WO2018212467A1 - Fusion protein having c-terminal sequence of lamprey-derived vlrb protein connected to hagfish-derived vlrb protein having hydrophobic tail domain removed therefrom, and use thereof - Google Patents

Abstract

The present invention relates to a multivalent antibody having an increased binding force with respect to a target antigen. The multivalent antibody has a fusion protein self-assembled into a multimer, the fusion protein having a C-terminus of a lamprey-derived variable lymphocyte receptor B (VLRB) protein connected to a hagfish-derived VLRB protein having a hydrophobic tail domain removed therefrom.

Description

Fusion protein linking the C-terminal sequence of seven growth fish-derived VLRB proteins to a fish-derived VLRB protein from which the hydrophobic tail domain has been removed and its use

The present invention relates to a fusion protein in which the C-terminal sequence of Chil-grown-derived VLRB protein is linked to a fish-derived VLRB protein from which a hydrophobic tail domain has been removed, and a use thereof.

In general, antibodies are known as immunoglobulin (Ig) proteins present in most vertebrates, and the antigen-specific binding ability of antibodies is widely used in the fields of biotechnology and medicine. However, antibodies have limitations such as limitation of binding area, limitation of selection of epitopes, complicated production process and the like. As a result, many researchers are working hard to find new antibody candidates.

In recent years, the presence of variable lymphocyte receptor (VLR) proteins that play a role similar to that of immunoglobulins responsible for the acquired immunity of larvae in hagfish and lamprey, the oldest vertebrates and invertebrates. VLR-A and VLR-C are known to play the same role as mammalian T-lymphocytes, and VLR-B plays the same role as B-lymphocyte types secreted from lymphocyte-like cells. VLR protein is formed by the combination of leucine-rich repeat (LRR) modules.The components include amino-terminus (N-terminus), SP (signal peptide), LRRNT (N-terminal capped LRR), and LRRVs (variable LRRs). , Connecting peptide (CP), C-terminal capped LRR (LRRCT), Stalk, and hydrophobic tail (HP) domains. Among them, the LRRV module, which recognizes the antigen, is known to include one to up to nine. It has been found that the VLR proteins, in which modules are combined by somatic rearrangement, theoretically possess more than about 10 ¹⁴ variants.

Since the identification of a new type of antibody, the VLR protein in blackfish and seven-growth fish, many attempts have been made to replace Ig protein. However, these attempts have been carried out using only the VLR protein of seven growth fish, which is a C-terminal (C-terminus) domain having a very different sequence among the structures of a seven growth fish or an eel-derived VLR protein having a generally similar structure. Because. Unlike the C-terminal portion of the VLR, which is highly hydrophobic by hydrophobic amino acids, the cysteine residues and Glycosylphosphatidylinositol (GPI) cleavage sequences that induce disulfide bonds exist at the C-terminus of the VLR. Due to the above characteristics, VLRs of seven-growth fishes form secreted octahedra or 10-mer VLR complexes.

In the present invention, the hydrophobic C-terminus is replaced with the C-terminus of the VLRB protein derived from Chilfish, while maintaining the site capable of binding to the antigen in the incubator antibody VLRB protein. To grant.

Meanwhile, Korean Patent Publication No. 2009-0024648 discloses a 'fusion method between LLR family proteins' and Korean Patent Publication No. 2016-0147787 discloses 'humanized variable lymphocyte receptor (VLR) and related compositions and uses'. However, there is no description of the fusion protein and its use in which the C-terminal sequence of the seven-growth fish-derived VLRB protein is linked to the fish-derived VLRB protein from which the hydrophobic tail domain of the present invention has been removed.

The present invention was derived from the above-described needs, and the present inventors have replaced the C-terminal hydrophobic tail domain with the C-terminus of the C-terminal antibody VLRB protein while maintaining the binding site with the antigen in the mutant fish VLRB protein. Fusion proteins were prepared. As a result of transforming the host cell with a recombinant vector comprising the nucleic acid encoding the fusion protein, it was confirmed that the fusion protein of the present invention was formed in the form of an octet or a dimer in a multimer form in the host cell. The present invention has been completed by confirming that the form of the eel antibody has a significantly higher binding ability to the target antigen as compared to the monomeric (monomer) eel antibody having the same antigen recognition site.

In order to solve the above problems, the present invention is to encode the C-terminal domain of the seven-growth VLRB protein linked to the 3'-end of the gene encoding a murine tail-derived variable lymphocyte receptor B (VLRB) protein Provided is a recombinant expression vector comprising a polynucleotide.

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

The present invention also provides a fusion protein produced by the host cell in which the hydrophobic tail domain has been removed, and the C-terminal domain of the CLR-derived VLRB protein and the CLS-derived VLRB protein are linked to each other.

In another aspect, the present invention provides a multivalent antibody having increased binding ability to a target antigen, characterized in that consisting of a multimer or octahedron multimer by the self-assembly of the fusion protein.

In addition, the present invention comprises the step of culturing the host cell transformed with the recombinant expression vector to obtain a multimer or octamer multimer of the fusion protein, a method for producing a multivalent antibody having increased binding capacity to the target antigen. To provide.

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

The present invention also provides a method for detecting a target antigen by treating the multivalent antibody with a target antigen-containing suspect sample.

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

The antibody of the present invention is not a simple fish eel antibody but has 8 or 10 binding sites with the antigen, thereby maximizing antigen-antibody binding ability, and by removing the hydrophobic tail domain, the expression rate is increased in the host cell. Stability also has the advantage of increased. Unlike mouse antibodies, the eel antibody consists of a single peptide, so that its genetic engineering is free and its applicability is infinite. Therefore, the eel antibody derived from the eel of the present invention utilizes high binding ability and specificity for antigens to develop and develop biomarkers. It may be useful.

1 is a vector map of pKINGeo / ccdB and pTEL used for wild type VLRB protein expression or VLRB fusion protein expression.

FIG. 2 shows Western blotting results (B) of the cell culture medium and cell lysates of HEK 293-F cells transduced with wild-type eel VLRB protein (A) and randomly selected wild-type eel VLRB protein.

3 is a schematic diagram for replacing the hydrophobic C-terminus of the blackfish VLRB protein with the C-terminal sequence of the seven-fish VLRB protein. Western blotting results (B) confirmed under reducing conditions.

Figure 4 shows the results (A and B) of the pTEL plasmid cloned from the VLR library derived from the musk Eels immunized with VHSV for the preparation of the musk VLRB fusion protein substituted with the C-terminal sequence of the seven-fish VLRB protein Cell culture medium of 293-F cells transfected with clones was confirmed by Western blotting (C).

5 is a result of confirming the VHSV specific binding capacity of each clone of the mudfish VLRB fusion protein substituted with the C-terminal sequence of seven growth fish VLRB protein by ELISA.

Figure 6 shows the presence of polymer formation of 43LC and 7LC selected as having the VHSV-specific binding capacity of the blackfish VLRB fusion protein clones substituted with the C-terminal sequence of seven growth fish VLRB protein (A), and other than VHSV The result of confirming the binding ability to the antigen of the kind by ELISA (B and C) and Western blotting results (D) using 43LC and 7LC as the primary antigen. 43LC or 7LC; Cell culture medium of 293-F cells transduced with a recombinant vector comprising a 43 clone or a 7 clone gene, AIV, which is substituted with the C-terminal sequence of the seven-fish VLRB protein; Avian influenza virus, HA; Hemagglutinin, AgX; Antigen no treatment, VNNV; Viral neuronecrosis virus, G protein; Surface glycoproteins of VHSV.

7 is a result of treating 43LC and 7LC in HINAE cells infected with the VHS virus and confirming the degree of response through immunocytochemical analysis.

FIG. 8 shows the results obtained by electrophoresis of monoclonal (mono 43) expressing only the LR VLRB portions of 43LC and 43LC under reducing and non-reducing conditions.

9 is a result of confirming the antigen-specific binding capacity of the monoclonal (mono 43) expressing only the LR VLRB portion of 43LC and 43LC through ELISA.

In order to achieve the object of the present invention, the present invention provides a C-terminal domain of Chil-growth-derived VLRB protein linked to the 3'-terminus of a gene encoding a murine tail-derived variable lymphocyte receptor B (VLRB) protein. Provided is a recombinant expression vector comprising a polynucleotide encoding.

In the recombinant expression vector of the present invention, the fish-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, and LRRNT (N-terminal). capped LRR), leucine-rich repeat (LRR), variable LRR modules (LRRVs), connecting peptide (CP), C-terminal capped LRR (LRRCT), and Stalk domains, which can be antigen-recognized LRRVs and LRRCT It is a VLRB protein that contains a domain and is capable of binding to a target antigen.

In the recombinant expression vector according to an embodiment of the present invention, the fish-derived VLRB protein from which the hydrophobic tail domain has been removed may be a signal peptide that is a murine immunoglobulin κ chain leader sequence, but is not limited thereto. If the signal peptide or sequence that can further enhance the extracellular secretion capacity of the recombinant protein can be used without limitation in its kind.

In the recombinant expression vector according to an embodiment of the present invention, the murine immunoglobulin κ chain leader sequence may be composed of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

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

In the recombinant expression vector of the present invention, the polynucleotide encoding the C-terminal domain of the seven-fish VLRB protein may comprise a nucleotide sequence represented by the nucleotide sequence of SEQ ID NO: 2. In addition, homologues of the above nucleotide sequences are included within the scope of the present invention. Specifically, the polynucleotide encoding the C-terminal domain of the seven-tailed VLRB protein is 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably the nucleotide sequence of SEQ ID NO: 2, respectively. For example, it may include a base sequence having 95% or more sequence homology. The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

The C-terminal domain of the seven-tailed VLRB protein has eight cystein (Cystein) residues and a GPI (Glycosylphosphatidylinositol) incision sequence. Due to these characteristics, the seven-growth VLRB protein is formed into a VLRB multimer of about 400 kDa, which is composed of 8-10 monomers, expressed on the cell surface and secreted into the serum.

The term “recombinant” refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes are modified and reintroduced into cells by artificial means.

The term "recombinant expression vector" means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In principle, any plasmid and vector can be used as long as it can replicate and stabilize in the host. An important feature of the expression vector is that it has an origin of replication, a promoter, a marker gene and a translation control element.

Expression vectors comprising genes encoding the fish-derived VLRB protein from which the hydrophobic tail domain of the present invention has been removed, polynucleotides encoding the C-terminal domain of the CLS-derived VLRB protein, and appropriate transcriptional / translational control signals are well known to those skilled in the art. Can be constructed by a method. Such methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence can be effectively linked to a suitable promoter in the expression vector to drive mRNA synthesis. Expression vectors may also include ribosomal binding sites and transcription terminators as translation initiation sites.

The recombinant expression vector may preferably include one or more selectable markers, but is not limited thereto. The marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells.

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

The host cell capable of continuously cloning and expressing the vector of the present invention in a prokaryotic cell can be used by any host cell known in the art, for example, Escherichia coli Rosetta, E. coli JM109, E. coli BL21, E. coli RR1, Escherichia coli LE392, Escherichia coli B, Escherichia coli X 1776, Escherichia coli Dα, Escherichia coli W3110, Bacillus sp. Strains such as Bacillus subtilis , Bacillus thuringiensis , Salmonella typhimurium , Enterobacteria and strains such as Serratia marcescens and various Pseudomonas species.

In the case of transforming a vector of the present invention into eukaryotic cells, yeast ( Saccharomyce) as a host cell. cerevisiae ), insect cells, human cells and animal cells (e.g., human embryonic kidney (HEK) 293, Chinese hamster ovary (CHO), W138, BHK, COS-7, HepG2, 3T3, RIN and MDCK cell lines) and plant cells This can be used.

The present invention also provides a fusion protein produced by the host cell in which a hydrophobic tail domain-removed mudfish-derived variable lymphocyte receptor B (VLRB) protein and a C-terminal domain of seven growth fish-derived VLRB proteins are linked.

The fusion protein according to the present invention is a fusion protein in which the C-terminal domain of the seven-growth VLRB protein is linked to the C-terminus of the stalk domain of the eel-derived VLRB protein.

The present invention also provides a multivalent antibody having increased binding ability to a target antigen, characterized in that the fusion protein is composed of a multimer or an octahedron multimer by self-assembly. The multivalent antibody of the present invention is composed of a multimeric form of an octopus VLRB fusion protein in which a C-terminal hydrophobic tail domain is substituted with a C-terminal sequence of seven growth fish while maintaining the binding ability to a target antigen. In addition, it is characterized by having a multimer form such as an octahedron or a dimer by the seven-growth C-terminal domain rich in cystein residues.

As used herein, the term multimer, composite or polymer may be used interchangeably.

The polyvalent antibody of the present invention is a multivalent antibody containing 8 or 10 VLRB proteins, which are blackfish antibodies, and the antigen binding ability is significantly increased.

The present invention also provides

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

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

(c) obtaining a multimer or an oligomeric multimer of the fusion protein from the cultured host cell of step (b) or a culture thereof, wherein the multivalent antibody having increased binding capacity to the target antigen is obtained. It provides a manufacturing method.

Specifically, the method for preparing the multivalent antibody of the present invention, immunization by injecting an immunogen or antigen into the eel, to extract the blood from the immunized eel and to separate the lymphocytes, extract the total RNA of the lymphocytes based on the mature VLRBs After securing the mRNA, a polymerase chain reaction was carried out using the template to prepare a cDNA pool of VLRBs except for the hydrophobic region at the carboxy terminus. Each clone of the cDNA prepared as described above was cloned into a pTEL vector (FIG. 1B) containing the C-terminal sequence of seven growth fish VLRB, and the C-terminus of the seven-fish VLRBs and seven growth fish VLRBs having different antigen recognition sequences were linked. A recombinant expression vector capable of expressing a fusion protein was constructed. Thereafter, 293-F cells were transfected with each recombinant expression vector, the transduced 293-F cells were cultured, and a culture solution was obtained to confirm reactivity with an immunogen or antigen by ELISA experiment. Finally, clones with excellent binding ability to the immunogen or antigen were selected, and the culture of the selected clones was Western blotting under non-reducing and reducing conditions to confirm that the fusion protein was formed into multimers. The immunogen or antigen is not limited thereto, and may be a virus, a microorganism, or the like.

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

The multivalent antibody of the present invention is composed of a multimeric form of an octamer or a dimer of a fusion protein in which a hydrophobic tail domain is substituted with the C-terminal sequence of a VLRB protein while maintaining the binding ability to a target antigen in a blackfish VLRB protein. It is a multivalent antibody which contains 8 or 10 blackfish VLRB proteins which can bind with a target antigen, and the antigen binding ability is markedly increased compared with a single body.

The present invention also provides a method for detecting a target antigen by treating the multivalent antibody with a target antigen-containing suspect sample.

In the target antigen detection method of the present invention, the antigen is not limited thereto, but may be a virus or a microorganism.

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

The target antigen detection method of the present invention can be carried out in an antigen-antibody reaction mode. In this case, it can be carried out using the 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 of a target antigen by carrying out according to conventional immunoassay methods. This immunoassay can be carried out according to various quantitative or qualitative immunoassay methods developed in the past.

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

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

The composition for detecting a target antigen of the present invention comprises a multivalent antibody consisting of multimers in the form of a multimer in the form of a mutton fish VLRB fusion protein substituted with a hydrophobic tail domain derived from seven growth fish while maintaining the binding ability to the target antigen. The kit of the present invention comprising a composition for the present invention is not limited thereto, but is preferably an immunoassay kit, and the immunoassay kit is direct-ELISA, indirect-ELISA, sandwich-ELISA, protein microarray, radioimmunoassay. (RIA: Radioimmunoassay) and the like.

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

Example 1 Analysis of Extracellular Secretion Efficiency of Wild-type Black Eel VLRB Protein

It was confirmed that the C-terminus of the hagfish VLRB protein is composed of amino acids having high hydrophobicity (FIG. 2A). Intact forms of VLRB genes from cDNA synthesized from the eel blood mRNA were amplified by PCR and cloned into pKINGeo / ccdB vector. Randomly selected plasmids were transfected into 293-F cell lines to express VLRB proteins. Two days after the transduction, the cell culture medium (supernatant) and cells (pellets) were separated and recovered separately. Cell culture medium was filtered with a 0.45 ㎛ membrane filter to remove debris and concentrated 10 times with lyophilizer. The recovered cells were treated with a cell lysate (Promega, USA) containing a protease inhibitor cocktail to induce intracellular proteins to elute. Cell culture solution and cell samples prepared as described above were electrophoresed by mixing in a loading buffer containing 1% β-mercaptoethanol (hereinafter, 2-ME). After electrophoresis, the VLRB protein was identified using a mouse anti-VLRB antibody 11G5 capable of detecting VLRB protein after transfer to a PVDF membrane. As a result, all of the eight clones selected were found to be recombinant VLRB protein in the cell pellet, but in the cell culture, only some clones showed VLRB protein (FIG. 2B). The results indicated that the efficiency of wild type musk VLRB protein secretion outside the cell is low.

Example 2 C-terminal Sequence Substitution of Blackfish VLRB

In the present invention, the C-terminal sequence of the eel VLRB is replaced with the C-terminal sequence of the larvae VLRB to overcome the low secretion efficiency of the eel VLRB protein to the outside of the cell and induce complex formation, thereby improving the binding ability of the VLRB antibody to the antigen. Was planned.

First, in order to establish a recombinant protein expression system through human cell lines, among the cell lines derived from human embryonic kidney (HEK), which is known to have the highest foreign gene expression rate, it is possible to grow even in suspension form and foreign protein. The 293-F cell line with a high expression level of was selected and used. In order to develop a vector system expressible in human cell lines, the inventors constructed a recombinant expression vector based on a pTracer-EF / V5-His (# V887-20, Invitrogen, USA) vector. First, secretion signal peptides were introduced for the separation of foreign proteins into cells. To this end, pSecTag2A (# V900-20, Invitrogen) murine (murine) Ig κ chain leader sequence for the polymerase chain reaction from the vector (polymerase chain reaction, PCR) to amplify and, VLRB Sfi I restriction to facilitate gene transfer the enzyme seat is connected to pull air to remove the Cm / ccdB gene, located at both ends from the vector pEF-DEST gateway (# 12285-011, Invitrogen) and cloned between the pTracer-EF / V5-His vector of the Kpn I and Not I pKINGeo / ccdB vector was constructed (FIG. 1A).

The LC sequence is then fused after the stalk portion of the randomly selected mutant VLRB gene by PCR technique to replace the C-terminal sequence of the mutant VLRB with the C-terminal sequence (lamprey C-term, hereinafter LC) of the chinchillary VLRB. (FIG. 3A).

Primer Set for C-terminal Fusion of Seven Growth Fish VLRB

Primer Name	Sequence (5 '→ 3')	SEQ ID NO:
Primer SP		CAGGTACCATGGAGACAGACACACTCCTG	3
Primer 1	TTGTGCAGGCGGGCTTTCCGCAGTCGCCACCGTCCGCGCACGCGTTCATGACACG	4
Primer 2	CGGAAAGCCCGCCTGCACAACTCTCCTGAACTGCGCGAATTTCCTCAGCTGCCT	5
Primer 3	TCAACGTTTCCTGCAGAGGGCGCAGGTCGAGCAGAGGCAGCTGAGGAAATTCGC	6

The PCR product was cloned into pKINGeo / ccdB vector and transduced with 293-F cell line to confirm expression patterns. As a result, the LC fusion musk VLRB recombinant protein was detected in both the cell culture medium and the cell lysate (FIG. 3B). In particular, it was confirmed that the amount of VLRB protein secreted out of the cell was higher than that of the wild-type musk VLRB. In addition, the results of electrophoresis under non-reducing conditions confirmed the VLRB of various types of complexes (dimer, tetramer, octet and depolymer) including monomer size.

Example  3. Chil-eok Substituted with C-terminal Sequences VLRB  Expression Trend Analysis of Fusion Proteins

Using the LC sequence, a pTEL vector containing the LC sequence was constructed to produce a library of blackfish VLRB proteins. The pTEL vector was constructed so that the cloned library-type VLRB genes could be expressed like the LC sequence by introducing an LC sequence instead of the existing V5 and 6 x His tag sequences using the pKINGeo / ccdB vector as a skeleton (FIG. 1B). ).

Total RNA was extracted from lymphocytes in the eel blood, and as a template, VLRBs cDNA in library form was obtained. The obtained VLRB gene was cloned into the pTEL vector, and then transformed into E. coli DH5α competent cells. The library was validated by identifying VLRB insertion genes of various sizes from randomly selected colonies (FIG. 4B). Randomly selected VLRB plasmids were each transduced with 293-F cell lines to confirm expression patterns by western blotting. Three days after the transduction, the cell cultures were collected and subjected to electrophoresis under non-reducing conditions without adding 2-ME. As a result, the expression of VLRB protein in 11 clones except 3 clones (3LC, 6LC, 14LC) was confirmed (FIG. 4C). In particular, double clones or more complexes were observed in all clones in which the LC fusion VLRB protein was expressed. From the above results, the present inventors confirmed that the LC-fused VLRB proteins produced in the pTEL vector secrete VLRB protein in cell culture at a high rate (78.6%) and most of them form a complex form.

Example  4. Target Antigen Specific Binding capacity  Fusion protein screening

Viral Hemorrhagic Septicemia virus (VHSV), the causative agent of viral hemorrhagic sepsis (VHS), is known to cause damage from farmed flounder at low temperatures in winter and spring, and the number of cases is increasing. The inventors of the present invention obtained a VLRB library from a mush that immunized VHSV to make a mush VLRB antibody against VHSV and cloned it into a pTEL vector. Recombinant plasmids were transformed with E. coli DH5α competent cells and then amplified and transduced with 293-F cell lines, respectively. ELISA was performed using cell culture solution 3 days after transduction to find VLRB antibodies specific for VHSV.

The experiment blocked multiwell plates coated with VHSV (200 ng) or HEL (20 ng) antigen with 3% skim milk solution and treated the transduced 293-F cell culture supernatant. Of the 96 clones, two clones with the highest reactivity to VHSV, 43LC and 7LC, were selected. High-purity DNA was extracted with DNA prep kits after two streaking procedures for further experiments with 7LC clones with non-specific binding capacity to both VHSV and HEL and 43LC with specific reactivity with VHSV. We analyzed the amino acid sequence of these two clones and analyzed the expression pattern from the 293-F cell line via western blotting.

Sequence analysis of the fusion proteins 43LC and 7LC

	43LC	7LC
LRR1	VLWLGGNKIPSLPHGVFD (SEQ ID NO: 7)	VLQLQGNKLQSLPSGVFD (SEQ ID NO: 14)
LRRv	KLTSLTLLSLHTNQLQSLPDGVFD (SEQ ID NO: 8)	KLTQLTYLSLSTNQLQSLPNGVFD (SEQ ID NO: 15)
LRRv	KLTSLTLLSLHTNQLQSLPSGVFD (SEQ ID NO: 9)	KLTQLTVLGLQTNQLKSVPDGVFD (SEQ ID NO: 16)
LRRv	KLTELKELRLYENKLQSLPHGVFD (SEQ ID NO: 10)	-
LRRv	KLTQQLKDLRLHQNQLKSVPDGVFD (SEQ ID NO: 11)	-
CP	RLTSLQTIYLYSNP (SEQ ID NO: 12)	RLTSLQKIYLYSNP (SEQ ID NO: 17)
LRRCT	WDCTCPGVDYLSRWLHTNSKKETSDSAKCSGSGFPVRSIICP (SEQ ID NO: 13)	WDCTCPGIRYFSEWINKHSGVVRDSSNNVNPDSAKCSGSGKPVRSIICP (SEQ ID NO: 18)

The two clones showed a significant difference in the LRRv module, which plays an important role in antigen recognition, and it was confirmed that 7LC had two LRRv modules compared to 43LC which included a total of four LRRv modules (Table 2). In addition, it was confirmed by Western blot that the molecular weight of the recombinant 43LC is greater than 7LC by the difference in the number of modules (Fig. 6A).

Since the two clones showed nonspecific binding ability in the ELISA, various antigens were used to examine their antigen specificity. Testing of reactivity of 43LC and 7LC clones against wells coated with VHSV, AIV (Avian Influenza Virus), HA (hemagglutinin), and HEL antigens and wells without antigens (AgX) revealed that 7LC Regardless of the type, it was confirmed that the clone had a high OD value and non-specific binding ability, whereas 43LC showed a slight OD value, but showed the highest binding capacity to VHSV and antigen-specific reactivity (FIG. 6B). In order to remove the non-specific reactivity observed above, ELISA was performed after treating 5% skim milk solution in a ratio of 1: 1 in 43LC and 7LC. As a result, the nonspecific binding of the 43LC clone having specific binding ability with VHSV was confirmed to be lower, and the 7LC clone was found to have little reactivity with all antigens (FIG. 6C). The binding ability of 43LC to VHSV antigen and VHSV surface glycoprotein (G protein) was confirmed by Western blotting to determine the site of VHSV antigen recognized by 43LC. Briefly, each antigen was transferred to a PVDF membrane and treated with 43LC and 7LC clones as primary antibodies, respectively, and with anti-mouse antibody (11G) capable of recognizing the stalk site of VLRB. As a result, no reaction was observed in the membrane treated with 7LC as the primary antibody, whereas VHSV and recombinant G protein were confirmed in the membrane treated with 43LC as the primary antibody (FIG. 6D). From the above results, it was found that the 43LC clone specifically binds to the G protein, which is the outer membrane protein of the VHS virus.

In addition, HINAE (Hirame natural embryo cell) cell line derived from flounder was infected with VHSV at a concentration of 1 × 10 ⁵ TCID50 / ml and the binding capacity of 43LC clone to the amplified VHS virus was confirmed by immunocytochemical analysis. As a result, as shown in FIG. 7, 43LC specifically bound to VHS virus, and green fluorescence was confirmed. Consistent with the previous results, no fluorescence was observed in the cells treated with 7LC.

Example  5. Target Antigen Specific Binding capacity  have Fusion protein unit  And antigen binding capacity of polymers

It was investigated whether the polymer was formed by the presence or absence of the LC site that induces the polymer formation of the 43LC clone. As shown in FIG. 8, the 43LC including the LC site and the mono 43 protein genetically deleted from the LC site were identified as complex and mono form, respectively, under non-reducing conditions without 2-ME treatment. It was confirmed that the results were all separated in a monolithic form under reducing conditions.

VISAV-specific binding ability of the two antibodies, mono 43 in the monoform form and 43LC in the polymer form, was examined by ELISA, and specifically high binding capacity to VHSV was observed only in the polymer 43LC (FIG. 9).

Muckfish-derived VLRB antibody, in which the hydrophobic tail domain of the present invention is substituted with the C-terminal sequence of the seven-tailed fish VLRB, was formed in complex form upon expression in transduced cells, and has the same extracellular secretion ability as compared to wild-type VLRB and the same antigen recognition. It has been shown that there is a markedly increased target antigen binding capacity compared to a monolith with sequence. VLRB antibodies selected through the method of the present invention may be used in various antibody-related experiments later, such as mouse antibodies.

Claims (10)

1. A recombinant expression vector comprising a polynucleotide encoding the C-terminal domain of a seven-growth VLRB protein linked to the 3'-terminus of a gene encoding a murine tail-derived variable lymphocyte receptor B (VLRB) protein having a hydrophobic tail domain removed.
2. According to claim 1, wherein the hydrophobic tail domain-derived eel-derived VLRB protein from the N- terminal to the C- terminal signal peptide (SP), N-terminal capped LRR (LRRNT), leucine-rich repeat (LRR), LRRVs (variable LRR modules), connecting peptide (CP), C-terminal capped LRR (LRRCT) and recombinant expression vector, characterized in that consisting of the Stalk domain.
3. The recombinant expression vector according to claim 2, wherein the fish-derived VLRB protein from which the hydrophobic tail domain has been removed has a signal peptide (SP) murine immunoglobulin κ chain leader sequence.
4. A host cell transformed with the recombinant expression vector of claim 1.
5. A fusion protein linked to a C-terminal domain of an eel-derived variable lymphocyte receptor B (VLRB) protein from a hydrophobic tail domain and a CLR-derived VLRB protein produced by the host cell of claim 4.
6. The multivalent antibody having increased binding ability to a target antigen, characterized in that the fusion protein of claim 5 is composed of an octahedron or a dimer multimer by self-assembly.
7. (a) transforming a host cell with the recombinant expression vector of claim 1;

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

   (c) obtaining a multimer of an octahedron or a dimer of a fusion protein from the host cell or the culture thereof cultured in the step (b), wherein the multivalent antibody having increased binding ability to the target antigen is Manufacturing method.
8. A multivalent antibody having increased binding capacity to a target antigen produced by the method of claim 7.
9. A method of detecting a target antigen by treating a multivalent antibody of claim 6 or 8 with a suspected sample containing a target antigen.
10. A composition for detecting a target antigen, comprising the multivalent antibody of claim 6 or 8 as an active ingredient.

Images