KR101607281B1 - A scFv gene and its recombinant protein of monoclonal antibody - Google Patents

Abstract

The present invention relates to a monoclonal antibody single-chain variable fragment capable of recognizing the change in concentration of myoglobin. More specifically, the present invention relates to a recombinant antibody having a nucleotide sequence of SEQ ID NO: 15 and an amino acid sequence of SEQ ID NO: 18 The recombinant antibody scFv is an antibody which is characterized by reversible reactivity and recognizes the change in myoglobin concentration, which is the initial biomarker of myocardial infarction, so that it can be used for continuous diagnostic methods .

Description

A scFv gene and its recombinant protein of monoclonal antibody, which express the monoclonal antibody short chain variable segment gene,

The present invention relates to the development of monoclonal antibody single-chain variable fragments (hereinafter, referred to as 'scFv') capable of recognizing the change in concentration of myoglobin. Specifically, myoglobin, an early biomarker of myocardial infarction, Derived myoglobin-specific scFv gene and a recombinant antibody expressing the same.

Acute myocardial infarction (AMI) among acute illnesses is one of the highest mortality worldwide. AMI is a disease caused by myocardial ischemia or decreased blood flow to the heart. It prevents normal recovery of normal function and homeostasis of cardiac cells, and ischemia beyond the long-term limit causes permanent damage or death of cardiac cells. (Boersma et al, 2003, Lancet . 361: 847-58).

To date, biomarkers for the diagnosis of AMI have been identified as creatine kinase-MB isoform (CK-MB) (Blomberg et al, 1975, Am . J. Med . 59: 464-69), (Ishikawa et al., 1997, Clin Chem 43:.. 467-75) , cardiac troponin-I (cTnI) (Antman et al, 1996, N. Engl J. Med 335:.. 1342-9), heart type fatty acid binding protein (HFABP) ( . Glatz et al, 1998, Biochim Biophys Acta 961:.. 148-52), (Kakoti et al, 2013, Biosens Bioelectron 43:... 400-11), glycogenphosphorylase BB (Krause et al, 1996, Mol Cell Biochem . 160-161: 289-295), and myoglobin (Hayes et al, 2009, Analyst . 134: 533-41). Among the five biomarkers, myoglobin is recognized as an appropriate biomarker for the early diagnosis of AMI because it is a biomarker that increases serum concentration within one hour after the onset of AMI (Bruins et al, 2013, Int J. Cardiol . : 5355-62).

In order to diagnose acute myocardial infarction and prevent recurrence, early detection and continuous monitoring of disease biomarkers are required. Medical diagnosis tools for detecting and monitoring biomarkers are mostly ELISA (enzyme-linked immunosorbent assay) kits It is based on immunological measurements using antibodies such as immunosensors. Immunological measurements can identify the biomarkers of the disease in the human serum, but most antibodies exhibit a very slow desorption reaction rate due to the formation of antibody-antigen complexes, (Peak et al, 1997, Biotechnol . Bioeng . 56: 221-31 ).

In recent years, antibodies that rapidly attenuate not only the adhesion to the antigen but also the desorption reaction have been developed in the early stages of immunization. Antibodies of this nature are called 'reversible antibodies' and are characterized by having a high attachment kinetics ( k _on ) and a desorption kinetics ( k _off ). Reversible reactive antibodies with these characteristics have the possibility of facilitating the continuous monitoring of the concentration of the analyte and the recycling of the immune sensor by varying the degree of antibody-antigen response according to the concentration of the antigen (Cho et al , 2011, Analyst 136:. 1374-9 ), (Peak et al, 2011, Analyst 136:. 4268-76), (Peak et al, 2013, Biosens Bioelectron 40:.. 38-44).

An anti-human myoglobin monoclonal antibody of the IgG type among reversible reactive antibodies was applied to a non-labeled microfluidic device capable of monitoring myoglobin as an antigen (Kim et al, 2012, Anal . Biochem . 420: 54-60 ). However, the hybridoma technique used for the production of an IgG type antibody requires several subcultures, has a disadvantage in that a long incubation period and a high production cost are required.

Peak et al, 1997, Biotechnol. Bioeng. 56: 221-31 Kim et al, 2012, Anal. Biochem. 420: 54-60

It is an object of the present invention to produce a scFv-type recombinant antibody gene which solves the problems of producing an IgG-type anti-human myoglobin monoclonal antibody and to express the recombinant antibody gene. It is also intended to realize a continuous diagnosis method of AMI using the reversible reactivity characteristic of recombinant antibody of scFv type.

In order to achieve the above object, the present invention provides a scFv-type recombinant antibody comprising the amino acid sequence of SEQ ID NO: 18, which binds to myoglobin.

The scFv recombinant antibody of the present invention is provided as an antibody suitable for continuous diagnosis of diseases as a reversible reactive antibody characterized by reversible reactivity according to the concentration of myoglobin.

The present invention provides a gene encoding said scFv recombinant antibody for expressing an scFv recombinant antibody. Genetic cloning is performed through sequencing of hybridoma cells and PCR to provide the gene.

The present invention provides an E. coli transformed with the above expression vector by preparing an expression vector containing the gene to express a gene encoding an scFv recombinant antibody.

The scFv-type recombinant antibody of the present invention is a reversibly reactive antibody having reversible reactivity according to the concentration of myoglobin, and can be used for continuous diagnosis of the AMI by detecting a change in the concentration of myoglobin, which is a biomarker of AMI.

In addition, the reversible reactivity of the scFv recombinant antibody of the present invention can be continuously diagnosed through an easy regeneration process, thereby reducing cost and shortening the production time. In addition, there is a correlation between the amino acid structure of the antibody and the reversibility Can be used for easy analysis.

1 shows electrophoresis results of PCR products of cDNA synthesized using hybridoma Myo2-7Ds cells.
Fig. 2 shows electrophoresis results of PCR products containing the scFv-Myo2-7Ds gene.
Fig. 3 shows the expression system of a recombinant antibody scFv fused with His-tag or His-tag and protein A. Fig. (A) is an expression system of a recombinant antibody scFv fused with a His-tag, and (B) is an expression system of a recombinant antibody scFv fused with a His-tag and a protein A.
Fig. 4 shows a cleavage map of the recombinant vector pET22b-scFv-Myo2-7Ds6xH.
Figure 5 shows electrophoresis results of PCR products containing the scFv-Myo2-7Ds6xH-ProA gene.
Fig. 6 shows a cleavage map of the recombinant vector pscFv-Myo2-7Ds6xH-ProA.
FIG. 7 shows the result of SDS-PAGE and western blotting after purification of the recombinant antibody scFv-Myo2-7Ds produced in E. coli. (A) is the result of SDS-PAGE, and (B) is the result of western blot.
FIG. 8 shows ELISA results of recombinant antibody scFv-Myo2-7Ds and monoclonal antibody IgG-Myo2-7Ds against immobilized myoglobin antigen.
FIG. 9 shows the results of reaction kinetics measurement by attachment and desorption of recombinant antibody scFv-Myo2-7Ds and monoclonal antibody IgG-Myo2-7Ds and myoglobin antigen. (A) is the result of kinetic measurement of scFv-Myo2-7Ds and (B) is the result of kinetic measurement of IgG-Myo2-7Ds.
FIG. 10 shows the repeated attachment and desorption reactions of the recombinant antibody scFv-Myo2-7Ds and the monoclonal antibody IgG-Myo2-7Ds, and shows the result of the reaction kinetics measurement thereof.

The recombinant antibody scFv recognizing the change in concentration of myoglobin of the present invention will be described in more detail.

Definitions of terms not defined in the present invention are understood to mean those commonly used by those skilled in the art.

In the present invention, 'reversible reactivity' means a fast attachment rate and a desorption rate at the same time as a general antibody having a fast attachment rate and a low desorption rate, so that not only an adhesion reaction between an antibody and an antigen but also a desorption reaction can be performed rapidly.

In the present invention, 'reversibly reactive antibody' means the above-mentioned reversible antibody.

The reversible antibody is an antibody having an attachment reaction rate ( k _on ) and a desorption reaction rate ( k _off ), specifically, k _on > 10 ⁵ M ^-1 s ^-1 , and k _off > 10 ^-3 s ^-1 . The reason why the reversible reactive antibody has the above characteristics is that the equilibrium of the antigen-antibody complex formation reaction according to the concentration change of the antigen can be rapidly transferred due to the rapid attachment and desorption rate. For the above reasons, the antigen-antibody complex is formed under the condition that the antigen exists, but the antigen easily separates by the washing solution without the antigen. Therefore, when the reversible reactive antibody is used, the antibody is regenerated even if only a small amount of the washing solution is used to desorb the antigen from the antibody, and the sample is continuously flown, and the reaction of the complex formation of the antigen- Can be changed.

The recombinant antibody scFv of the present invention is an antibody that binds myoglobin as an antigen and comprises V _H and V _L and a linker connecting them. Specifically, SEQ ID NO: 18 represents the entire amino acid sequence of the scFv, SEQ ID NO: 19 represents the amino acid sequence of V _L , and SEQ ID NO: 20 represents the amino acid sequence of V _H. GGGGSGGGGSGGGGS in SEQ ID NO: 18, which is a sequence from 119 to 134, represents an amino acid sequence of a linker linking V _H and V _L , wherein the linker may be composed of any amino acid sequence, It is not limited to the position. The DNA base sequence of the V _H and V _L shows a very high homology (Homology) the domain of Mouse-derived V _H and V _L. The following Table 1 is the same as the amino acid sequence of SEQ ID NO: 18, and specifically shows the characteristics of each part of the amino acid sequence of the scFv recombinant antibody. More specifically, Table 1 below shows the CDR (complementarity determining region) portion as well as the amino acids of V _H , V _L and linker.

[Table 1]

The recombinant antibody scFv of the present invention is characterized by having reversible reactivity, wherein the reversibility reacts with changes in the concentration of myoglobin.

The recombinant antibody scFv of the present invention is a reversible reactive antibody, suggesting the possibility of being used for continuous diagnosis of diseases based on immunological measurement. In particular, it is possible to facilitate the continuous monitoring of the concentration change of the substance to be analyzed as well as the recycling of the immune sensor by the reversible reactivity characteristic of the scFv. More specifically, the scFv activates myoglobin, an early biomarker of AMI, as an antigen, thereby forming an antigen-antibody complex with myoglobin, enabling early diagnosis of AMI, desorption of myoglobin with decreasing concentration, Continuous diagnosis can be made possible.

The gene encoding the recombinant antibody scFv of the present invention is a gene comprising SEQ ID NO: 15, which is produced through complementary DNA synthesized from hybridoma cells. The results of the analysis of the above gene sequences show very high homology with the nucleotide sequences of V _H and V _L genes of monoclonal antibody IgG.

In order to express a gene encoding the recombinant antibody scFv of the present invention, a recombinant vector containing the gene and Escherichia coli transformed with the vector are used. Specifically, the vector is ligated to an expression vector by restriction enzyme treatment after the gene is prepared, and the vector is transformed into E. coli and recovered. The recovered vector is transformed into Escherichia coli, and the recombinant antibody scFv of the present invention is produced by selecting and culturing the strain transformed with the vector.

The following examples are provided for the purpose of illustrating the present invention and are not to be construed as limiting the scope of the present invention.

Example 1: Sequence analysis of scFv-Myo2-7Ds6xH antibody variable region using Myo2-7 Ds hybridoma cells

Hybridoma Myo2-7Ds cells (Kim et al, 2012, Anal ) prepared by fusing myeloma and splenocyte obtained from immunized mice by injecting 50 ㎕ of immunogen two times at intervals of 2 weeks . Biochem 420:. by using the Total RNA embellish separated by using a tri-sol (Trizol) reagent (Invitrogen, Carlsbad, USA) from 54-60) as a template was synthesized complementary DNA (hereinafter 'cDNA'). The reaction was carried out using a Generacer kit (Invitrogen). A forward primer (SEQ ID NO: 1, generacer kit) used for cDNA synthesis was used as a forward primer for amplifying a DNA fragment containing a variable region of a heavy chain and a light chain. In order to amplify a DNA fragment containing a variable region of the heavy chain through the cDNA, a mouse-derived IgG antibody (V00818, J00475, V00786, X01857, J00453, V00825, V00763, J00479, X00915 obtained from Immunogenetics (IMGT.org) ), The amino acid sequences of heavy chains were analyzed and classified into five groups according to amino acid sequence homology. The amino acid sequences (Group 1, ESQSFPNVF; group 2, ESARNPTIY; group 3, GDKKEPDMF; group 4, ASIRNPQLY; group 5, AKTTPPSVY) to prepare reverse primers (SEQ ID NOS: 2-6), which are degenerate primers encoding the corresponding amino acid sequences. In order to amplify the DNA fragment containing the variable region of the light chain through the cDNA, reverse primer CL-kappa and lambda-Rv primers, which are degenerate primers, are referenced to the amino acid sequences conserved in the constant regions of mouse IgG kappa and lambda light chain (SEQ ID NOS: 7 and 8). The prepared primers are shown in Table 2 below.

[Table 2]

To 0.5 μl of the synthesized cDNA, 10 pmole / 2.5 μl of the forward generer primer and 10 pmole / 2.5 μl of each reverse primer prepared as shown in Table 2 were used to perform PCR. The electrophoresis results of the PCR products are shown in Fig. Lane 1 is a result of amplification with the combination of SEQ ID NOs: 1 and 2, lane 2 is a combination of SEQ ID NOs: 1 and 3, lane 3 is a combination of SEQ ID NOs: 1 and 4, lane 4 is a combination of SEQ ID NOs: 1 and 5, 5 is a combination of SEQ ID NOs: 1 and 6, lane 6 is a combination of SEQ ID NOs: 1 and 7, and lane 7 is a combination of SEQ ID NOs: 1 and 8, respectively. In the reaction using the reverse primer CH-Gp5-Rv, the variable region of the heavy chain was successfully amplified at about 500 bp in the reaction using the reverse primer CL-kappa-Rv in the variable region of the light chain (Fig. 1). The PCR conditions are as described in Table 3 below.

[Table 3]

The PCR product was recovered and cloned into a PCR 2.1 Top 10 vector (Invitrogen), followed by transformation into a host cell, E. coli Top 10 (Invitrogen). Recombinant plasmids containing the PCR product were recovered using the obtained transformants. DNA sequencing analysis was performed with reference to the IMGT database, and each PCR product showed very high homology with the V _H and V _L domains derived from the mouse.

<Example 2> Construction and cloning of scFv-Myo2-7Ds gene using nucleotide sequence analysis data

A primer for the preparation of a scFv-Myo2-7Ds recombinant antibody was constructed by referring to the nucleotide sequence analysis data of the V _H and V _L gene fragments. First, the forward primer scFv-Mb2-7H-Nc-fw (SEQ ID NO: 9) and the reverse primer scFv-Mb2-7H-link-rev (SEQ ID NO: 10) were prepared to amplify the V _H gene fragment. Next, the forward primer scFv-Mb2-7L-link-fw (SEQ ID NO: 11) and the reverse primer scFv-Mb2-7L-Xh-rev (SEQ ID NO: 12) were prepared to amplify the V _L gene fragment. The primers prepared for amplification of each variable region gene fragment are shown in Table 4 below.

[Table 4]

ScFv-Mb2-7H-Nc-fw in Table 4 was used as a forward primer and scFv-Mb2-7H-link-rev in Table 4 was used as a reverse primer for the cDNA of Example 1, carry out the reaction to obtain a PCR product (V _H -Linker). Similarly, scFv-Mb2-7L-link-fw of Table 4 was used as a forward primer and scFv-Mb2-7L-Xh-rev of Table 4 was used as a reverse primer for the cDNA of Example 1, The reaction was performed to obtain a PCR product (Linker-V _L ). The two PCR products, V _H -Linker and Linker-V _L, were mixed and subjected to 7 cycles of PCR reaction without primer according to the above Table 3. The two PCR products were assembled. Afterwards, the forward primer scFv-Mb2-7H-Nc-fw and the reverse primer scFv-Mb2-7L-Xh-rev were added to the reaction mixture to amplify the full-sized gene fragment assembled into one. 23 cycles of PCR were performed according to Table 3 to obtain a PCR product containing the scFv gene encoding only the variable region of the anti-human myoglobin monoclonal antibody provided by the present invention. It was confirmed by electrophoresis that the size of the PCR product was about 700 bp (SEQ ID NO: 16). The DNA fragment of the scFv gene was digested with NcoI and XhoI and ligated with a pET-22b (+) expression vector (Novagen) treated with the same restriction enzyme, followed by transformation into E. coli Top10 to obtain a recombinant vector pET22b- scFv-Myo2-7Ds6xH was recovered. The results of the electrophoresis are shown in FIG. 2, the expression system of the recombinant scFv is shown in FIG. 3- (A), and the cleavage map of the vector of pET22b-scFv-Myo2-7Ds6xH is shown in FIG.

Example 3 Preparation and Cloning of scFv-Myo2-7Ds Gene Fused with Protein A Tag for Improvement of Purification Process

To improve the purification process, a gene of scFv-Myo-27Ds (Fig. 3- (B)) into which a protein A tag is fused is prepared. The forward primer MbscFv-Nh-Fw (SEQ ID NO: 13) and the reverse primer MbscFv-Nc-Rv (SEQ ID NO: 14) were prepared for the production of the gene fused with the protein A tag. The primers are shown in Table 5 below.

[Table 5]

PCR was carried out according to the above Table 3 using the forward primer MbscFv-Nh-Fw and the reverse primer MbscFv-Nc-Rv for the pET22b-scFv-Myo2-7Ds6x HT of Example 1 above. A PCR product containing the gene encoding the scFv-Myo2-7Ds was obtained through the PCR, and the size of the PCR product was confirmed to be about 700 bp by electrophoresis. The results are shown in FIG. The PCR products were ligated to plg20m-sc1 vector (Brigido et al, 1993, J. Immunol . 150: 469-79) digested with NheI and NcoI and treated with the same restriction enzymes . Transformation was performed on E. coli Top10 Respectively.

Recombinant vectors were recovered from the obtained transformants, and clones without PCR error were selected by DNA sequencing analysis and named pscFv-Myo2-7Ds6xH-ProA. The pscFv-Myo2-7Ds6xH-ProA vector was constructed so that six Histidine residues and Protein A genes were sequentially positioned at the C-terminal region of the scFv-Myo2-7Ds gene (SEQ ID NO: 17) A map is shown in Fig.

Example 4 Production and Purification of scFv-Myo2-7Ds6 Antibody

4-1: Production of recombinant antibodies

The thus-prepared recombinant vector pscFv-Myo2-7Ds6xH-ProA was transformed into E. coli BL21 (DE3) (Novagen), followed by confirmation of resistance to ampicillin, and strains transformed with the vector were selected. The selected transformants were inoculated in 3 L of LB liquid medium containing ampicillin (100 μg / ml) and 0.2% glycerol, cultured at 37 ° C., and incubated with Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at a final concentration of 0.2 mM and cultured at 30 占 폚 for 15 hours.

4-2: Purification of Recombinant Antibody

The E. coli cultured in Example 4-1 was centrifuged at 8 DEG C for 20 minutes at 4 DEG C to collect the cells. The recovered cells were suspended in TST buffer (50 mM Tris-HCl (pH 7.6), containing 150 mM NaCl and 0.05% Tween-20), and the suspended cells were ultrasonically pulverized. Lt; / RTI &gt; for 20 minutes. The recovered cell lysate was injected into a column filled with IgG sepharose 6 fast flow resin (GE healthcare, Uppsala, Sweden) for purification and washed with 10 times volume of TST buffer solution. The recombinant scFv-Myo2-7Ds6xH-ProA antibody was eluted with 0.5 M acetate buffer (pH 3.4) and neutralized with 1 M Tris-HCl buffer (pH 9.1). The eluate fractions were concentrated using an Amicon Ultra-4 filter unit (Millipore, Temecula, USA) and dialyzed against phosphate buffered saline (PBS) buffer. The scFv-Myo2-7Ds6xH-ProA antibody was stored at 4 DEG C for 1 week to induce autocleavage of the ProA tag, and then subjected to size exclusion chromatography (GPC) using a Sephadex HR-200 column (GE healthcare) The scFv-Myo2-7Ds6xH antibody of 30 kDa size was isolated and concentrated, and the purification was completed by dialysis with PBS buffer solution.

The results of the electrophoresis of the scFv-Myo2-7Ds purified from E. coli BL21 (DE3) transformed with the recombinant vector pscFv-Myo2-7Ds6xH-ProA are shown in FIG. 7, SM is the size marker, lane 1 is the cell lysate obtained by ultrasonication of the transformant, lane 2 is the purified concentrate obtained after purification by IgG affinity chromatography, lane 3, The supernatant obtained after the above Autocleavage, lane 4 is a scFv-Myo2-7Ds recombinant antibody obtained after Size-Exclusion Chromatography. As shown in FIG. 7, it was confirmed that the recombinant antibody was efficiently purified through IgG affinity chromatography and Autocleavage induction and size exclusion chromatography, and the size of the scFv-Myo2-7Ds on SDS-PAGE was about 30 kDa and showed a value similar to the molecular weight that can be deduced from the amino acid sequence of scFv-Myo2-7Ds (SEQ ID NO: 18).

Example 5 Measurement of Affinity of Recombinant Antibody Using Enzyme-linked Immunosorbent Assay (ELISA)

The affinity of the scFv-Myo2-7Ds recombinant antibody of the present invention to the myoglobin antigen was measured and compared with the affinity of IgG-Myo2-7Ds monoclonal antibody using an EIA / RIA (immunoassay / radioimmunoassay) 8-well strip (Bovine serum alvumin) / PBS (phosphate buffered saline) buffer solution (pH 7.4) at 37 ° C for 1 hour, and then incubated at 37 ° C for 1 hour. Then, 0.1 μg of human myoglobin antigen Respectively. The wells immobilized with the antigens were washed 5 times with 1% BSA / PBST buffer solution containing 0.1% Tween 20, and the scFv-Myo2-7Ds recombinant obtained according to Example 4-2 Antibody and IgG-Myo2-7Ds monoclonal antibody were diluted to the predetermined concentration, and the antigen-antibody reaction was induced in the wells at 37 ° C for 1 hour. To measure the amount of antibody reacted with the antigen, anti-Mouse IgG secondary antibody conjugated with HRP (horseradish peroxidase) diluted 1: 2500 with Anti-Histag Mouse primary antibody diluted at a ratio of 1: 500 Promega, Madison, USA) at 37 ° C for 1 hour. Subsequently, 200 μl of TMB (tetramethylbenzidine) substrate was added to the wells, and color development was induced at room temperature for 15 minutes. Then, absorbance was measured at 450 nm using a microplate reader.

The affinity of the scFv-Myo2-7Ds recombinant antibody and the IgG-Myo2-7Ds monoclonal antibody measured by ELISA is shown graphically in Fig. 8. The affinity of scFv-Myo2-7Ds antibody is similar to that of IgG-Myo2-7Ds antibody And the affinity was maintained.

<Example 6> Reversibility of reaction was confirmed by reaction kinetics analysis

A non-labeled sensor system, Octet red device (ForteBio, MenloPa, USA), was used to measure the reaction kinetics for the attachment and desorption reactions of the scFv-Myo2-7Ds recombinant antibody obtained according to Example 4-2 above. First, the myoglobin antigen was diluted to 5 ㎍ / ㎖ with PBS buffer, and immobilized on the surface of Aminopropylsilane disposal sensor tip (ForteBio) for 15 minutes at 30 ° C. The extra sensor tip surface without antigen immobilization was 0.5 % Casein in PBS buffer solution. The sensor tip immobilized with myoglobin was immersed in a 1 μg / ml antibody solution diluted in a 0.5% Casein-PBS buffer solution to induce the reaction, and then the reaction was induced in a PBS buffer solution containing 0.5% casein The desorption reaction was induced again.

The reaction kinetics of the scFv-Myo2-7Ds recombinant antibody using Octet red are shown in FIG. From the results shown in FIG. 9, the antibody solution of the tip immobilized with the antigen showed an increase in signal due to the antigen-antibody reaction. When the antibody was immobilized in the 0.5% Casein-PBS buffer without antibody, Of the reaction was observed.

The scFv-Myo2-7Ds can also be confirmed to have reversible reactivity characteristics such as IgG-Myo2-7Ds by measuring the reversibility of the scFv-Myo2-7Ds. Table 6 below shows the binding kinetics constant comparison of IgG-Myo2-7Ds and scFv-Myo2-7Ds, and it is shown in Fig. 10 that repetitive attachment and desorption are possible like IgG-Myo2-7Ds.

[Table 6]

<110> KOREA UNIVERSITY <120> a scFv gene and its recombinant protein of monoclonal antibody <130> 01-01 <160> 20 <170> Kopatentin 2.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward generacer primer <400> 1 cgactggagc acgaggacac tga 23 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer CH-Gp1-Rv <400> 2 gaagacattt gggaaggact gactctc 27 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer CH-Gp2-Rv <400> 3 gtagatggtg ggatttctcg cagactc 27 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer CH-Gp3-Rv <400> 4 gaacatatca ggttccttct tatcacc 27 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer CH-Gp4-Rv <400> 5 gtagagctga gggttcctga tagaggc 27 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer CH-Gp5-Rv <400> 6 atagacngat ggggstgtyg ttktrgc 27 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer CL-kappa-Rv <400> 7 gatggataca gttggtgcag catcagc 27 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer CL-lambda-Rv <400> 8 gaswgwkggm gwrgacttgg gctgrcc 27 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer scFv-Mb2-7H-Nc-fw <400> 9 agtaccatgg cccagatcca gttggtgcag 30 <210> 10 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> reverse primer scFv-Mb2-7H-link-rev <400> 10 gccacctgaa ccgccaccac cgctaccgcc accgccgatg ttttgatgac ccaaactcca 60                                                                           60 <210> 11 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> forward primer scFv-Mb2-7L-link-fw <400> 11 ggtagcggtg gtggcggttc aggtggcggc ggtagcgatg ttttgatgac ccaaactcca 60                                                                           60 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer scFv-Mb2-7L-Xh-rev <400> 12 agcatcctcg agttttattt ccag 24 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer MbscFv-Nh-fw <400> 13 cagccagccg ctagccagat ccagttg 27 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer MbscFv-Nc-Rv <400> 14 gttagcagcc ggatctccat ggtggtg 27 <210> 15 <211> 744 <212> DNA <213> Artificial Sequence <220> <223> scFv-Myo2-7Ds DNA sequence <400> 15 cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60 tcctgcaagg cttctggtta taccttcaca gactattcaa tacactgggt gaggcaggct 120 gccaacatat 180 gcagatgact tcacgggacg gtttgccttc tctttggaaa cctctgccag caccgcctat 240 ttgcagatca ccaacctcaa aaatgaggac acggctacat atttctgtgc tagacgattt 300 actacgacta cctggtttgc ttactggggc caagggactc tggtcactgt ctctgcaggc 360 ggtggcggta gcggtggtgg cggttcaggt ggcggcggta gcgatgtttt gatgacccaa 420 actccactct ccctgcctgt cagtcttgga gatcaagcct ccatctcttg cagatctagt 480 cagaacattg tacatagtag tggaaacacc tatttagaat ggtaccttca gaaaccaggc 540 cagtctccaa ggttcctgat ctataaagtt tccaaccgat tttctggggt cccagacagg 600 ttcagtggca gtggatcagg gacagatttc acactcaaga tcagcagagt ggaggctgag 660 gatctgggag tttattactg ctttcaaggt tcacatgttc cgtacacgtt cggagggggg 720 accaagctgg aaataaaact cgag 744 <210> 16 <211> 783 <212> DNA <213> Artificial Sequence <220> <223> scFv-Myo2-7Ds6xH DNA sequence <400> 16 cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60 tcctgcaagg cttctggtta taccttcaca gactattcaa tacactgggt gaggcaggct 120 gccaacatat 180 gcagatgact tcacgggacg gtttgccttc tctttggaaa cctctgccag caccgcctat 240 ttgcagatca ccaacctcaa aaatgaggac acggctacat atttctgtgc tagacgattt 300 actacgacta cctggtttgc ttactggggc caagggactc tggtcactgt ctctgcaggc 360 ggtggcggta gcggtggtgg cggttcaggt ggcggcggta gcgatgtttt gatgacccaa 420 actccactct ccctgcctgt cagtcttgga gatcaagcct ccatctcttg cagatctagt 480 cagaacattg tacatagtag tggaaacacc tatttagaat ggtaccttca gaaaccaggc 540 cagtctccaa ggttcctgat ctataaagtt tccaaccgat tttctggggt cccagacagg 600 ttcagtggca gtggatcagg gacagatttc acactcaaga tcagcagagt ggaggctgag 660 gatctgggag tttattactg ctttcaaggt tcacatgttc cgtacacgtt cggagggggg 720 accaagctgg aaataaaact cgagcaccac caccaccacc atggactggt tccgcgtgga 780 tct 783 <210> 17 <211> 975 <212> DNA <213> Artificial Sequence <220> <223> scFv-Myo2-7Ds6xH-ProA DNA sequence <400> 17 cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60 tcctgcaagg cttctggtta taccttcaca gactattcaa tacactgggt gaggcaggct 120 gccaacatat 180 gcagatgact tcacgggacg gtttgccttc tctttggaaa cctctgccag caccgcctat 240 ttgcagatca ccaacctcaa aaatgaggac acggctacat atttctgtgc tagacgattt 300 actacgacta cctggtttgc ttactggggc caagggactc tggtcactgt ctctgcaggc 360 ggtggcggta gcggtggtgg cggttcaggt ggcggcggta gcgatgtttt gatgacccaa 420 actccactct ccctgcctgt cagtcttgga gatcaagcct ccatctcttg cagatctagt 480 cagaacattg tacatagtag tggaaacacc tatttagaat ggtaccttca gaaaccaggc 540 cagtctccaa ggttcctgat ctataaagtt tccaaccgat tttctggggt cccagacagg 600 ttcagtggca gtggatcagg gacagatttc acactcaaga tcagcagagt ggaggctgag 660 gatctgggag tttattactg ctttcaaggt tcacatgttc cgtacacgtt cggagggggg 720 accaagctgg aaataaaact cgagcaccac caccaccacc atggactggt tccgcgtgga 780 tctggtgatc cgaaagctga caacaaattc aacaaagaac aacaaaatgc tttctatgaa 840 atcttacatt tacctaacct aaatgaagaa caacgcaatg gtttcatcca aagcttaaaa 900 gatgacccaa gccaaagcgc taacctttta gcagaagcta aaaagctaaa tgatgcacaa 960 gcaccaaaag cttaa 975 <210> 18 <211> 246 <212> PRT <213> Artificial Sequence <220> <223> scFv-Myo2-7Ds6 amino acid sequence <400> 18 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu   1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr              20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Lys Trp Met          35 40 45 Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe      50 55 60 Thr Gly Arg Phe Ala Phe Ser Leu Val Thr Ser Ala Ser Thr Ala Tyr  65 70 75 80 Leu Gln Ile Thr Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys                  85 90 95 Ala Arg Arg Phe Thr Thr Thr Thr Trp Phe Ala Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly         115 120 125 Ser Gly Gly Gly Gly Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser     130 135 140 Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser 145 150 155 160 Gln Asn Ile Val His Ser Ser Gly Asn Thr Tyr Leu Glu Trp Tyr Leu                 165 170 175 Gln Lys Pro Gly Gln Ser Pro Arg Phe Leu Ile Tyr Lys Val Ser Asn             180 185 190 Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr         195 200 205 Asp Phe Thr Leu Asn Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val     210 215 220 Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Tyr Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys                 245 <210> 19 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL regions of scFv-Myo2-7Ds6 amino acid sequence <400> 19 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly   1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser              20 25 30 Ser Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser          35 40 45 Pro Arg Phe Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro      50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile  65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly                  85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 20 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> VH regions of scFv-Myo2-7Ds6 amino acid sequence <400> 20 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu   1 5 10 15 Thr Val Lys Ile Ile Ser Cys Lys Ala Ser Gly Tyr Phe Thr Asp Tyr              20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Lys Trp Met          35 40 45 Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe      50 55 60 Thr Gly Arg Phe Ala Phe Ser Leu Val Thr Ser Ala Ser Thr Ala Tyr  65 70 75 80 Leu Gln Ile Thr Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys                  85 90 95 Ala Arg Arg Phe Thr Thr Thr Thr Trp Phe Ala Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ala         115

Claims (9)

A recombinant antibody in the form of scFv comprising SEQ ID NO: 18 and binding to myoglobin. The method according to claim 1,
The V _L of said recombinant antibody comprises SEQ ID NO: 19,
Wherein the V _H of the recombinant antibody comprises SEQ ID NO: 20. 3. The recombinant antibody of claim 2, wherein the recombinant antibody has reversible reactivity. 4. The recombinant antibody of claim 3, wherein the reversible reactivity is by myoglobin concentration. A method for continuously measuring the change in myoglobin concentration using a recombinant antibody of any one of claims 1 to 4 or a recombinant antibody in which the recombinant antibody is immobilized on a carrier. A gene encoding a recombinant antibody of any one of claims 1 to 4. 7. The method according to claim 6, wherein the gene is a gene comprising SEQ ID NO: 15 9. A recombinant vector comprising the gene according to claim 7. 8. The Escherichia coli transformed with said vector according to claim 7.

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