KR20230038903A - Fusion protein comprising foldon domain linked to hagfish VLRB protein with deleted hydrophobic tail domain and uses thereof - Google Patents

Abstract

The present invention relates to a recombinant expression vector including: a polynucleotide encoding a V5 epitope sequentially linked to the 3'-end of a gene encoding a hagfish-derived variable lymphocyte receptor B (VLRB) protein from which the hydrophobic tail domain containing a sequence specific to a target antigen has been removed; a linker coding sequence; and a polynucleotide encoding a foldon domain. The present invention also relates to a host cell transformed with the recombinant expression vector, a fusion protein produced by the host cell, in which a hagfish-derived VLRB protein from which the hydrophobic tail domain has been removed and a foldon domain are linked, and a multivalent antibody with increased binding ability to a target antigen, characterized in that the fusion protein is formed into a trimer through self-assembly. The present invention shows higher antigen binding ability.

Description

Fusion protein comprising foldon domain linked to hagfish VLRB protein with deleted hydrophobic tail domain and uses thereof}

The present invention relates to a fusion protein in which a foldon domain is linked to a hagfish-derived VLRB protein having a hydrophobic tail domain removed, and a use thereof.

VLR (variable lymphocyte receptor) identified in lamprey and hagfish, the jawless vertebrates, the lowest vertebrates, rearranges 1 to 12 LRR (leucine rich repeat) modules (somatic rearrangement) It is a polypeptide made by doing the adaptive immune function like an antibody. This is the only antigen that does not use the immunoglobulin structure found so far as a single polypeptide that is small in size compared to the large mammalian antigen receptor (about 150 kDa in the case of immunoglobulin), which is formed as a multi-domain complex. As a receptor, it can be a substitute for an existing antibody.

The VLRB protein has the advantage that its diversity is arithmetically greater than about 10 ¹⁴ and is much more stable to changes in the surrounding environment such as pH and temperature. Therefore, it is believed that using the hagfish's adaptive immune system, it is possible to produce customized antibodies for a specific desired antigen more cheaply, easily, and quickly, and it will be a strong competitive alternative antibody candidate that can compete head-on with various existing antibody markets. do.

Meanwhile, Korean Patent Registration No. 1934578 discloses 'a fusion protein in which a mouse antibody-derived Fc domain is linked to a hagfish-derived VLRB protein having a hydrophobic tail domain removed and a use thereof', and Korean Patent Registration No. 1972894 discloses 'a hydrophobic tail domain A fusion protein in which the C-terminal sequence of the lamprey-derived VLRB protein is linked to the removed hagfish-derived VLRB protein and its use is disclosed, but the present invention 'fusion protein in which the hydrophobic tail domain is removed and the foldon domain is linked to the hagfish-derived VLRB protein There is nothing described about 'proteins and their uses'.

The present invention was derived from the above needs, and the inventors of the present inventors maintained the site capable of binding to an antigen in the hagfish antibody VLRB (variable lymphocyte receptor B) as it is, and the hydrophobic tail of the carboxy-terminal region that determines the three-dimensional structure. By removing the domain coding gene and introducing a foldon domain capable of forming a trimer (the carboxy terminal domain of the fibritin protein of bacteriophage T4) to prepare a VLRB fusion protein having higher antigen binding ability and a smaller molecular weight, the present invention completed.

In order to solve the above problems, the present invention is a V5, which is sequentially linked to the 3'-end of a gene encoding a hagfish-derived variable lymphocyte receptor B (VLRB) protein from which a hydrophobic tail domain containing a sequence specific to a target antigen has been removed. polynucleotides encoding epitopes; linker coding sequence; and a polynucleotide encoding a foldon domain.

In addition, the present invention provides a host cell transformed with the recombinant expression vector.

In addition, the present invention relates to a hagfish-derived VLRB protein produced by the host cell and having a hydrophobic tail domain containing a target antigen-specific sequence removed; V5 epitope; linker; and a fusion protein in which foldon domains are sequentially linked.

In addition, the present invention provides a multivalent antibody with increased binding ability to a target antigen, characterized in that the fusion protein is composed of a trimeric multimer by self-assembly.

In addition, the present invention comprises (a) transforming a host cell with the recombinant expression vector; (b) culturing the transformed host cell of step (a); and (c) obtaining a trimer of the fusion protein from the cultured host cell or its culture medium in step (b).

In addition, the present invention provides a multivalent antibody having increased binding ability to a target antigen prepared by the above method.

In addition, the present invention provides a method for detecting a target antigen by treating a suspected sample containing the target antigen with the multivalent antibody.

In addition, the present invention provides a composition for detecting a target antigen containing the multivalent antibody as an active ingredient.

In general, it is known that when the antigen-binding site of an antibody is increased by one, the Kd value of binding force to the antigen is increased by 10 times. The trimeric hagfish antibody developed in the present invention has three antigen-binding sites, one more site than general immunoglobulins, and a molecular weight smaller than about 30 kDa. Therefore, since it has a smaller size and stronger antigen-binding ability than general immunoglobulins, it is considered that it can be widely used in various studies using antibodies.

1 is a schematic diagram of the preparation of a trimeric fusion protein in which a foldon domain is linked to a hagfish-derived VLRB protein from which a hydrophobic tail domain has been removed.
2 is a vector map used for expression of a trimeric fusion protein.
Figure 3 confirms the prepared trimeric fusion protein on SDS-PAGE under non-reducing or reducing conditions. This is the result.
4 shows the result of analyzing the enhanced antigen-specific binding ability of the trimeric fusion protein by ELISA.

In order to achieve the object of the present invention, the present invention is sequentially linked to the 3'-end of a gene encoding a hagfish-derived variable lymphocyte receptor B (VLRB) protein from which a hydrophobic tail domain containing a sequence specific to a target antigen has been removed. , a polynucleotide encoding the V5 epitope; linker coding sequence; and a polynucleotide encoding a foldon domain.

In the recombinant expression vector of the present invention, the foldon domain is the C-terminal domain of T4 fibritin of bacteriophage T4 fibritin, adopts a β-propeller conformation, and automatically can be tri-merged with The scope of the foldon domain according to the present invention includes a peptide having the amino acid sequence of SEQ ID NO: 1 and functional equivalents thereof. "Functional equivalent" means at least 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more of the amino acid sequence of SEQ ID NO: 1 as a result of addition, substitution or deletion of amino acids. It refers to a peptide having a sequence homology of % or more and exhibiting substantially the same activity as the peptide represented by SEQ ID NO: 1. By "substantially homogeneous activity" is meant the activity of forming trimeric multimers by self-assembly.

In addition, in the recombinant expression vector of the present invention, the V5 epitope may preferably consist of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

In addition, in the recombinant expression vector of the present invention, the linker may be a GS (Gly-Ser) linker or a GPP (Gly-Pro-Pro) linker, preferably consisting of the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4. It may be, but is not limited thereto.

The V5 epitope and linker sequence according to the present invention were used so that the trimeric structure formation site does not interfere with the three-dimensional structure of the antigen binding site of the VLRB protein when connecting the VLRB protein and the foldon domain.

In addition, the hagfish-derived VLRB protein from which the hydrophobic tail domain is removed has a signal peptide (SP) from the N-terminus to the C-terminus, LRRNT (N-terminal capped LRR), LRR (leucine-rich repeat), LRRVs (variable LRR modules) ), CP (connecting peptide), LRRCT (C-terminal capped LRR), and Stalk domains, and contains LRRVs and LRRCT domains, which are antigen recognition sites, so that it is a VLRB protein capable of binding to a target antigen. In other words, the hagfish-derived VLRB protein of the present invention is a hagfish-derived VLRB protein in which only the C-terminal hydrophobic tail domain is removed from the structure of the wild-type VLRB protein.

In the recombinant expression vector according to an embodiment of the present invention, the gene encoding the hagfish-derived VLRB protein from which the hydrophobic tail domain has been removed may be a gene encoding a VLRB protein having the highest binding ability to a target antigen, , from a VLRB cDNA library of hagfish immunized with the target antigen.

The term “recombinant” refers to a cell that replicates, expresses a heterologous nucleic acid, or expresses a peptide, a protein encoded by a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene segments not found in the cell's native form, either in sense or antisense form. A recombinant cell may also express a gene found in the cell in its natural state, but the gene has been reintroduced into the cell by artificial means as a modified one.

The term “recombinant expression vector” refers to a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In general, any plasmid and vector may be used provided that it is capable of replicating and stabilizing in the host. An important characteristic of the expression vector is that it has an origin of replication, a promoter, a marker gene and a translation control element.

An expression vector comprising a gene encoding the hagfish-derived VLRB protein from which the hydrophobic tail domain is removed, a polynucleotide encoding a foldon domain, and appropriate transcription/translation control signals can be constructed by a method well known to those skilled in the art. The method includes in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, and the like. The DNA sequence can be effectively linked to a suitable promoter in an expression vector to direct mRNA synthesis. In addition, the expression vector may include a ribosome binding site and a transcription terminator as a translation initiation site.

The recombinant expression vector may preferably include one or more selectable markers, but is not limited thereto. The marker is a nucleic acid sequence having a characteristic that can be selected by a conventional chemical method, and includes all genes capable of distinguishing transformed cells from non-transformed cells.

The present invention also provides a host cell transformed with the recombinant expression vector.

Any host cell known in the art may be used as a host cell capable of continuously cloning and expressing the vector of the present invention while being stable in a prokaryotic cell, for example, Escherichia coli Rosetta, Escherichia coli JM109, Escherichia coli BL21, Escherichia coli RR1, Escherichia coli LE392, Escherichia coli B, Escherichia coli X 1776, Escherichia coli Dα, Escherichia coli W3110, Bacillus subtilis ( Bacillus subtilis ), Bacillus thuringiensis ( Bacillus thuringiensis ), Salmonella typhimurium ( Salmonella typhimurium ), Enterobacteriaceae and strains such as Serratia marcescens and various Pseudomonas species; and the like.

In addition, when the vector of the present invention is transformed into a eukaryotic cell, as a host cell, yeast ( Saccharomyce cerevisiae ), insect cells, human cells and animal cells (eg, HEK (Human embryonic kidney) 293, CHO (Chinese hamster ovary) , WI-38, BHK (Baby hamster kidney), COS-7, HepG2, 3T3, RIN and MDCK cell lines) and plant cells may be used.

The present invention also relates to a hagfish-derived variable lymphocyte receptor B (VLRB) protein produced by the host cell, from which a hydrophobic tail domain containing a target antigen-specific sequence has been removed; V5 epitope; linker; and a fusion protein in which foldon domains are sequentially linked.

The fusion protein according to the present invention has a V5 epitope at the C-terminus of the stalk domain of hagfish-derived VLRB protein; Gly-Ser (GS) linker and Gly-Pro-Pro (GPP) linker; It is a fusion protein in which foldon domains are sequentially connected.

The present invention also provides a multivalent antibody with increased binding ability to a target antigen, characterized in that the fusion protein is composed of a trimeric multimer by self-assembly.

The multivalent antibody of the present invention is composed of a trimeric multimeric form of a fusion protein in which a hagfish-derived VLRB protein from which the hydrophobic tail domain is removed while maintaining the binding ability to a target antigen and a foldon domain are linked, the foldon domain is located in the center, It is characterized in that the hagfish-derived VLRB protein has a structure that protrudes to the outside.

The multivalent antibody of the present invention contains three VLRB proteins, which are hagfish antibodies, and thus has significantly increased antigen-binding ability.

The present invention also

(a) transforming a host cell with the recombinant expression vector;

(b) culturing the transformed host cell of step (a); and

(c) a method for producing a multivalent antibody with increased binding ability to a target antigen, comprising the step of obtaining a fusion protein trimer from the cultured host cell or its culture medium in step (b).

Specifically, the method for producing the polyvalent antibody of the present invention is to inject an immunogen or antigen into hagfish to immunize them, collect blood from the immunized hagfish to isolate lymphocytes, extract total RNA from the lymphocytes, and, based on this, produce mature VLRBs. After securing the mRNA, a polymerase chain reaction was performed using it as a template to prepare a cDNA pool of VLRBs excluding the hydrophobic region at the C-terminus. A recombinant expression vector (FIG. 2) capable of expressing a fusion protein linked to each clone of the cDNA prepared as described above and the foldon domain was constructed. Thereafter, 293-F cells were transformed with each recombinant expression vector, and the transformed 293-F cells were cultured, and then the culture medium was obtained and the reactivity with the immunogen or antigen was confirmed through an ELISA experiment. Finally, clones having excellent binding ability to the immunogen or antigen were selected, and the cultures of the selected clones were subjected to Western blotting under non-reducing and reducing conditions to confirm that multimers were formed. Various types of immunogens or antigens such as viruses, microorganisms, and proteins may be used.

The present invention also provides a multivalent antibody having increased binding ability to a target antigen prepared by the above method.

The multivalent antibody of the present invention is composed of a trimeric multimer form of a hagfish-derived VLRB protein from which the hydrophobic tail domain has been removed and a fusion protein in which a foldon domain is linked while maintaining the binding ability to a target antigen, and can bind to a target antigen. It is a multivalent antibody that contains three hagfish VLRB proteins and has significantly increased antigen-binding ability.

The present invention also provides a method of detecting a target antigen by treating a suspected sample containing the multivalent antibody with the target antigen.

In the target antigen detection method of the present invention, the sample may be food, water, a solution containing specific or unspecified microorganisms, tissue, cells, blood, serum, plasma, saliva, etc., but is not limited thereto.

The target antigen detection method of the present invention may be carried out in an antigen-antibody reaction method. In this case, it can be carried out using a multivalent antibody of the present invention that specifically binds to the target antigen to be detected. The present invention can be used to detect the presence or absence of a target antigen by performing it according to a conventional immunoassay method. Such an immunoassay can be performed according to various quantitative or qualitative immunoassay methods previously developed.

Detection of the conjugate between the multivalent antibody of the present invention and the target antigen may be performed through an indirect ELISA (direct enzyme linked immunosorbent assay) or a sandwich ELISA method, but is not limited thereto. In the indirect ELISA method and the sandwich-ELISA method, the final enzyme activity measurement or signal measurement may be performed according to various methods known in the art. Detection of these signals allows qualitative or quantitative analysis of the multivalent antibodies of the present invention.

The present invention also provides a composition for detecting a target antigen containing the multivalent antibody as an active ingredient.

The composition for detecting a target antigen of the present invention contains, as an active ingredient, a multivalent antibody composed of a trimeric multimer form of a hagfish-derived VLRB protein from which the hydrophobic tail domain has been removed while maintaining the binding ability to the target antigen, and a fusion protein with a foldon domain linked thereto. Therefore, it can be used for target antigen detection based on antigen-antibody reaction.

The terms 'multimer' or 'polymer' used herein may be used interchangeably.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited to the following examples.

Example 1. Preparation of trimeric fusion proteins

Among hagfish's wild-type VLRB (variable lymphocyte receptor B) genes, the C-terminal gene, which forms a spherical polymer and encodes a strong hydrophobic region, was deleted and the foldon domain peptide (SEQ ID NO: 1) forming a triplex was linked. Primer sets shown in Table 1 were ordered and synthesized.

Primer information used in the present invention Primer name Base sequence (5'→3') Igk Xba I Fwd caTCTAGAATGGAGACAGACACACTCCTGC (SEQ ID NO: 12) stalk FLD link Rev CCACCACCAGAACCACCACCACCAGAACCACCACCGGTACGCGTAGAATCGAGACCGAGGAGAGGGTTAGGGATAGGCTTACCACTACCTGGGCCCCAGAGGCCCGCGTTC (SEQ ID NO: 13) stalk FLD link Fwd (SEQ ID NO: 14) GGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGATCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGCCCTCCCGGACCTCCTGGACCCCCTGGACCCCCCGGG FLD stop Rev TCACAGAAAGGTGCTCAGCAGCACCCACTCGCCGTCCTTCCTCACGTAGGCCTGGCCGTCTCTGGGGGCCTCGGGGATGTAGCCGCTCCCGGGGGGTCCAGGGG (SEQ ID NO: 15)

First, PCR products were obtained using the previously discovered OmpA-specific hagfish antibody gene as a template using Igk Xba I Fwd and stalk FLD link Rev. In addition, in order to link the foldon domain to the hagfish antibody C-terminus, two primer sets, i.e., stalk FLD link Fwd and FLD stop Rev, were made single-stranded at 98 ° C and cooled slowly at room temperature to induce annealing of the two primers did The obtained two types of PCR products were diluted to about 10 ng each, and the two PCR products were linked using Igk Xba I Fwd and FLD stop Rev as primer sets, and the PCR conditions were as follows: 1st step (95℃ for 3 min) > 2nd step (95℃ for 20 sec > 60℃ for 10 sec > 72℃ for 1 min) repeat 32 cycles > 3rd step (72℃ for 5 min).

The finally obtained PCR product was hydrolyzed with Xba I, cloned into the Xba I/Pme I site of the pTracer EF/A vector (Invitrogen, USA) and inserted, and a recombinant vector was selected by DNA sequencing to obtain the pOmpVLR-FLD vector. was produced (Fig. 2). The produced hagfish antibody gene is linked to two Sfi I sites to facilitate substitution with other types of antigen-specific hagfish antibody genes. GS linker and GPP linker were connected to secure structural distance during linkage, and finally, a foldon domain capable of forming a triplex was connected. The recombinant vector, pOmpVLR-FLD, expresses a foldon domain-linked fusion protein, and it is expected that a trimeric OmpA-specific VLRB polymer protein will finally be produced. The constructed vector and the hagfish antibody vector pOmpVLR, which do not contain foldon domains and are expressed as monomers, were transduced into HEK 293-F human cell line (Gibco, USA) for 4 hours with Lipofectamine 2000 (Invitrogen), respectively, and then cultured in an expression medium. did After 72 hours of transduction, the culture supernatant was collected and subjected to Western blotting and ELISA for antigen-specific binding ability.

Poldon Domain Fusion Anti-OmpA VLRB Antibody Amino Acid Sequence regions amino acid sequence LRRNT CPSRCSCSGTTVSCNSRSLTSVPSGFPASTT (SEQ ID NO: 5) LRR1 VLHLGGNKLQSIPSGVFD (SEQ ID NO: 6) 4 LRRVs KLTQLTRLDLDDNQLKFLPNGVFDKLTKLTILGLETNQLQSLPSGVFDNLSKLKELYLYSNKLQSLPSGLFDKLTQLTNLQLYSNQLKSVPDGVFD (SEQ ID NO: 7) CP RLTSLQYIYLYSN (SEQ ID NO: 8) LRRCT PWDCTCPGIRYLSEWINKNSGIVYYYDGSHSVDPDSAKCSGSGKPVRSIICP (SEQ ID NO: 9) stalk TTTTTTTTTTMPTTTTLPTTTKMSMVKVPLVPPEAFGRVMNA (SEQ ID NO: 10) Joint SfiI GLWGPGS (SEQ ID NO: 11) V5 epitopes GKPIPNPLLGLDST (SEQ ID NO: 2) GS linker RTGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 3) GPP linker GPPGPPGPPGPPGS (SEQ ID NO: 4) foldon domain GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 1)

Example 2. Identification of trimeric fusion proteins

The cloned hagfish-derived wild-type VLRB is a monoclonal hagfish antibody (mono Ab) with the C-terminus removed and a trimeric hagfish antibody (FLD Ab) linked to a foldon domain, and expressed in SDS-PAGE under non-reducing conditions as mono- and tri-mers, respectively. It was confirmed by Western blot. Western blotting was performed using a monoclonal antibody (Korean Patent No. 2171950) that specifically binds to the stalk region of hagfish VLRB. In addition, it was confirmed that the trimeric hagfish antibody (FLD Ab) was also reduced to a monomeric form under the reducing condition treated with 2% β-mercaptoethanol (FIG. 3).

Example 3. Verification of enhanced antigen-specific binding ability of trimeric fusion proteins

The antigen-specific binding ability of the anti-OmpA monoclonal hagfish antibody (OmpA-mono) or the trimeric hagfish antibody (OmpA-FLD) was verified by ELISA. In order to determine whether the antibody selectively binds to the antigen, as a result of treating mono and FLD on an ELISA plate coated with BSA (bovine serum albumin), it was confirmed that neither antibody binds (BSA-mono, BSA-FLD in FIG. 4). On the other hand, on the ELISA plate coated with the OmpA protein antigen, it was confirmed that the mono antibody and the FLD antibody bind to the antigen. An increase was observed (Fig. 4).

<110> EarwynTech <120> Fusion protein comprising foldon domain linked to hagfish VLRB protein with deleted hydrophobic tail domain and uses its <130> PN21255 <160> 15 <170> KoPatentIn 3.0 <210> 1 <211> 27 <212> PRT <213> artificial sequence <220> <223> foldon domain <400> 1 Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys 1 5 10 15 Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu 20 25 <210> 2 <211> 14 <212> PRT <213> artificial sequence <220> <223> V5 epitope <400> 2 Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr 1 5 10 <210> 3 <211> 31 <212> PRT <213> artificial sequence <220> <223> gs linker <400> 3 Arg Thr Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 30 <210> 4 <211> 14 <212> PRT <213> artificial sequence <220> <223> GPP linker <400> 4 Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Ser 1 5 10 <210> 5 <211> 31 <212> PRT <213> artificial sequence <220> <223> anti-OmpA VLRB_NRRNT <400> 5 Cys Pro Ser Arg Cys Ser Cys Ser Gly Thr Thr Val Ser Cys Asn Ser 1 5 10 15 Arg Ser Leu Thr Ser Val Pro Ser Gly Phe Pro Ala Ser Thr Thr 20 25 30 <210> 6 <211> 18 <212> PRT <213> artificial sequence <220> <223> anti-OmpA VLRB_LRR1 <400> 6 Val Leu His Leu Gly Gly Asn Lys Leu Gln Ser Ile Pro Ser Gly Val 1 5 10 15 Phe Asp <210> 7 <211> 96 <212> PRT <213> artificial sequence <220> <223> anti-OmpA VLRB_4LRRVs <400> 7 Lys Leu Thr Gln Leu Thr Arg Leu Asp Leu Asp Asp Asn Gln Leu Lys 1 5 10 15 Phe Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Lys Leu Thr Ile Leu 20 25 30 Gly Leu Glu Thr Asn Gln Leu Gln Ser Leu Pro Ser Gly Val Phe Asp 35 40 45 Asn Leu Ser Lys Leu Lys Glu Leu Tyr Leu Tyr Ser Asn Lys Leu Gln 50 55 60 Ser Leu Pro Ser Gly Leu Phe Asp Lys Leu Thr Gln Leu Thr Asn Leu 65 70 75 80 Gln Leu Tyr Ser Asn Gln Leu Lys Ser Val Pro Asp Gly Val Phe Asp 85 90 95 <210> 8 <211> 13 <212> PRT <213> artificial sequence <220> <223> anti-OmpA VLRB_CP <400> 8 Arg Leu Thr Ser Leu Gln Tyr Ile Tyr Leu Tyr Ser Asn 1 5 10 <210> 9 <211> 52 <212> PRT <213> artificial sequence <220> <223> anti-OmpA VLRB_LRRCT <400> 9 Pro Trp Asp Cys Thr Cys Pro Gly Ile Arg Tyr Leu Ser Glu Trp Ile 1 5 10 15 Asn Lys Asn Ser Gly Ile Val Tyr Tyr Tyr Asp Gly Ser His Ser Val 20 25 30 Asp Pro Asp Ser Ala Lys Cys Ser Gly Ser Gly Lys Pro Val Arg Ser 35 40 45 Ile Ile Cys Pro 50 <210> 10 <211> 42 <212> PRT <213> artificial sequence <220> <223> anti-OmpA VLRB_stalk <400> 10 Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Met Pro Thr Thr Thr Thr 1 5 10 15 Leu Pro Thr Thr Thr Lys Met Ser Met Val Lys Val Pro Leu Val Pro 20 25 30 Pro Glu Ala Phe Gly Arg Val Met Asn Ala 35 40 <210> 11 <211> 7 <212> PRT <213> artificial sequence <220> <223> SfiI site <400> 11 Gly Leu Trp Gly Pro Gly Ser 1 5 <210> 12 <211> 30 <212> DNA <213> artificial sequence <220> <223> primer <400> 12 catctagaat ggagacagac acactcctgc 30 <210> 13 <211> 111 <212> DNA <213> artificial sequence <220> <223> primer <400> 13 ccaccaccag aaccaccacc accagaacca ccaccggtac gcgtagaatc gagaccgagg 60 agagggttag ggataggctt accactacct gggccccaga ggcccgcgtt c 111 <210> 14 <211> 111 <212> DNA <213> artificial sequence <220> <223> primer <400> 14 ggtggtggtt ctggtggtgg tggttctggt ggtggtggat ctggtggtgg tggttctggt 60 ggtggtggtt ctggccctcc cggacctcct ggaccccctg gaccccccgg g 111 <210> 15 <211> 104 <212> DNA <213> artificial sequence <220> <223> primer <400> 15 tcacagaaag gtgctcagca gcacccactc gccgtccttc ctcacgtagg cctggccgtc 60 tctgggggcc tcggggatgt agccgctccc ggggggtcca gggg 104

Claims (10)

A polynucleotide encoding a V5 epitope sequentially linked to the 3'-end of a gene encoding a hagfish-derived variable lymphocyte receptor B (VLRB) protein from which a hydrophobic tail domain containing a sequence specific to a target antigen has been removed; linker coding sequence; and a polynucleotide encoding a foldon domain. The method of claim 1, wherein the V5 epitope consists of the amino acid sequence of SEQ ID NO: 2, the linker consists of the amino acid sequences of SEQ ID NOs: 3 and 4, and the foldon domain consists of the amino acid sequence of SEQ ID NO: 1 Characterized in that A recombinant expression vector to be. According to claim 1, wherein the hagfish-derived VLRB protein from which the hydrophobic tail domain has been removed is a signal peptide (SP) from the N-terminus to the C-terminus, LRRNT (N-terminal capped LRR), LRR (leucine-rich repeat), LRRVs (variable LRR modules), CP (connecting peptide), LRRCT (C-terminal capped LRR) and a recombinant expression vector characterized by consisting of a Stalk domain. A host cell transformed with the recombinant expression vector of claim 1. VLRB (variable lymphocyte receptor B) protein produced by the host cell of claim 4, derived from hagfish, from which a hydrophobic tail domain containing a sequence specific to a target antigen has been removed; V5 epitope; linker; and a fusion protein in which foldon domains are sequentially linked. A multivalent antibody with increased binding ability to a target antigen, characterized in that the fusion protein of claim 5 is composed of a trimeric multimer by self-assembly. (a) transforming a host cell with the recombinant expression vector of claim 1;
(b) culturing the transformed host cell of step (a); and
(c) a method for producing a multivalent antibody with increased binding ability to a target antigen, comprising the step of obtaining a trimer of a fusion protein from the cultured host cell or its culture medium in step (b). A multivalent antibody having increased binding ability to a target antigen prepared by the method of claim 7. A method of detecting a target antigen by treating a suspected sample containing the target antigen with the multivalent antibody of claim 6 or 8. A composition for detecting a target antigen containing the multivalent antibody of claim 6 or claim 8 as an active ingredient.

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