KR101972899B1 - Fusion protein comprising human C4bp oligomerization domain linked to hagfish VLRB protein with deleted hydrophobic tail domain and uses thereof - Google Patents

Abstract

The present invention includes a polynucleotide encoding a human-derived C4b-binding protein oligomerization domain linked to the 3 ' -terminal of a gene encoding a wild-type VLRB (variable lymphocyte receptor B) protein from which a hydrophobic tail domain has been removed A recombinant expression vector, a host cell transformed with the recombinant expression vector, a fusion protein in which the humanized C4bp oligomerization domain is linked to the VLRB protein derived from the horseshoe, from which the hydrophobic tail domain has been removed, produced by the host cell, The present invention relates to a polyvalent antibody having increased binding ability to a target antigen. The fusion antibody derived from a falconer of the present invention can be usefully used for the development and diagnosis of a biomarker.

Description

A fusion protein comprising a C4bp oligomerization domain and a VLRB protein derived from a hippocampus from which a hydrophobic tail domain is removed, and a use thereof, and a fusion protein comprising the C4bp oligomerization domain and a hydrophobic tail domain.

The present invention is characterized in that a fusion protein in which a hydrophobic tail domain-deleted VLRB (variable lymphocyte receptor B) protein is linked to a human-derived C4b-binding protein oligomerization domain and that the fusion protein is composed of a multimer by self assembly To multivalent antibodies with increased binding to the target antigen.

Since the introduction of monoclonal antibody production technology in the 1970s, mammalian antibodies have grown in vast quantities around the world in the areas of biomarker development, disease diagnosis and treatment. In recent years, the market for antibody-drug conjugates is growing very rapidly and the market size is expected to reach $ 396 million in 2013 and $ 3 trillion in 2018. However, the field of new antibody candidates has already been monopolized by some advanced countries. Domestic companies are trying to reduce costs by developing improved antibodies and developing biosimilar antibody drugs rather than finding new antibody candidates. Common antibodies, which are mainly derived from mice, have been consistently pointed out problems such as limitations of epitopes, limitations of binding area, relatively large molecular weights, and costly overheads of complex production processes. In order to solve these problems, studies on the development of various types of antibodies and artificial antibodies have been conducted. However, since there are no economically and commercially competitive candidates yet, The development of more innovative alternative antibodies is urgent.

Recently, the possibility of developing a new antibody which shows much higher antigen binding ability than mouse antibody using the acquired immune system of hagfish or lamprey has been suggested through research. Eagle and Seven Eel are the only living beings of the evolutionarily the lowest vertebrate, the Jawless vertebrate. Recently, it has been shown that their innate immune and acquired immunity are composed of mechanisms similar to Jawed vertebrate. Three different types of VLR (variable lymphocyte receptors) -A, B and C from their lymphocyte-like cells Found. VLR-A and VLR-C play a role as T-lymphocytes in mammals, and VLR-B acts as a B-lymphocyte type secreted from lymphocyte-like cells. In particular, VLRB proteins, which play a role in the role of antibodies in mammals, are differentiated from mammalian immunoglobulins by inducing diversity through gene assembly, but they are expressed as a single polypeptide with a much simpler structure, 10 It is possible to form more than ¹⁴ combinations, and it is more stable to the change of surrounding environment such as pH and temperature. Therefore, it is predicted that using the acquired immune system of Eagle can produce a customized antibody against a specific antigen at a lower cost, easily, and rapidly, and thus become a competitive competitive alternative antibody candidate against the existing antibody market.

An important factor in protein detection technology based on antigen-antibody binding is to maintain the specific binding between the antibody protein and the target antigen stably with high binding force. The most commonly used mouse antibody, for example, has a Y-type structure with two binding sites for antigen. On the other hand, the udder's antibody has a U-shaped horseshoe structure in the case of a monolayer and does not act as an antibody in a monolayer. In the case of Chiloong eel, it has a pentagonal multimeric structure similar to the IgM form of mammalian mammals. In the case of the wild-type VLRB antibody found in previous studies of the present inventors, about 20 to 30 monoliths exhibit a spherical shape in a spherical form having a specific shape. However, in the case of the spermatozoa of this spherical form, the expression rate is significantly reduced due to the high hydrophobic part located at the C-terminus of the wild type VLRB when it is expressed in an artificial environment such as the human cell line HEK 293 Unlike the naturally occurring horseshoe IgG, it has a disadvantage in that it is difficult to investigate the binding ability with the antigen by ELISA or immunoblotting.

Accordingly, in the present invention, the C-terminal region gene that determines the three-dimensional structure is removed while maintaining the site capable of binding to the antigen in the Vera Antibody VLRB protein, and a peptide domain To give a higher expression rate and a higher antigen binding capacity.

US Patent No. 9243047 discloses' Influenza nucleoprotein vaccines' that link virus antigens to the C4bp oligomerization domain of chickens. Korean Patent Publication No. 2016-0147787 discloses' humanized variable lymphocyte receptor (VLR) Related compositions and uses' have been disclosed. However, there is no description about a fusion protein in which the hydrophobic tail domain of the present invention is linked to the C4bp oligomerization domain, and a polyvalent antibody consisting of a multimer of the fusion protein.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a gene encoding the VLRB protein of the egg yolk antibody, which retains the binding site with the antigen and deletes the hydrophobic tail domain at the C- The oligomerization domain of C4bp was ligated to produce a fusion protein. As a result of transforming the host cell with a recombinant vector containing the nucleic acid encoding the fusion protein, it was confirmed that the fusion protein of the present invention was formed into a multimer of the 7-complex in the host cell, The present inventors have completed the present invention by confirming that the antibody has a significantly higher binding capacity to a target antigen than a monomelicoma antibody having the same antigen recognition site.

In order to solve the above problems, the present invention provides a human-derived C4b-binding protein oligomerization domain linked to the 3'-end of a gene coding for a wild-type VLRB (variable lymphocyte receptor B) protein from which a hydrophobic tail domain has been removed Lt; / RTI > polynucleotide encoding a recombinant expression vector.

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

The present invention also provides a fused protein produced by the host cell, wherein the humanized C4bp oligomerization domain is linked to the VLRB protein derived from the horseshoe, from which the hydrophobic tail domain has been removed.

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

In addition, the present invention provides a method for preparing a polyvalent antibody having increased binding affinity to a target antigen, comprising culturing the transformed host cell to obtain a fusion of a fusion protein.

In addition, the present invention provides a multivalent antibody with increased binding affinity to a target antigen produced by the method.

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

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

The multivalent antibody consisting of a multimer of a fused protein in which the hydrophobic tail domain of the present invention is linked to the VLRB protein derived from the horsetail-derived C4bp oligomerization domain has seven binding sites for the antigen, thereby maximizing the antigen-antibody binding ability, The removal of the tail domain increases the expression rate in the host cell and also increases the stability of the antibody itself. Unlike the mouse antibody, the feline antibody is composed of a single peptide and is free of genetic manipulation. Therefore, the fused antibody of the present invention may be usefully used for the development and diagnosis of biomarkers.

Figure 1 shows the structure of the wild-type VLRB protein of Eagle. SP, signal peptide; LRR, leucine-rich repeat; LRRNT, N-terminal capped LRR; LRRVs, variable LRR modules; CP, a connecting peptide; LRRCT, C-terminal capped LRR; HC, hydrophobic tail domain. A box in the amino acid sequence means a hydrophobic amino acid.
Fig. 2 is a schematic of the development of a vector system for producing the fusion protein of the present invention. Fig. 2 shows a pSHepta vector obtained by cloning a human-derived C4bp oligomerization domain.
Fig. 3 is a schematic of the development of a vector system for producing the fusion protein of the present invention. Fig. 3 shows the pSHepta / VLRB vector in which the horseshoe VLRB protein having the hydrophobic C-terminal deleted is cloned.
Figure 4 shows the Western blotting results of VLRB clone 74 monolith (m74) in the C4bp oligomerization domain unbonded form and VLRB clone 74 (Shepta / 74) in the C4bp oligomerization domain linked form.
FIG. 5 shows the result of Western blotting of β-mercaptoethanol (2-ME) treated at various concentrations to a sample of VLRB clone 74 (Shepta / 74) in which a C4bp oligomerization domain is linked.
Figure 6 shows the results of western blotting of another randomly selected VLRB-C4bp clone 43 showing the tendency of the polymer to separate into monomers with increasing 2-ME treatment concentration.
7 is a schematic diagram of a process for producing a fusion protein of the present invention that specifically binds to avian influenza virus (AIV).
FIG. 8 shows the binding activity of recombinant VLRB-C4bp to the AIV antigen by ELISA.
Figure 9 shows the results of Western blotting under non-reducing and reducing conditions of selected clones (1-94, 2-61 and 3-43) with AIV antigen-specific binding potency.
FIG. 10 shows the results of ELISA for the ability of the selected clones having the AIV antigen-specific binding ability to bind at different concentrations.
FIG. 11 shows the result of dot blotting of the binding reaction of the selected clones having AIV antigen-specific binding ability to various kinds of antigens. AIV, avian influenza virus; VHSV, viral hemorrhagic sepsis virus; HA, hemagglutinin; HEL, HEL protein.
Figure 12 shows the binding potency of VLRB-C4bp clone 1-94 to various subtypes of AIV through dot blotting. VHSV, viral hemorrhagic sepsis virus; VNNV, viral neurotic virus; Inactivated H9N2, viruses that have been heated with MS96 virus for 5 minutes; MS96 H9N2, VI13 H9N2; ADL H9N2, H9N2 type AIV; H6N2, H6N2 type AIV; VI14 H4N2, H4N2 AIV.
Fig. 13 shows the results of the non-reducing and reducing conditions of the 293-F cell culture supernatant producing the VLRB-C4bp fusion protein (1-94 clone) having AIV-specific binding ability and the mono VRLB protein in which the C4bp domain was deleted from the protein Western blotting results.
Figure 14 shows AIV and VHSV (negative control) of 293-F cell culture supernatants producing VLRB-C4bp fusion protein (1-94 clone) with AIV-specific binding potency and mono VRLB protein with C4bp domain deleted from the protein, The binding ability to the antigen was analyzed by ELISA.

In order to accomplish the object of the present invention, the present invention relates to a C4b-binding oligomerization of human C4bp linked to the 3'-terminal of a gene coding for a wild-type VLRB (variable lymphocyte receptor B) protein from which a hydrophobic tail domain has been removed Lt; RTI ID = 0.0 > polynucleotide < / RTI >

In the recombinant expression vector of the present invention, the horseshoe-derived VLRB protein from which the hydrophobic tail domain is removed has a signal peptide (SP) at the N-terminus, a signal peptide (LRRNT) at the N-terminus capped LRR, leucine-rich repeat (LRR), variable LRR modules, CP (connecting peptide), LRRCT (C-terminal capped LRR), and Stalk domains. The antigen recognition sites, LRRVs and LRRCT Domain and is capable of binding to the target antigen. In other words, the hare-originated VLRB protein of the present invention is a horseshoe-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 coding for the VLRB protein derived from Euphorbia, from which the hydrophobic tail domain has been removed, may be a gene coding for the VLRB protein having the best binding ability to the target antigen , Or from the VLRB cDNA library of hagfish that has been immunized with the target antigen.

In the recombinant expression vector of the present invention, the range of the oligomerization domain of the human-derived C4bp includes a peptide having the amino acid sequence of SEQ ID NO: 1, and functional equivalents thereof. Is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, more preferably 95% or more, Or more homologous to a peptide having substantially the same activity as the peptide of SEQ ID NO: 1. &Quot; Substantially homogenous activity " means an activity of self-assembly to form a multimer of a 7-mer.

Human-derived C4bp (C4b-binding protein) is a protein that acts as a classical complement and a major inhibitor of the lectin pathway. This protein is composed of oligomerization of seven isoforms, and the polymer is cleaved through the internal disulfide bonds of the cysteine (Cys) residues in the amino acid sequence 540-597 of the C-terminus of each homologous protein There is a characteristic to form.

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, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

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

The expression vector containing the gene coding for the wild-type VLRB protein from which the hydrophobic tail domain of the present invention has been removed, the polynucleotide encoding the human-derived C4bp oligonucleotide domain and the appropriate transcription / translation regulation signal is constructed by a method known in the art . Such methods include in vitro recombinant DNA technology, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be effectively linked to appropriate promoters in the expression vector to drive mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

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

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

As a host cell capable of continuously cloning and expressing the vector of the present invention in a stable and prokaryotic cell, any host cell known in the art may be used, and examples thereof include Escherichia coli Rosetta, Escherichia coli JM109, Escherichia coli BL21, RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli Dα, E. coli W3110, Bacillus subtilis (Bacillus subtilis), Bacillus Chuo ringen cis Bacillus strain, Salmonella typhimurium (Salmonella typhimurium), such as (Bacillus thuringiensis), Serratia marcescens and various enterococci such as Pseudomonas species and strains.

When the vector of the present invention is transformed into eukaryotic cells, yeast ( Saccharomyce cerevisiae ), insect cells, human cells and animal cells (for example, HEK (Human embryonic kidney) 293, CHO (Chinese hamster ovary) , W138, BHK, COS-7, HepG2, 3T3, RIN and MDCK cell lines) and plant cells.

The present invention also provides a fusion protein produced by the host cell, wherein the humanized C4bp oligomerization domain is linked to the VLRB protein derived from the horseshoe, from which the hydrophobic tail domain has been removed.

The fusion protein according to the present invention is a fusion protein in which the N-terminal of the oligomerization domain of human-derived C4bp is linked to the C-terminus of the stalk domain of the Eagle's VLRB protein.

The present invention also provides a multivalent antibody with increased binding affinity to a target antigen, characterized in that the fusion protein consists of a multimer of heptamer by self-assembly. The multivalent antibody of the present invention is composed of a multimerized form of a fusion protein in which the VLRB protein derived from Eagle's follicle and the oligomerization domain of human-derived C4bp are linked to each other while the hydrophobic tail domain is maintained while retaining the binding ability to the target antigen. The oligomerization domain of C4bp is located at the center, and the VLRB protein derived from Euphorbia is protruded to the outside.

The term multimer or polymer as used herein can be used interchangeably.

The multivalent antibody of the present invention contains seven VLRB proteins, which are ubiquitous antibodies, and is a polyvalent antibody having a significantly increased antigen binding ability.

The present invention also relates to

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

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

(c) obtaining a heptamer of the fusion protein from the host cell cultured in step (b) or a culture thereof; and (c) culturing the multivalent antibody having increased binding ability to the target antigen, to provide.

Specifically, the method for producing the multivalent antibody of the present invention comprises immunizing a mouse with an immunogen or an antigen, isolating lymphocytes by collecting the blood of the immunized eel, extracting the total RNA of the lymphocytes, and extracting mature VLRBs After securing mRNA, a polymerase chain reaction was performed using this as a template to prepare a cDNA pool of VLRBs excluding the C-terminal hydrophobic region. Each clone of the prepared cDNA was cloned into a pSHepta vector containing a C4bp oligomerization domain base sequence to produce a recombinant expression capable of expressing a fusion protein in which VLRBs having different antigen recognition sequences and a human C4bp oligomerization domain were linked Vector (FIG. 3). Then, 293-F cells were transformed with each of the recombinant expression vectors, and the transformed 293-F cells were cultured. Then, a culture solution was obtained and the reactivity with the immunogen or antigen was confirmed by ELISA. Finally, clones with excellent binding strength to immunogens or antigens were selected and Western blotting of the cultures of the selected clones was carried out under non-reducing and reducing conditions to confirm that multimer was formed. Immunogens or antigens can be used in various kinds of viruses, microorganisms, proteins and the like.

The present invention also provides an increased antibody binding to the target antigen produced by the method.

The multivalent antibody of the present invention is composed of a multimer of a seven-membered fusion protein in which the VLRB protein derived from Eagle's follicle and the oligomerization domain of human-derived C4bp are linked while the binding ability to the target antigen is maintained, , Which is a polyvalent antibody with a significantly increased antigen binding ability.

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

In the target antigen detection method of the present invention, the sample may be a solution, a tissue, a cell, a blood, a serum, a plasma, a saliva, etc., including, but not limited to, food, water, and specific or unspecified microorganisms.

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

Detection of the conjugate of the multivalent antibody of the present invention and the target antigen can be performed by an indirect enzyme linked immunosorbent assay (ELISA) or a sandwich ELISA method, but is not limited thereto. Determination of the activity or signal of the final enzyme in the indirect ELISA method and the sandwich-ELISA method can be carried out according to various methods known in the art. Detection of such a signal enables qualitative or quantitative analysis of the polyvalent antibody of the present invention.

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

The composition for detecting target antigens of the present invention comprises a multivalent antibody consisting of a multimerized form of a fusion protein in which the uberoid-derived VLRB protein in which the hydrophobic tail domain is removed and the oligomerization domain of human-derived C4bp are linked, while retaining the binding ability to the target antigen The kit for immunoassay includes a direct-ELISA, an indirect-ELISA, a sandwich, and an immunoassay kit. The immunoassay kit according to the present invention is not limited to the kit, -ELISA, protein microarray, radioimmunoassay (RIA), and the like.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1. Preparation of fusion protein of human VLRB protein and human-derived C4bp oligomerization domain

The wild-type variable lymphocyte receptor B (VLRB) protein of the hagfish contains a strong hydrophobic region at the C-terminus. Therefore, when the recombinant protein is expressed through the human-derived cell line HEK 293-F, There is a problem that it is significantly reduced. Thus, in the present invention, a fusion protein is prepared by linking the oligomerization domain of human-derived C4bp capable of eliminating a strong hydrophobic region of the hagfish VLRB protein and capable of forming a multimer, and confirming whether the fusion protein produced forms a multimer .

First, the primers shown in Table 1 below were prepared to synthesize the oligomerization domain of the human-derived C4bp gene (GenBank ID: 416733, amino acids 540 to 597).

PCR primer set for C4bp gene synthesis Primer name The base sequence (5 '- > 3') C4bp Spe I_2 Sfi I Fwd cacactagtGGCCACCGGGGCCAATAATTGGCCTCTGGGGCCcaggtagttgggagacccccgaaggctgtg (SEQ ID NO: 2) C4bp con Rev tcctctgggtttgggagacactgcatgagtcttttgcctgtgagcacttgttcacagccttcgggggtctcc (SEQ ID NO: 3) C4bp con Fwd tgtctcccaaacccagaggatgtgaaaatggccctggaggtatataagctgtctctggaaattgaacaactg (SEQ ID NO: 4) C4bp con stop Rev ttatagttctttatccaaagtggattgtcttgcgctgtctctctgtagttccagttgttcaatttccagag (SEQ ID NO: 5)

PCR was carried out using C4bp Spe I_2 Sfi I Fwd (SEQ ID NO: 2) and C4bp con Rev (SEQ ID NO: 3), C4bp con Fwd (SEQ ID NO: 4) and C4bp con stop Rev ), the PCR products were mixed, again C4bp Spe I_2 Sfi I Fwd (SEQ ID NO: 2) and after performing a C4bp con stop Rev (SEQ ID NO: 5) was carried out the second PCR with primer set. After obtaining PCR products of the final degree of 220 bp, hydrolysis with Spe I and purchased pTracer EF / A vector (Invitrogen, USA), recombinant by DNA sequencing after insert cloned into the Spe I / Pme I site The vector was selected to construct a pSHepta vector (Fig. 2). Total RNA was randomly obtained from Eagle's lymphocytes and amplified from the signal peptide (SP) to stalk in the horseshoe antibody VLRB protein as a template. A randomly selected one clone (VLRB 74) of the amplification product, and hydrolysis with Sfi I and then, by cloning the Sfi I site of the production cost pSHepta vector was produced finally pSHepta / 74 vector (Figure 3) . The recombinant vector expresses a fusion protein in which the C-terminal hydrophobic region (HC) of the VLRB gene is deleted and the C4bp oligomerization domain is bound, and the VLRB polymer protein of the 7-combination is finally expected to be produced by the oligomerization domain . The constructed pSHepta / 74 was transformed into HEK 293-F human cell line (Gibco, USA) with Lipofectamine 2000 (Invitrogen) for 4 hours and regenerated in the expression medium. After 72 hours of transformation, the culture supernatant was recovered and Western blotting was performed.

As a result, as shown in FIG. 4, the culture solution (m74) of the cells transformed with the recombinant vector expressing only the VLRB protein to which the C4bp oligomerization domain was not bound was cultured in a non- The reduction condition and the reduction condition treated with 1% of 2-ME showed that the VLRB protein was secreted into the extracellular form in a monolithic form. On the other hand, the culture medium (SHepta / 74) transformed with the recombinant vector expressing the VLRB fusion protein in which the C4bp oligomerization domain is connected has a bulk of 300-400 kDa in the non- bulky protein form, and it was confirmed to be degraded into a monolithic form under reducing conditions treated with 1% 2-ME. In order to investigate more closely, it was confirmed that when 2-ME was treated by concentration, it was decomposed into a unit form in the polymer (7 combination) depending on the 2-ME treatment concentration (FIG. 5). In addition, 74 clones as well as randomly selected 43 clones were screened for the complete form of VLRB-2 in the non-reducing conditions without 2-ME treatment, the partial reduction with 0.05% 2-ME and the reduction with 5% A C4bp polymer (7 combination), a sequentially degraded form in the form of a ladder, and a band in the form of a complete monolith were observed (Fig. 6).

Example 2 Preparation of avian influenza virus-specific fusion protein and analysis of antigen binding ability of fusion antibody

In order to produce the target antigen-specific horseshoe VLRB antibody, the wild-type avian influenza virus (H9N2 type) was immunized 4 times at 2-week intervals and the lymphocytes were recovered to extract the total RNA and prepare the hatchlings VLRB cDNA library 7). Each VLRB cDNA was cloned into the pSHepta vector prepared above (pSHepta / VLRB cDNA library). Approximately 1,000 plasmid DNAs expressing different VLRB clones were transformed into HEK 293-F cells. After 72 hours of transformation, the supernatant of the cell culture was recovered and reacted with 96-well plates coated with wild-type avian influenza virus (H9N2 type) particles (200 ng / well) to perform enzyme-linked immunosorbent assay .

ELISA analysis showed that the VLRB clone with different antigen binding sites and the oligomerization domain of C4bp were fused to the 96-well plate treated with the culture supernatant of the transfected cells containing the fusion protein in the form of a polymer, Mouse 11G5 antibody recognizing the stalk region and anti-mouse IgG antibody conjugated with HRP were sequentially treated with TMB / H ₂ O ₂ , and the absorbance was measured.

As a result, VLRB-C4bp clones selected as having excellent antigen-binding ability through avian influenza virus-specific ELISA experiments were 1-94, 2-79, 2-61, 3-43, 3-89 and 6-62 clones 8). These clones did not show any reactivity to the Viral Hemorrhagic Septicemia Virus (VHSV) (Fig. 11). The clones that showed relatively high reactivity to avian influenza viruses were clones 1-94, 2-61 and 3-43, while the remainder showed low reactivity to the same antigen. To confirm that the fusion proteins of the selected 1-94, 2-61, 3-43 clones formed polymers due to the C4bp oligomerization domain, cultivation of HEK 293-F cell line transformed with the plasmid DNA recovered from each clone The supernatant was recovered and subjected to SDS-PAGE under conditions in which 2-ME was treated or untreated, and western blotting was performed using an 11G5 antibody capable of recognizing the stalk of hagfish VLRB. As a result, it was confirmed that when the 2-ME was not treated (non-reducing condition) in all three clones as observed in Fig. 9, the polymer was successfully secreted. The number of LRRV modules in the clones was found to be 5, 4, and 5, respectively, in the 1-94, 2-61, and 3-43 clones. The culture supernatant recovered from 293-F cells transformed with plasmid DNA recovered from clones 1-94, 2-61, 2-79, 3-43, 3-89 and 6-62 was treated with 5 As a result of observing the specific reactivity with avian influenza virus by diluting with 25, 125 and 125 times, it was observed that the binding ability was increased depending on the concentration of culture supernatant as shown in Fig.

The binding ability of the selected falcatellar antibodies was again verified by dot blotting. First, virus (AIV 200 ng, VHSV 200 ng) and a single protein (HA 20 ng, HEL 20 ng) were coated on the PVDF membrane, respectively. (HA), which is well known as the main envelope protein of avian influenza virus (Fig. 11). While the VLR18 clone used as a negative control did not show any reactivity to all antigens, each of the selected fusion antibodies showed reactivity to avian influenza virus as well as ELISA results. However, it did not show any reactivity with hemagglutinin. In addition, it was confirmed that the specific binding ability to the MS96 (H9N2 type) strain injected into the antigen using the 1-94 clone showing the highest reactivity among the selected antibodies is shown (FIG. 12). Specifically, the 1-94 clone showed no reactivity to the H9N2 avian influenza virus boiled for 5 minutes. Thus, we could confirm that the selected frying pan conjugate antibody binds to the surface of avian influenza virus but does not bind to the denatured virus.

Example 3. Analysis of antigen binding ability of VLRB monolith and VLRB-C4bp polymer

The present inventors sought to analyze the difference in antigen binding ability between the ubiquitin-derived antibody VLRB protein monoclonal antibody having the same antigen binding site and oligomerization domain-linked polymer. First, only the VLRB portion of the VLRB-C4bp 1-94 clone showing the best binding ability to the AIV antigen in Example 2 was recloned. VLRB monomers (mono VLRB 1-94) and VLRB-C4bp 1-94 clones deficient in the oligomerization domain expressed respectively in 293-F cells were subjected to Western blotting under reducing and non-reducing conditions to confirm whether or not the polymer was formed 13) and the ability to bind to wild-type avian influenza virus (H9N2 type) by ELISA showed that the polymer-type VLRB-C4bp 1-94 fusion antibody significantly improved antigen-specific binding (Fig. 14).

From the above results, it was found that although the binding ability to the target antigen was low in the monomorphic form, the binding ability to the target antigen could be increased several tens times or more when it was fused with the oligomerization domain and formed into the polymer form.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY          IMMUCELL CO., LTD. <120> Fusion protein comprising human C4bp oligomerization domain          linked to hagfish VLRB protein with deleted hydrophobic tail          domain and uses thereof <130> PN17035 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 58 <212> PRT <213> Homo sapiens <400> 1 Trp Glu Thr Pro Glu Gly Cys Glu Gln Val Leu Thr Gly Lys Arg Leu   1 5 10 15 Met Gln Cys Leu Pro Asn Pro Glu Asp Val Lys Met Ala Leu Glu Val              20 25 30 Tyr Lys Leu Ser Leu Glu Ile Glu Gln Leu Glu Leu Gln Arg Asp Ser          35 40 45 Ala Arg Gln Ser Thr Leu Asp Lys Glu Leu      50 55 <210> 2 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 cacactagtg gccaccgggg ccaataattg gcctctggg cccaggtagt tgggagaccc 60 ccgaaggctg tg 72 <210> 3 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tcctctgggt ttgggagaca ctgcatgagt cttttgcctg tgagcacttg ttcacagcct 60 tcgggggtct cc 72 <210> 4 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tgtctcccaa acccagagga tgtgaaaatg gccctggagg tatataagct gtctctggaa 60 attgaacaac tg 72 <210> 5 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ttatagttct ttatccaaag tggattgtct tgcgctgtct ctctgtagtt ccagttgttc 60 aatttccaga g 71

Claims (9)

A recombinant expression vector comprising a polynucleotide encoding a human-derived C4b-binding protein oligomerization domain linked to the 3'-end of a gene encoding a horseshoe-derived variable lymphocyte receptor B (VLRB) protein from which the hydrophobic tail domain has been removed . 2. The method of claim 1, wherein the VRRB protein from which the hydrophobic tail domain is removed comprises a signal peptide (SP), an N-terminal capped LRR, an LRR (leucine-rich repeat) (variable LRR modules), a connecting peptide (CP), a C-terminal capped LRR (LRRC), and a Stalk domain. A host cell transformed with the recombinant expression vector of claim 1. A fusion protein produced by the host cell of claim 3, wherein the hydrophobic tail domain-deleted horsetail-derived variable lymphocyte receptor B (VLRB) protein and the human-derived C4b-binding protein oligomerization domain are linked. A multivalent antibody having increased binding affinity to a target antigen, characterized in that the fusion protein of claim 4 is made by self assembly of a multimer of heptamer. (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 heptamer of the fusion protein from the host cell cultured in step (b) or a culture thereof; and (c) obtaining a heptamer of the fusion protein from the host cell cultured in step (b) or a culture thereof. An antibody of increased binding to a target antigen produced by the method of claim 6. A method for detecting a target antigen by treating the multivalent antibody of claim 5 or 7 with a suspected sample containing a target antigen. A composition for detecting a target antigen comprising the polyvalent antibody of claim 5 or 7 as an active ingredient.

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