KR20110051778A - Novel immobilizing fusion protein for effective and oriented immobilization of antibody on surfaces - Google Patents

Abstract

PURPOSE: A novel fusion protein for a novel antibody fixation is provided to effectively attach to a solid substrate and to detecting an antigen. CONSTITUTION: A fusion protein for antibody fixation contains a mussel adhesive protein having an amino acid sequence of sequence number 6 with Staphylococcus aureus-derived protein A or an amino acid of protein A fragment and sequence number 6. The protein A has an amino acid of sequence number 7. A bio-chip for immune response contains the fusion protein for antibody fixation. A method for detecting an antibody comprises: a step of preparing a biochip containing the fusion protein; a step of fixing the antibody to an antigen; a step of reacting a sample to the bio-chip; and a step of measuring antigen-antibody reaction.

Description

Novel immobilizing fusion protein for effective and oriented immobilization of antibody on surfaces

The present invention relates to a novel antibody immobilized fusion protein, comprising a protein A derived from Staphylococcus aureus or a fragment of the protein A and a fusion protein comprising an mussel adhesive protein. will be.

Antibody-based bioassay is one of the main techniques for analyzing the interaction between biomaterials due to the high specificity and affinity of the antibody for antigen. For this purpose, the antibody is immobilized on solid substrates such as gold, glass, and polymer based surfaces, and the interaction of the antigen with the antibody is measured. Therefore, it is very important to efficiently immobilize the antibody on the solid substrate.

Many techniques have been developed for immobilizing antibodies on solid substrates, of which the physical adsorption method was used first. However, this method has been pointed out that the attached antibody exhibits a random orientation and degenerates very badly (Ferretti S et al., Self-assembled monolayers: a versatile tool for the formulation of bio-surfaces). Trends Anal.Chem., 19; 530-540, 2000).

Covalent immobilization is an antibody immobilization technique for solving the above problems. However, although the direct covalent immobilization technique of the antibody has somewhat improved the problem of the physical adsorption method, it has been pointed out that the attached antibody still exhibits random orientation.

As described above, the antigen-binding ability of an antibody having any orientation is shown to be reduced by two to three times as compared to the antigen-binding ability of an antibody having a preferred orientation (Cho IH et. Al., Site-directed biotinylation of antibodies). for controlled immobilization on solid surfaces.Anal. Biochem. 365; 14-23, 2007: Kang JH et. al., Improving immunobinding using oriented immobilization of an oxidized antibody.J. Chromatogr. A 116; 9-14, 2007).

Improved shared immobilization methods have been developed to solve the above problem. For example, methods have been developed for immobilizing antibodies to site-specific using carbohydrates or disulfide bonds of antibodies (Neves-petersen MT et. Al., Photonic activation of disulfide bridges achieves oriented protein immobilization on biosensor surfaces.Protein Sci. 15: 343-351, 2006).

However, the above method maintains the desired orientation by immobilizing the antibody on the surface in a site-specific manner, but since the antibody has to undergo a pretreatment process such as carbohydrate oxidation process or breaking disulfide bonds before the antibody is immobilized on the surface, the antibody is modified. (Modification) there is a problem that the antigen binding ability of the antibody is reduced.

Therefore, there is an urgent need to develop a method for immobilizing an antibody on a solid substrate without modifying the antibody at all.

Accordingly, the present inventors have completed the present invention by discovering that a protein A derived from Staphylococcus aureus or a fragment of the protein A and a fusion protein including a mussel adhesive protein can effectively fix an antibody. .

Accordingly, the present invention provides a protein A or a fragment of the protein A derived from Staphylococcus aureus and a fusion protein for immobilizing an antibody comprising a mussel adhesive protein having an amino acid sequence of SEQ ID NO: 6 The purpose.

It is another object of the present invention to provide a biochip for an immune reaction containing the antibody immobilized fusion protein.

The present invention also provides a method for preparing a biochip, immobilizing an antibody against an antigen to be detected on the biochip, reacting a sample with a biochip including the immobilized antibody, and whether an antigen-antibody reaction occurs. An object of the present invention is to provide an antigen detection method comprising the step of measuring.

In order to solve the above problems, the present invention is a protein A or a fragment of the protein A derived from Staphylococcus aureus or an antibody comprising a mussel adhesive protein (mussel adhesive protein) having an amino acid sequence of SEQ ID NO: 6 Provided is a fusion protein for immobilization.

In another aspect, the present invention provides a biochip for immune reaction containing the antibody immobilized fusion protein.

The present invention also provides a method for preparing a biochip, immobilizing an antibody against an antigen to be detected on the biochip, reacting a sample with a biochip including the immobilized antibody, and whether an antigen-antibody reaction occurs. It provides an antigen detection method comprising the step of measuring.

Hereinafter, the present invention will be described in more detail.

The antibody immobilization fusion protein of the present invention includes a protein A derived from Staphylococcus aureus or a fragment of the protein A and a mussel adhesive protein (MAP) having an amino acid sequence of SEQ ID NO: 6.

Protein A derived from Staphylococcus aureus may include, but is not limited to, an amino acid sequence of SEQ ID NO: 7;

The fragment of protein A is not limited thereto, but may preferably have a sequence consisting of 50 to 500 amino acids contiguous among the amino acid sequences of SEQ ID NO. 7, more preferably 58 to 500 amino acids. have. If the fragment of protein A is less than 50 antibody immobilization efficiency is low, the fragment of protein A is preferably composed of a sequence consisting of 50 to 500 amino acids.

More preferably, the fragment of the protein A is an A domain having an amino acid sequence of SEQ ID NO: 8, a B domain having an amino acid sequence of SEQ ID NO: 9, a C domain having an amino acid sequence of SEQ ID NO: 10, and an amino acid of SEQ ID NO: 11 At least one selected from the group consisting of a D domain having a sequence and an E domain having an amino acid sequence of SEQ ID NO: 12, and more preferably, having a B domain having an amino acid sequence of SEQ ID NO: 9 and an amino acid sequence of SEQ ID NO: 10 It may be one or more selected from the group consisting of C domain, and most preferably may have an amino acid sequence of SEQ ID NO: 13. The amino acid sequence of SEQ ID NO: 13 is the amino acid sequence of BC domain to which B and C domains of Staphylococcus aureus-derived protein A are bound (Ulhen M. et. Al., Stapylococcal protein A consists of five IgG-binding domains.Eur J. Biochem. 156; 637-643, 1986).

The antibody immobilization fusion protein of the present invention may be a protein A or a fragment of protein A coupled to the N terminal or C terminal of the mussel adhesive protein (MAP) having the amino acid sequence of SEQ ID NO: 6. Preferably, the antibody immobilization fusion protein of the present invention may be prepared by fusing the BC domain of protein A having the amino acid sequence of SEQ ID NO: 13 to the N or C terminus of the mussel adhesive protein having the amino acid sequence of SEQ ID NO: 6, More preferably, the antibody immobilization fusion protein of the present invention may be prepared by fusing the BC domain of protein A having the amino acid sequence of SEQ ID NO: 13 to the N terminus of the mussel adhesive protein having the amino acid sequence of SEQ ID NO: 6, Most preferably, the antibody immobilization fusion protein of the present invention may have an amino acid sequence of SEQ ID NO.

In order to prepare the fusion protein for immobilizing the antibody of the present invention by binding the protein A or fragment of protein A to the mussel adhesive protein, a method for preparing a fusion protein used in the art may be used, and the present invention is not particularly limited. Do not. However, preferably, the gene of the protein A or fragment of protein A is inserted into the vector into which the gene of the mussel adhesive protein is inserted, injected into the transformant, and then induced to express the fusion protein of the present invention. It can manufacture.

In one embodiment of the present invention prepared primers SEQ ID NO: 3 and SEQ ID NO: 4 based on the sequence of the mussel adhesive protein of SEQ ID NO: 6 and amplified by PCR, inserting the amplified mussel adhesive protein gene into the vector pET- After preparing the MAP, primers of SEQ ID NO: 1 and SEQ ID NO: 2 were prepared based on the B domain and C domain sequences of protein A, and amplified by PCR, and the amplified BC domain gene was inserted into the pET-MAP vector to insert pET- Construct a BC-MAP vector. At this time, a histidine tag is fused to the C-terminus of the BC-MAP fusion protein to facilitate further purification (see <Example 1> to <Example 2>).

A vector prepared as described above may be inserted into a transformant, preferably Escherichia coli, and expressed, and a 23.5 kDa BC-MAP fusion protein may be prepared through SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). See Example 3).

The antibody-immobilized fusion protein of the present invention can immobilize the antibody in a preferred direction so that the antibody and antigen can bind well. Specifically, in the antibody immobilization fusion protein of the present invention, a protein A and a fragment of the protein A that can specifically bind to the FC region of the antibody and an mussel adhesive protein having an activity capable of adhering to various solid substrates are fused. The antibody can be immobilized in a direction capable of binding to the antigen.

In one experimental example of the present invention, the antibody immobilization activity of the antibody-immobilized fusion protein of the present invention was very excellent compared with the case of using only the mussel adhesive protein and the fragment of the protein A (BC domain). 0.9% when used, 4.4% when using only the fragment of Protein A, but when using the antibody-immobilized fusion protein of the present invention shows a breakthrough antibody immobilization efficiency of 20% (see <Experimental Example 1>).

Therefore, the antibody-immobilized fusion protein of the present invention can induce a mussel adhesive protein to be effectively attached to a solid substrate so that protein A or a fragment of protein A can be effectively bound to the antibody, and the antibody bound thereto is capable of binding to the antigen. Direction can be immobilized (see FIG. 1).

Meanwhile, the biochip for immune reaction of the present invention contains the antibody immobilized fusion protein of the present invention.

The biochip for immune reaction is not limited thereto, but preferably, the antibody-immobilized fusion protein of the present invention may be immobilized on a solid substrate.

The immobilized fusion protein of the present invention is immobilized on a solid substrate by the mussel adhesive protein included in the fusion protein, and the solid substrate may be used in a biochip because the mussel adhesive protein may be adhered to the solid substrate without limitation. If it is, it is not limited. However, preferably, commonly used polymers or gels such as gold, glass, modified silicon, tetrafluoroethylene, polystyrene, and polypropylene may be used. The surface of the substrate may also be surface treated with polymers, plastics, resins, carbohydrates, silicas, silica derivatives, carbons, metals, inorganic glasses and films. The substrate not only serves as a support but also provides a place for the binding reaction between the immobilized antibody and the antigen to occur. The size of the substrate and the position, size, and shape of the substrate to be fixed on the substrate may vary depending on the purpose of the analysis, a spotting machine, a scanner, and the like.

Meanwhile, the antigen detection method of the present invention comprises the steps of preparing the biochip of the present invention, immobilizing an antibody against the antigen of detection on the biochip, reacting a sample with a biochip containing the immobilized antibody. And measuring the antigen-antibody reaction.

In addition to using the biochip of the present invention, a method known in the art may be applied to the method for detecting an antigen in the present invention. Specifically, an immobilized antibody on the biochip of the present invention is collected from a sample, preferably a patient. Antigens can be detected by reacting with a blood or body fluid or the like to confirm antigen-antibody reaction. In order to confirm the antigen-antibody reaction, different chromophores may be used depending on the type of antibody, and at the same time, various types of antigens may be searched using fluorescence of various colors.

The antigen to be detected is not particularly limited as all bioactive materials detectable by immunoassay methods, for example, autoantibodies, ligands, natural extracts, peptides, proteins, metal ions, synthetic drugs , Natural drugs, metabolites, genomes, viruses and virus-producing materials, and bacteria and viruses-producing materials may be one or more selected from the group consisting of, but is not limited thereto. The target sample for measuring the concentration or the concentration of such an antigen may be used without any treatment or diluted with a suitable buffer solution. After incubation for a suitable time, the target material that has not yet been bound to the capture antibody and the other material in the sample that cannot be bound may be further washed and removed before performing the next step.

On the other hand, the antibody immobilization fusion protein expression vector of the present invention comprises a promoter and a polynucleotide encoding the fusion protein of the present invention operably linked thereto. The polynucleotide may be a protein A or a fragment of the protein A and a mussel adhesive protein having an amino acid sequence of SEQ ID NO: 6, but is not limited thereto. Preferably, the polynucleotide may have a nucleotide sequence of SEQ ID NO: 15. Always SEQ ID NO: 15 is the nucleotide sequence of the polynucleotide encoding the protein fused to the BC domain of the protein A and the mussel adhesive protein.

The antibody-immobilized fusion protein expression vector of the present invention may most preferably be a vector having a cleavage map described in 2a.

The 'promoter' refers to a DNA sequence that controls the expression of a nucleic acid sequence operably linked in a particular host cell, the promoter is not limited to this, if used for transformation, preferably can function in Escherichia coli A promoter can be used and more preferably a T7 promoter can be used.

Also, “operably linked” means that one nucleic acid fragment is combined with another nucleic acid fragment so that its function or expression is affected by another nucleic acid fragment.

In addition, the vector of the present invention may further include any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating termination of transcription and translation.

Meanwhile, the transformant of the present invention is transformed with the vector of the present invention, and the transformant is not limited thereto, but may preferably be Escherichia coli.

 The 'transformation' means that the DNA is introduced into the host so that the DNA can be replicated as an extrachromosomal factor or by chromosomal integration.

As the transformation method using the vector, a method known in the art may be used, and preferably, a thermal shock method may be used (Doskocil J et. Al., Increased transformation frequency in E. coli. Biochem Biophys Res Commun 82: 477 -483, 1978).

The antibody-immobilized fusion protein of the present invention may induce a mussel adhesive protein to be effectively attached to a solid substrate so that Protein A or a fragment of Protein A can be effectively bound to the antibody, and the antibody bound thereto is capable of binding to the antigen. Since it can be immobilized, it can be usefully used for biochips and antigen detection methods for immune reactions.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1>

BC  Protein and BC - MAP Fusion protein  Expression vector production

<1-1> BC  Construction of Protein Expression Vectors

In order to construct a vector for BC protein expression, PCR amplification using the BC-N primer of SEQ ID NO: 1 and the BC-C primer of SEQ ID NO: 5 using a genome obtained from Staphylococcus aureus (Accession Number: ATCC 6538) as a template It was. The amplified gene was inserted into the NdeI and XhoI restriction sites of the pET22b (+) (Novagen, USA) vector to prepare a pET-BC vector as described in FIG. 2B.

The BC protein was to include a histidine tag at the C-terminal to facilitate separation, purification and identification.

<1-2> BC - MAP  Construction of Protein Expression Vectors

In order to prepare a vector for expressing BC-MAP fusion protein, first, a pET-MAP vector was constructed. pMDG05 (D.S. Hwang et al., 2004, Applied and Environmental Microbiology, 70, 3352-3359) was amplified by using a MAP-N primer of SEQ ID NO: 3 and a MAP-C primer of SEQ ID NO: 4 as a template. The amplified gene was inserted into NcoI and XhoI restriction enzyme sites of pET22b (+) (Novagen, USA) to prepare pET-MAP vector.

In order to insert the DNA sequence encoding the BC protein into the prepared pET-MAP, the genome of Staphylococcus aureus (ATCC 6538) was used as a template, using the BC-N primer of SEQ ID NO: 1 and the BC-C primer of SEQ ID NO: 2 PCR amplification. The amplified gene was inserted into NdeI and NcoI restriction enzyme sites of pET-MAP to prepare pET-BC-MAP as described in FIG. 2A (see FIG. 2A).

The BC-MAP fusion protein was able to include a histidine tag in the C-terminal to facilitate separation, purification and identification.

< Example  2>

BC  Protein and BC - MAP Fusion proteins  Preparation of the transformant to express

To prepare transformants expressing BC protein and BC-MAP fusion protein, Escherichia coli TOP10 (Invitrogen), which is commonly used for conventional cloning, and Escherichia coli BL21 (DE3), which were used for protein expression, were prepared using CaCl ₂ buffer. Competent cells were made, and the plasmids were inserted into the host cells by a thermal shock method that was left at 42 ° C. for 2 minutes. Subsequently, ampicillin (Sigma) was used for the characteristics of the plasmid for selection of transformed colonies.

In addition, the vectors prepared in Example 1 were to include the T7 promoter, which is a widely used E. coli expression promoter, and induced expression using IPTG (isopropylthio-β-D-galactoside, Sigma, USA).

< Example  3>

SDS - PAGE Protein Expression Verification and Purification

LB medium to which ampicillin was added at a concentration of 50 g / ml was used as a medium for expressing the strains of <Example 2>. For protein expression, IPTG, an inducer, was added (1 mM) when the absorbance of the culture medium was about 0.8-0.9 at 600 nm.

The strain was recovered after incubating in a 37 ° C. incubator for 10 hours. The cultured cells were centrifuged at 4000 rpm for 10 minutes, the supernatant was removed, the cells were recovered, suspended in pH 8.0 lysis solution (lysis buffer, 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole), and then disrupted using an ultrasonic sonicator. It was. In order to confirm the expression of the BC protein and BC-MAP fusion protein, a portion of the crushed sample was divided into whole cell lysate, soluble fraction and insoluble fraction. The remaining whole cell lysate was centrifuged at 9000 rpm for 50 minutes to recover only the supernatant. The supernatant was purified by separating the BC protein and BC-MAP fusion protein expressed in a water-soluble aliquot using a his-tag column.

Each aliquot and purified sample were diluted in buffer for SDS-PAGE (0.5M Tris-HCl (pH 6.8), 10% glycerol, 5% SDS, 5% beta-mercaptoethanol, 0.25% bromophenol blue) After boiling for 5 minutes at 100 ℃ denatured. This sample was electrophoresed on a 15% SDS-poly acrylamide gel and the results are shown in FIGS. 4A and 4B.

As shown in FIG. 3a and FIG. 3b, it can be seen that the BC protein and BC-MAP fusion protein are mostly expressed in water-soluble fractions, and that the BC protein and BC-MAP fusion protein are purified to about 95% purity.

< Experimental Example  1>

BC - MAP Fusion protein  Antibody Immobilization Activity

In order to evaluate the antibody immobilization ability of the BC-MAP fusion protein, a crystal oscillator microbalance apparatus (model name: QCM200, manufactured by Stanford Research Systems) was used. The crystal oscillator microbalance is a device for measuring the mass of the material adsorbed per unit area by measuring the frequency change of the quartz crystal (quartz crystal). Therefore, the interactions occurring on the surface can be analyzed by the frequency change.

Prior to the experiment, the gold-coated crystal oscillator was exposed to ultraviolet light for 20 minutes to clean it, and a sample solution and a carrier solution (1X phosphophate buffered saline (PBS (pH7.4)) were removed using a 250ul injection loop. Injection at 10 ul / min.

Mussel adhesive protein (0.2mg / ml) was used as a control to determine whether BC-MAP fusion protein affects the binding of antibodies. The concentration is higher than BC protein and BC-MAP fusion protein. Concentration was used. BC protein (0.12 mg / ml) and BC-MAP fusion protein (0.2 mg / ml) used the same molar concentration (8.5 μM) .Anti-DsRed antibody used as an antibody was 0.13 μM (20 μg). / ml) was used.

The antibody binding efficiency was evaluated by comparing the number of molecules per unit area of the antibody bound to the gold surface coated with BC protein and BC-MAP fusion protein versus the number of molecules per unit area of the BC protein and BC-MAP fusion protein bound to the gold surface. The efficiency evaluated the well-oriented BC domain on bare gold, which of BC protein and BC-MAP fusion protein aligned to the surface. If the BC domains are well aligned to the surface, the probability of binding to the antibody increases, and the higher the probability, the better the interaction between the antibody and the antigen. Good interaction between the antibody and antigen means that the antibody is immobilized in the proper direction.

Specifically, a quartz coated crystal oscillator is mounted in a flow cell, and the crystal oscillator waits until the frequency of the crystal oscillator stabilizes. Then, when the frequency of the crystal oscillator stabilizes, the mussel adhesive protein, BC protein and BC-MAP fusion protein were put at 10 ul / min so that mussel adhesive protein, BC protein and BC-MAP fusion protein were bound to the surface thereof, and the experimental results are described in FIGS. 4A and 4C.

4A and 4C, the frequency of 70 Hz of mussel adhesive protein (MAP) was changed and the number of molecules bound to the gold surface per unit area was 5.13 * 10 ¹³ (molecule / cm ² ). The frequency of 23 Hz was varied and the number of molecules bound to the gold surface per unit area was 1.69 * 10 ¹³ (molecules / cm ² ). However, in the case of BC-MAP fusion protein, the frequency of 38 Hz was changed, and the number of molecules bound to the gold surface per unit area was 1.78 * 10 ¹³ (molecules / cm ² ).

In addition, 0.1% bovine serum albumin was fused with mussel adhesive protein, BC protein and BC-MAP fusion to prevent the adsorption of nonspecific antibodies to unreacted sites after treatment of mussel adhesive protein, BC protein and BC-MAP fusion protein. The protein was coated on the coated surface. After treatment with bovine serum albumin, 20 ug / ml of anti-DsRed antibody was reacted and the results are shown in FIGS. 4B and 4C.

4B and 4C, the mussel adhesive protein-coated surface was reacted with an anti-DsRed antibody, followed by a frequency change of 7 Hz, and the number of molecules per unit area of 4.96 * 10 ¹¹ (molecules / cm ² ) bound to BC protein. The frequency of 10.5Hz was changed to the surface coated with, and the number of molecules per unit area of 7.45 * 10 ¹¹ (molecules / cm ² ) was combined after the anti-DsRed antibody was reacted. A frequency of 48 Hz was changed on the surface coated with BC-MAP fusion protein, and the number of molecules per unit area of 3.40 * 10 ¹² (molecules / cm ² ) was combined after the anti-DsRed antibody was reacted.

Taken together, mussel adhesive protein, BC-MAP fusion protein and BC protein showed 0.9%, 20% and 4.4% antibody binding efficiency, respectively.

Thus, BC-MAP fusion protein, compared to mussel adhesive protein (MAP) or BC protein, allows the BC domain to be well aligned to the surface, thereby increasing the probability of binding to the antibody and improve the interaction between the antibody and the antigen. It can be seen.

1 is a schematic diagram comparing the difference of antibody binding ability on the surface treated with BC protein and BC-MAP fusion protein.

2A is a cleavage map of the pET-BC-MAP vector expressing BC-MAP fusion protein.

2B is a cleavage map of the pET-BC vector expressing BC protein.

3A is a SDS-PAGE gel photograph confirming the expression of a 23.5 kDa BC-MAP fusion protein (M: molecular weight labeling, 1: whole cell lysate, 2: water-soluble aliquots, 3: insoluble solution, and 4: purified sample) ).

Figure 3b is a SDS-PAGE gel picture confirming the expression of BC protein of 14.5kDa size (M: molecular weight labeling, 1: whole cell lysate, 2: water-soluble aliquot, 3: insoluble solution and 4: purified sample).

Figure 4a is a graph showing the frequency changes of mussel adhesive protein (MAP), BC-MAP fusion protein and BC protein bound to the gold surface.

Figure 4b is a graph showing the frequency change of the antibody bound to the mussel adhesive protein (MAP), BC-MAP fusion protein and BC protein coated on the gold surface

4C is a chart showing the surface density of mussel adhesive protein (MAP), BC-MAP fusion protein and BC protein bound to the gold surface and the surface density of antibodies bound to the protein coated surface.

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Novel immobilizing fusion protein for effective and oriented          immobilization of antibody on surfaces <130> DPP20095435 <160> 15 <170> KopatentIn 1.71 <210> 1 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> forward primer for BC protein <400> 1 gcgccatatg gcggataaca aattcaacaa agaacaac 38 <210> 2 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for BC protein <400> 2 gcgcccatgg ttttggtgct tgagcatcgt ttagc 35 <210> 3 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> forward primer for MAP protein <400> 3 gggcccatgg ggtggcggag ggagctctga agaatataaa g 41 <210> 4 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for MAP protein <400> 4 cgcgctcgag gctgctgccg ccataatatt ttttatag 38 <210> 5 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for BC protein <400> 5 gcgcctcgag ttttggtgct tgagcatcgt ttagc 35 <210> 6 <211> 75 <212> PRT <213> mussel adhesive protein <400> 6 Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His   1 5 10 15 Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr              20 25 30 Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys          35 40 45 Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg      50 55 60 Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser  65 70 75 <210> 7 <211> 500 <212> PRT <213> Staphylococcus aureus <400> 7 Met Lys Lys Lys Asn Ile Tyr Ser Ile Arg Lys Leu Gly Val Gly Ile   1 5 10 15 Ala Ser Val Thr Leu Gly Thr Leu Leu Ile Ser Gly Gly Val Thr Pro              20 25 30 Ala Ala Asn Ala Ala Gln His Asp Glu Ala Gln Gln Asn Ala Phe Tyr          35 40 45 Gln Val Leu Asn Met Pro Asn Leu Asn Ala Asp Gln Arg Asn Gly Phe      50 55 60 Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Gly  65 70 75 80 Glu Ala Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys Ala Asp Ala Gln                  85 90 95 Gln Asn Asn Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile Leu             100 105 110 Asn Met Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser         115 120 125 Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys     130 135 140 Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Asn Asn Phe Asn Lys 145 150 155 160 Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn                 165 170 175 Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser             180 185 190 Gln Ser Ala Asn Leu Leu Ser Glu Ala Lys Lys Leu Asn Glu Ser Gln         195 200 205 Ala Pro Lys Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe     210 215 220 Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly 225 230 235 240 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu                 245 250 255 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp Asn             260 265 270 Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu         275 280 285 Pro Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys     290 295 300 Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu 305 310 315 320 Asn Asp Ala Gln Ala Pro Lys Glu Glu Asp Asn Asn Lys Pro Gly Lys                 325 330 335 Glu Asp Asn Asn Lys Pro Gly Lys Glu Asp Asn Asn Lys Pro Gly Lys             340 345 350 Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys         355 360 365 Glu Asp Asn Lys Lys Pro Gly Lys Glu Asp Asn Lys Lys Pro Gly Lys     370 375 380 Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys 385 390 395 400 Glu Asp Gly Asn Gly Val His Val Val Lys Pro Gly Asp Thr Val Asn                 405 410 415 Asp Ile Ala Lys Ala Asn Gly Thr Thr Ala Asp Lys Ile Ala Ala Asp             420 425 430 Asn Lys Leu Ala Asp Lys Asn Met Ile Lys Pro Gly Gln Glu Leu Val         435 440 445 Val Asp Lys Lys Gln Pro Ala Asn His Ala Asp Ala Asn Lys Ala Gln     450 455 460 Ala Leu Pro Glu Thr Gly Glu Glu Asn Pro Phe Ile Gly Thr Thr Val 465 470 475 480 Phe Gly Gly Leu Ser Leu Ala Leu Gly Ala Ala Leu Leu Ala Gly Arg                 485 490 495 Arg Arg Glu Leu             500 <210> 8 <211> 58 <212> PRT <213> Staphylococcus aureus <400> 8 Ala Asp Asn Asn Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile   1 5 10 15 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln              20 25 30 Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ser Glu Ala          35 40 45 Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys      50 55 <210> 9 <211> 58 <212> PRT <213> Staphylococcus aureus <400> 9 Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile   1 5 10 15 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln              20 25 30 Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala          35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys      50 55 <210> 10 <211> 58 <212> PRT <213> Staphylococcus aureus <400> 10 Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile   1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln              20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala          35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys      50 55 <210> 11 <211> 59 <212> PRT <213> Staphylococcus aureus <400> 11 Ala Asp Asn Asn Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile   1 5 10 15 Ile Asn Met Pro Asn Leu Asn Glu Ala Gln Asn Arg Asn Gly Phe Ile              20 25 30 Gln Ser Leu Lys Ile Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu          35 40 45 Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys      50 55 <210> 12 <211> 59 <212> PRT <213> Staphylococcus aureus <400> 12 Ala Asp Ala Gln His Asp Glu Ala Gln Gln Asn Ala Phe Tyr Glu Ile   1 5 10 15 Ile Asn Met Pro Asn Leu Asn Glu Glu Gln Asn Arg Asn Gly Phe Ile              20 25 30 Gln Ser Leu Lys Ile Asp Pro Ser Gln Ser Ala Asn Leu Leu Ser Glu          35 40 45 Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys      50 55 <210> 13 <211> 116 <212> PRT <213> Staphylococcus aureus <400> 13 Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile   1 5 10 15 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln              20 25 30 Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala          35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn      50 55 60 Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu  65 70 75 80 Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro                  85 90 95 Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala             100 105 110 Gln Ala Pro Lys         115 <210> 14 <211> 206 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein <400> 14 Met Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu   1 5 10 15 Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile              20 25 30 Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu          35 40 45 Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp Asn Lys Phe      50 55 60 Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn  65 70 75 80 Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp                  85 90 95 Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp             100 105 110 Ala Gln Ala Pro Lys Pro Trp Gly Gly Gly Gly Ser Ser Glu Glu Tyr         115 120 125 Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser Gly Gly     130 135 140 Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys Tyr Tyr 145 150 155 160 Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly Lys Tyr                 165 170 175 Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys Lys             180 185 190 Tyr Tyr Gly Gly Ser Ser Leu Glu His His His His His His         195 200 205 <210> 15 <211> 618 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding fusion protein <400> 15 atggcggata acaaattcaa caaagaacaa caaaatgctt tctatgaaat cttacattta 60 cctaacttaa acgaagaaca acgcaatggt ttcatccaaa gcttaaaaga tgacccaagc 120 caaagcgcta accttttagc agaagctaaa aagctaaatg atgcacaagc accaaaagct 180 gacaacaaat tcaacaaaga acaacaaaat gctttctatg aaattttaca tttacctaac 240 ttaactgaag aacaacgtaa cggcttcatc caaagcctta aagacgatcc ttcagtgagc 300 aaagaaattt tagcagaagc taaaaagcta aacgatgctc aagcaccaaa accatggggt 360 ggcggaggga gctctgaaga atataaaggc ggctattatc cgggcaacac ctaccattat 420 cattctggcg gcagctatca tggctctggc tatcatggcg gctataaagg caaatattat 480 ggcaaagcga aaaaatatta ttataaatat aaaaacagcg gcaaatataa atatctgaaa 540 aaagcgagaa aatatcatag aaaaggctat aaaaaatatt atggcggcag cagcctcgag 600 caccaccacc accaccac 618  

Claims (16)

A protein A derived from Staphylococcus aureus or a fragment of the protein A and a fusion protein for immobilizing an antibody comprising a mussel adhesive protein having an amino acid sequence of SEQ ID NO: 6. According to claim 1, wherein the protein A is an antibody immobilized fusion protein having an amino acid sequence of SEQ ID NO: 7. The fusion protein for antibody immobilization according to claim 1, wherein the fragment of Protein A is a fragment having a sequence consisting of 50 to 500 amino acids consecutive from the amino acid sequence of SEQ ID NO. According to claim 3, wherein the fragment of protein A is A domain having an amino acid sequence of SEQ ID NO: 8, B domain having an amino acid sequence of SEQ ID NO: 9, C domain having an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: An antibody immobilized fusion protein selected from the group consisting of a D domain having an amino acid sequence of E and an E domain having an amino acid sequence of SEQ ID NO: 12. The fusion protein for antibody immobilization according to claim 4, wherein the fragment of Protein A is at least one selected from the group consisting of a B domain having an amino acid sequence of SEQ ID NO: 9 and a C domain having an amino acid sequence of SEQ ID NO: 10. The antibody immobilized fusion protein of claim 5, wherein the fragment of Protein A is a BC domain having an amino acid sequence of SEQ ID NO: 13. 7. According to any one of claims 1 to 6, wherein the fusion protein is a protein A or a fragment of the protein A at the N terminal or C terminal of the mussel adhesive protein (mussel adhesive protein) having the amino acid sequence of SEQ ID NO: 6 A conjugated antibody fusion protein for immobilization. The fusion protein for antibody immobilization of claim 7, wherein the fusion protein has an amino acid sequence of SEQ ID NO. The biochip for immune reaction containing the antibody immobilized fusion protein of claim 1. The biochip of claim 8, wherein the fusion protein is immobilized on a solid substrate. Preparing a biochip of claim 9; Immobilizing the antibody against the antigen to be detected on the biochip; Reacting a sample with a biochip comprising the immobilized antibody, and Antigen detection method comprising the step of measuring the antigen-antibody reaction. A vector for expressing a fusion protein for antibody immobilization comprising a promoter and a polynucleotide encoding the fusion protein of claim 1 operably linked thereto. The vector of claim 12, wherein the polynucleotide encoding the fusion protein of claim 1 has a nucleotide sequence of SEQ ID NO: 15. The vector of claim 12, wherein the vector has a cleavage map described in FIG. 2A. A transformant transformed with the vector of any one of claims 12-14. The transformant of claim 15, wherein the transformant is Escherichia coli.

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