in the figure, the absorbance change when using a fusion protein containing a GUS mutant is 0.0096 (min ^-1 ), and the absorbance change when using a fusion protein containing wild-type GUS is 0.0188 (min ^-1 )Met. From these values, the molar extinction coefficient of the reaction product p-nitrophenol (18.3 mM ⁻¹ cm ⁻¹ ), and the protein weight, the specific activities of both fusion proteins were calculated according to the Lambert Beer method. The specific activity of the fusion protein containing wild-type GUS was 0.55 μmolmin ^-1 mg ^-1 , whereas the specific activity of the fusion protein containing the GUS mutant was 0.043 μmolmin ^-1 mg ^-1 The activity of about 1/13 of the fusion protein was maintained.

In the method using a defective mutant such as the OS-ECIA method described above, a problem has been pointed out that the enzyme activity of the mutant is significantly lower than that of the wild type. For example, in Non-Patent Document 1 (T. Yokozeki, H. Ueda, R. Arai, W. Mahoney and T. Nagamune. Anal. Chem. 74, 2500-2504 (2002)), the activity of the mutant is wild type. It is described that the activity is reduced to 6.0 × 10 ⁻³ times (0.6%) at the maximum, and Mohler WA, Blau HM (1996) Proc. Natl. Acad. Sci. USA 93: 12423- 12427 describes that the mutant is 25-200 times weaker than the wild type when compared to intracellular activity.

Considering the low activity of the mutant in the conventional method, the above-mentioned activity exhibited by the fusion protein containing the GUS mutant is very high.

All publications, patents and patent applications cited in this specification shall be incorporated into the present specification as they are.

Since the present invention is useful for disease diagnosis, food inspection, environmental analysis, etc., it can be used in industrial fields related to these.

Claims (8)

A fusion protein comprising a mutant of an enzyme that is activated by formation of a multimer, a linker peptide that binds to the mutant of the enzyme, and a V _H region or a V _L region of an antibody that binds to the linker peptide, A fusion protein characterized in that the mutant is a mutant into which a mutation that reduces the binding affinity between monomers is introduced. The fusion protein according to claim 1, wherein the mutant of the enzyme activated by formation of a multimer is a mutant of β-glucuronidase. The fusion protein according to claim 2, wherein the mutant of β-glucuronidase is a mutant in which the 516th methionine and the 517th tyrosine in the amino acid sequence of Escherichia coli β-glucuronidase are substituted with other amino acids. The fusion according to claim 2, wherein the mutant of β-glucuronidase is a mutant in which the 516th methionine in the amino acid sequence of Escherichia coli β-glucuronidase is replaced with lysine and the 517th tyrosine is replaced with glutamic acid. protein. The fusion protein according to any one of claims 2 to 4, wherein the fusion protein contains one or two β-glucuronidase mutants. A nucleic acid encoding the fusion protein according to any one of claims 1 to 5. A method for detecting an antigen in a sample, wherein the sample comprises a V _H region of an antibody, and the fusion protein according to any one of claims 1 to 5 and the _VL region of an antibody. A method for detecting an antigen, comprising a step of contacting with the fusion protein according to any one of the above, and a step of detecting formation of a multimer by a change in enzyme activity. The fusion protein according to any one of claims 1 to 5 comprising a _VH region of an antibody and the fusion protein according to any one of claims 1 to 5 comprising a _VL region of an antibody. An antigen detection kit.

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