KR20180114859A - Method for preparing polymerized antigen protein with high immunogenicity and Plant for implementing the same - Google Patents

Abstract

The present invention relates to a method for preparing polymerized antigen protein with high immunogenicity and plant for the same. More specifically, the present invention relates to a method for preparing polymerized antigen protein with immunogenicity and plant for the same wherein the method comprises: step (a) of preparing transgenic plant expressing a chimeric antigen comprising an immune response domain, comprising antigenic protein, a target binding domain, comprising Fc antibody fragment, and a polymerization domain, comprising a tailpiece derived from IgM or IgA; and step (b) of obtaining protein from the transgenic plant. By the method comprising step (a) and step (b), a massive amount of target protein can be produced in a safe and economic manner. The polymerized antigen protein with immunogenicity prepared by the method of the present invention has a large four-dimensional structure and thus has an excellent immunity boosting effect, and has excellent antibody generation capability in a host animal even without the use of an immune adjuvant. Therefore, the probability of industrial use of the polymerized antigen protein with immunogenicity is high.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for preparing a highly immunogenic multimeric antigen protein,

The present invention relates to a method for producing a high immunogenicity multimeric antigen protein and a plant for the same, and more particularly, to a method for producing a high immunogenicity multimeric antigen protein, comprising the steps of (a) A chimeric antigen comprising a target binding domain comprising a Fc antibody fragment and a polymerization domain comprising an IgM or IgA-derived tailpiece, Producing a transgenic plant; And (b) obtaining a protein from the transgenic plant, and a method for producing the immunogenic multimeric antigen protein, and a plant therefor.

Vaccines are drugs used to generate immune responses to antigens in order to protect against pathogenic infections. Recently developed vaccines mainly use recombinant proteins as antigens. The recombinant protein is less toxic and safe than the live vaccine or live attenuated vaccine but has low immunogenicity, so immunoadjuvants are used together to produce sufficient immunity to the infection. An immunosuppressant is a vaccine additive that can stimulate an immune response to a vaccine antigen without inducing a specific antigen-antibody immune response, thereby leading to enhanced immunity. And the 'adjuvare' with.

Immunostigents currently approved in Europe and USA for use in vaccines include aluminum salts, MF59, AS03 and AS04. Aluminum salt, developed in 1926 as an adjuvant for the diphtheria toxoid vaccine, is now the most widely used immunosuppressant and has been used almost exclusively in human vaccines over the last 80 years. Aluminum salts are widely used in various vaccines and are thought to be very safe, but they are believed to cause allergic reactions and also have neurotoxicity. In addition, the antibody - mediated humoral immune response is strongly induced, but the cellular immune response is hardly induced, and there is a disadvantage that cryopreservation is impossible. As described above, adjuvant is used for vaccination (or vaccination), which causes various side effects such as autism spectrum disorders (ASD) and allergy There is a problem that needs to be addressed for adjuvant free vaccines.

On the other hand, protein therapeutics have recently emerged as an important class of highly effective drugs and Fc-based fusion proteins (Fc-fusions) in which the Fc domain of the IgG isotype antibody (Ab) is bound to other proteins are the most promising . The Fc region of Ab is an antigen (Ag) transporter in an adjuvant for protective immunity due to its ability to activate specific Fc [gamma] R targeting activation and / or inhibitory signal transduction pathways that set thresholds for balanced immunoreaction Are being studied more and more. However, the existing approach of transferring Ag to immune cells using Fc has the disadvantage that it can not cross-link with a large number of Fc [gamma] Rs due to the monomer structure. That is, in improving cell signaling, But it can not do so. Most Fc [gamma] Rs (except Fc [gamma] RI) can only interact with high binding forces to Ab, thereby providing the immune complex as an immune complex (antigen-antibody complex, IC). Thus, although attempts have been made to polymerize Fc-fusions fused with Ag and Fc regions, they have not been substantially accomplished. According to David NA Mekhaiel et al., (2011) K1 animal cells, the polymeric Fc-fusions were significantly less immunogenic than their dimeric or monomeric counterparts, indicating that the important Fc-receptors (1999)). In addition, it has been reported that the ability to interact is reduced (David NA Mekhaiel et al., Polymeric human Fc-fusion proteins with modified effector functions, Scientific Reports 1, Article number: 124 (2011)). As described above, when the Ag-linked Fc-fusions are polymerized, immunogenicity is lower than that of their monomers.

In addition, as the protein industry grows, recombinant proteins are being produced in a variety of expression systems depending on the purpose of use and production scale. There are various types of expression systems according to the biological host. Typical examples include microorganisms such as Escherichia coli and yeast, animal cells, and the like. In the case of glycoproteins, animal cells, which are highly eukaryotic cells, are used as host cells. However, in the case of animal cells, most of the non-glycoproteins are used as host cells because of the disadvantage of high production cost, low yield of protein production and difficult cultivation. In particular, the E. coli expression system is widely known as an easy and economical method of gene manipulation. However, when it is intended to produce proteins derived from eukaryotic cells such as cytokines, post-translational modifications such as glycosylation are impossible, and secretion of proteins into the culture medium is difficult. In many proteins, disulfide bonds disulfide bond is not properly folded and thus it is produced in the form of an insoluble protein such as an inclusion body and is not active.

Therefore, the present inventors investigated the production of an adjuvant free vaccine. When the Ag-binding Fc-fusion was induced to express the endoplasmic reticulum and expressed in plants, the Fc-fusion The present inventors have confirmed that they are not only expressed in multimers forming a macromolecular structure but also cause a high immune response without an adjuvant, thus completing the present invention.

Accordingly, it is an object of the present invention to provide a kit for the detection and quantification of (a) an immune response domain comprising an antigenic protein, a target binding domain comprising an antibody Fc fragment (Fc antibody fragment) Preparing a transgenic plant expressing a chimeric antigen comprising a polymerisation domain comprising a tailpiece; And (b) obtaining a protein from the transgenic plant, an immunogenic multimeric antigen protein prepared by the method, and a vaccine composition comprising the same.

Another object of the present invention is to provide an immunoassay kit comprising an immune response domain comprising an antigenic protein, a target binding domain comprising an antibody Fc fragment (Fc antibody fragment), and an IgM or IgA-derived tailpiece A method for producing a transgenic plant producing an immunogenic multimeric antigen protein comprising transforming a plant cell with a recombinant vector constructed so as to express a chimeric antigen, Plants.

(A) an immune response domain comprising an antigenic protein; a target binding domain comprising an Fc fragment of an antibody; Producing a transgenic plant expressing a chimeric antigen comprising a polymerisation domain comprising an IgM or an IgA-derived tailpiece; And (b) obtaining a protein from the transgenic plant, an immunogenic multimeric antigen protein prepared by the method, and a vaccine composition comprising the same.

In order to accomplish another object of the present invention, the present invention provides an immune response domain comprising an antigenic protein, a target binding domain comprising an antibody Fc fragment (Fc antibody fragment), and an IgM Or a chimeric antigen comprising an IgA-derived tailpiece, in a plant cell, comprising the steps of: (a) preparing a transgenic plant that produces an immunogenic multimeric antigen protein comprising a recombinant vector constructed to express a chimeric antigen comprising an IgA-derived tailpiece And a plant produced by the method.

Hereinafter, the present invention will be described in detail.

(A) an immune response domain comprising an antigenic protein, an antibody Fc  A target binding domain comprising a fragment (Fc antibody fragment), and IgM  or IgA  Comprising a tailpiece < RTI ID = 0.0 > Massification  The term " polymer " Chimeric  Preparing a transgenic plant expressing a chimeric antigen; And

(b) obtaining a protein from the transgenic plant, wherein the immunogenicity Multimeric  A method for producing an antigen protein is provided.

In step (a), a transgenic plant expressing a chimeric antigen constituting the sequence so as to be expressed by a method unique to the present invention is prepared.

The term " chimeric antigen " in the present invention refers to any sequence (hereinafter, referred to as " chimeric antigen ") for inducing plant-specific multimerization with a backbone of a fusion of an antigen and an antibody Fc fragment (I) to (iii): (i) an antigenic protein comprising an antigenic protein, and (ii) an antigenic protein, A target binding domain (TBD) comprising an immune response domain (IRD), (ii) an Fc antibody fragment, and (iii) a tailpiece derived from IgM or IgA. A polymerization domain (PD).

The immune response domain (IRD) in (i) above includes a part or all of the antigenic protein that induces an actual immune response, namely humoral and / or cellular immunity (T cell response) Quot; The antigenic protein refers to an antigenic substance of the polypeptide or protein class.

The term ' antigen ' in the present invention refers to a molecule that induces a sensitive and / or immunoreactive state as it enters contact with the appropriate cells and is capable of binding to immune cells and / or antibodies of such sensitized subjects in vivo or in vitro, It refers to all substances that react. The term " antigen " in the present invention can be collectively used in the same sense as the term " immunogen ", and preferably promotes the host immune system to cause secretion, humoral and / Quot; means a molecule comprising one or more epitopes that can be made. The term "antigenic" or "immunogenicity" as used herein means the property of the antigen or immunogen, and refers to a property causing secretion, humoral and / or cellular immune response.

The term 'immune response' is a biological phenomenon that exists in the animal body and is a biological phenomenon that distinguishes various substances or living organisms invading from the outside from oneself and removes this intruder. These surveillance systems for self defense are largely accomplished by two mechanisms: humoral immunity and cellular immunity. Humoral immunity is mediated by antibodies present in the serum. Antibodies have an important function in binding and intercepting invading external antigenic substances. On the other hand, cellular immunity is formed by several kinds of cells belonging to the lymphatic system, and these cells are responsible for directly destroying the invading cells or tissues. Thus, humoral immunity is mainly effective against external substances such as bacteria, viruses, proteins, and complex carbohydrates existing outside the cell, and cellular immunity exerts its functions in various parasites, tissues, intracellular infections, cancer cells and the like. These double defense systems are mainly performed by two kinds of lymphocytes such as B cells and T cells. B cells produce antibodies and T cells participate in cellular immunity. The immune response by these B cells or T cells is an immune system that reacts to the intracellular antigens but acts only when the same antigen is present or repeatedly invades. Thus, such an immune response is a specific response to a particular antigen. In addition to these antigen-specific immune responses, there is also a kind of natural immune response that directly attacks and destroys attack cells even if the body has no exposure to any antigen. Such immune responses include neutrophil, macrophage, natural killer (NK) Is involved, and it is characterized by exhibiting various functions without being restricted to the kinds of cells to be attacked.

The term 'epitope' refers to the simplest form of the epitope on the complex antigen molecule, which is a specific part of the antigen recognized by the antibody or T cell receptor.

The antigen of the present invention includes, but is not limited to, polypeptides or proteins, non-protein molecules, and fragments thereof. Preferably, an antigen of the invention refers to a polypeptide or protein and fragments thereof.

The antigens of the present invention may be immunogenic substances known to those skilled in the art and include, but are not limited to, bacterial antigens or epitopes, fungal antigens or epitopes, plant antigens or epitopes, filamentous antigens or epitopes, viral antigens or epitopes, (Cancer) cell antigen or epitope, a toxin antigen or epitope, a chemical antigen or epitope and an autoantigen or epitope.

The antigen of the present invention may preferably be a tumor-associated antigen. The tumor-associated antigen is not limited to a tumor (or cancer) -associated antigen known to those skilled in the art. For example, the tumor-associated antigen may be a breast cancer antigen, ovarian cancer antigen, prostate cancer antigen, cervical cancer antigen, pancreatic cancer antigen, lung cancer antigen, , Antigens related to multiple myeloma, antigens associated with non-Hodgkin's lymphoma, antigens associated with chronic lymphocytic leukemia, or colorectal cancer antigens.

More specifically, the tumor-associated antigen is selected from the group consisting of A33; ADAM-9; ALCAM; B1; BAGE; Beta-catenin; CA125; Carboxypeptidase M; CD5; CD19; CD20; CD22; CD23; CD25; CD27; CD28; CD32B; CD36; CD40; CD45; CD46; CD56; CD79a; CD79b; CD103; CD154; CDK4; CEA; CTLA4; Cytokeratin 8; EGF-R; Ephrin receptor; ErbB1; ErbB3; ErbB4; GAGE-1; GAGE-2; GD2; GD3; GM2; gplOO; HER-2 / neu; Human papillomavirus-E6; Human papillomavirus-E7; Integrin alpha-V-beta-6; JAM-3; KID3; KID31; KSA (17-1A); LUCA-2; MAGE-1; MAGE-3; MARCH; MUC-1; MUM-1; N-acetylglucosaminyltransferase; Oncostatin M (oncostatin receptor beta); p15; PIPA; PSA; PSMA; RAAG10; ROR1; SART; sTn; TES7; TNF-a receptor; TNF- [beta] receptor; TNF-y receptor; Transferrin receptors; VEGF receptor, GA733, or prostatic acid phosphatase (PAP).

The antigen of the present invention may preferably be GA733, a prostate cancer cell surface specific protein, or prostate acid phosphatase (PAP).

The GA733 is an epithelial cell adhesion molecule (EpCAM: 17-1A antigen, KSA, EGP40, GA733-2, ks 1-4 and also esa). EpCAM is a surface glycoprotein expressed by simple epithelial cells and tumorous cells derived therefrom. EpCAM molecules appear on the cell surface from healthy tissue, but their expression is upregulated in malignant tissues. EpCAM functions to adhere to epithelial cells in an aligned and highly ordered form (Litvinov, J Cell Biol. 1997, 139, 1337-1348). The GA733 of the present invention may preferably be a polypeptide represented by SEQ ID NO: 1.

The PAP is one of the enzymes that hydrolyze phosphoric acid monoester under acidic conditions and is produced in prostate epithelial cells and is the most abundant glycoprotein in prostate tissue. The PAP of the present invention may preferably be a polypeptide represented by SEQ ID NO: 3.

In the above (ii), the target binding domain (TBD) comprises a CH2 domain and / or a CH3 domain derived from at least one antibody Fc fragment and is capable of binding to an antigen-presenting cell (APC) Quot;

In the present invention, the term 'antibody' is used collectively with 'immunoglobulin' (hereinafter referred to as 'Ig') and is a generic term of proteins involved in bioimmunity by selectively acting on an antigen. The antibody consists of two pairs of light and heavy chains. The light and heavy chains of such antibodies are polypeptides composed of several domains. In whole antibodies, each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region includes the heavy chain constant domains CH1, CH2 and CH3 (antibody classes IgA, IgD and IgG) and optionally the heavy chain constant domain CH4 (antibody classes IgE and IgM). Each light chain comprises a light chain variable domain (VL) and a light chain constant domain (CL). The variable domains VH and VL can be subdivided into more conserved, hypervariable regions called complementarity determining regions (CDRs) scattered within sites called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 arranged at the amino-terminus carboxy-terminal (Janeway, CA, Jr. et al. (2001) Immunobiology., 5th ed., Garland Publishing; and Woof, J., Burton, D., Nat Rev Immunol 4 (2004) 89-99). Two pairs of heavy and light chains (HC / LC) can specifically bind to the same antigen. Thus, the whole antibody is a bivalent, monospecific antibody.

There are five types of mammalian antibody heavy chains designated by the Greek letters alpha, delta, epsilon, gamma and mu (Janeway, C.A., Jr., (2001) Immunobiology. 5th ed., Garland Publishing). The type of heavy chain present defines the class of antibodies; These chains are found in IgA, IgD, IgE, IgG and IgM antibodies, respectively (Rhoades R. A., Pflanzer RG (2002) Human Physiology, 4th ed., Thomson Learning). The distinct heavy chains are different in size and composition; ? and? contain approximately 450 amino acids, while? and? have approximately 550 amino acids. Each heavy chain has two regions, a constant region and a variable region. Constant sites are identical in all antibodies of the same isotype but different in different isotype antibodies. The heavy chain?,? And? Have a constant domain consisting of three constant domains CH1, CH2 and CH3 (in one line), and a hinge site to add flexibility (Woof, J., Burton, D., Nat Rev Immunol 4 2004) 89-99); The heavy chain [mu] and [epsilon] have a constant region consisting of four constant domains CH1, CH2, CH3 and CH4 (Janeway, C.A., Jr., et al., (2001) Immunobiology., 5th ed., Garland Publishing). The variable region of the heavy chain is different for antibodies produced by different B cells but identical for all antibodies produced by single B cells or B cell clones. The variable region of each heavy chain is approximately 110 amino acids in length and consists of a single antibody domain. There are only two types of light chains in mammals called lambda (λ) and kappa (κ). The light chain has two consecutive domains, one constant domain CL and one variable domain VL. The approximate length of the light chain is 211 to 217 amino acids.

Unless otherwise specified in the specification of the present invention, it is understood that IgG is described as a representative basic structure of an antibody.

The Fc fragment of the present invention may be derived from any one selected from the group consisting of IgG, IgA, IgD, IgE and IgM, and may preferably be an IgG-derived Fc fragment. The IgG may again be divided into IgG1, IgG2, IgG3 and IgG4, and the Fc fragment of the present invention may most preferably be an Fc fragment derived from IgG1.

The term " Fc fragment " in the present invention is a fragment obtained when an immunoglobulin (Ig) molecule is broken down into papain, and includes a light chain variable region (VL), a constant region (CL), a heavy chain variable region (VH) 1 (CH1) is removed. That is, the Fc fragment means a dimer of two CH2-CH3 chains, and the two chains form a dimer structure by a disulfide bond. In addition, the Fc fragment may comprise all or part of the hinge region peptide in the heavy chain constant region. It may also be an extended Fc fragment comprising part or all of the heavy chain constant region 1 (CH1) and / or the light chain constant region 1 (CL1), so long as it has substantially equivalent or improved effects to the native form. It may also be a fragment in which some long amino acid sequences corresponding to CH2 and / or CH3 have been removed.

The antibody Fc fragment may also be an antibody Fc fragment that is derived from the same species as the host (subject) to which the molecule or composition containing the chimeric antigen is to be administered, or is heterologous to the host. For example, if the host is a human, the antibody Fc fragment may be derived from a human antibody and the heterologous antibody Fc fragment may be a non-human mammalian animal such as a cow, goat, pig, mouse, rabbit, hamster, rat , Guinea pig or mouse-derived antibody Fc fragment.

The antibody Fc fragment of the present invention is not limited in its kind and amino acid sequence as long as it is an antibody Fc fragment peptide known to a person skilled in the art. For example, it may be a polypeptide represented by SEQ ID NO: 5 (Fc fragment sequence of human IgG1) , Or a polypeptide represented by SEQ ID NO: 7 to which a hinge region is added to the above sequence.

The term " antigen presenting cell " of the present invention refers to an antigen-inducible event that functions predominantly by internalizing an antigen, processing an antigen, and presenting an antigenic epitope to a lymphocyte in the context of a major histocompatibility complex (MHC) ≪ / RTI > The interaction between APC and the antigen is an essential step in the induction of immunity because it allows the lymphocytes to contact and recognize the antigenic molecule and be activated. Exemplary APCs include macrophages, monocytes, Langerhans cells, interdigitated dendritic cells, follicular dendritic cells, and B cells.

The 'target binding domain (TBD)' of the present invention includes CH2 and CH3 domains derived from at least one antibody Fc fragment, and thus can bind to an Fc receptor on APC. The antibody Fc fragment has an Fc receptor binding site and binds to the Fc receptor (such as FcγR) on the APC at the Fc receptor binding site.

The immune response domain (IRD) and the target binding domain (TBD) can be linked directly or indirectly by genetic fusion means. Thus, the chimeric antigen of the present invention includes the use of a linking molecule that links an IRD to a TBD. Illustrative linker molecules include lysine zipper, and biotin / avidin. For example, the other linker that can be used for the chimeric antigen is a peptide sequence. Such peptide linkers are generally about 2 to about 40 amino acids in length (e.g., about 4 to 10 amino acids). Exemplary peptide linkers include the amino acid sequence 'SRPQGGGS'. Other linkers are known in the art and are generally rich in glycine and / or alanine, taking into account the flexibility between the regions they connect.

The chimeric antigens of the invention may be monomeric (i. E. They contain a single unit comprising IRD and TBD) or dimeric. For the dimeric chimeric antigens, see US 8,465,745; US 8,029,803 and Korean Patent 10-1054851 can be referred to.

In the above (iii), a polymerization domain (PD) comprising a tailpiece derived from IgM or IgA comprises at least one of the IgM-derived tail peptide sequence or the IgA-derived tail fragment sequence and is present in the tailpiece Refers to a moiety that forms a disulfide bond between a cysteine residue (cysteine) and induces polymerization or macromolecularization.

The IgM-derived tail peptide sequence is derived from the IgM C-terminus and can induce multimerization. As long as it is a sequence known in the art, the sequence composition is not particularly limited and may be, for example, a peptide represented by SEQ ID NO: 9 .

The IgA-derived tail peptide sequence is derived from the IgA C-terminal and can induce the massification. The sequence composition is not particularly limited as long as it is a sequence known in the art. For example, it may be a peptide represented by SEQ ID NO: 11 .

The chimeric antigens of the present invention preferably comprise (i) an immune response domain (IRD) comprising an antigenic protein in the C-terminal direction in the N-terminal direction; (ii) a target binding domain (TBD) comprising an antibody hinge region, a CH2 domain and a CH3 domain; And a polymerizing domain (PD) comprising an IgM or IgA-derived tailpiece are successively linked by peptide linkage.

In the step (a) of the present invention, in order to express the antigen protein in a unique manner of the present invention for inducing plant-specific multimerization, the above-described chimeric antigen is expressed by an ER retention signal sequence (or an ER retention signal peptide). < / RTI > The type of the endoplasmic reticulum storage induction sequence is not limited as long as it is a plant endoplasmic reticulum storage induction sequence known to those skilled in the art. See WO 2009158716 and the following publications and the like; Pagny et al., Signals and mechanisms for protein retention in the endoplasmic reticulum, Journal of Experimental Botany, Vol. 50, No. 331, pp. 157-64, February 1999.

The ERK storage inducible sequence of the present invention may preferably be KDEL (SEQ ID NO: 13), HDEL (SEQ ID NO: 14) and 1 to 5 amino acids added to the sequence (for example SEKDEL of SEQ ID NO: 15, The KEL of SEQ ID NO: 16, the KEL of SEQ ID NO: 17, the SEHDEL of SEQ ID NO: 18, etc.), and most preferably the KDEL of SEQ ID NO:

By inserting the nucleotide coding for the endoplasmic reticulum storage induction sequence into a specific gene (chimeric antigen expression gene in the present invention), an endoplasmic reticulum storage induction sequence such as KDEL can be exposed at the end of the amino acid sequence of the final product. This induces the produced protein to be present in the endoplasmic reticulum in the transformed cell without being secreted outside the plant cell. The protein produced in the host cell into which the specific gene is introduced is post-translationally modified by a sequence such as KDEL stored in the endoplasmic reticulum and can be made in the plant. This plays an important role in solving the problems caused by the increase of the expression level of intracellular antibody protein and the difference of interspecies sugar structure which plays an important role in the interspecific immune response.

The site of insertion of the ERS (KDEL) is not limited as long as it does not affect the immunogenicity or antibody binding ability of the chimeric antigen. The site of insertion of the endoplasmic reticulum storage induction sequence is not limited thereto, but may preferably be the C-terminal region of the target binding domain comprising the antibody Fc fragment.

In the present invention, the plant-specific multimerization is induced by specific combination of an endoplasmic reticulum storage induction sequence and an IgM tail piece or an IgA tail piece sequence. The term " plant-specific multimerization " in the present invention means that a protein is polymerized through a specific mechanism that exists only in plant cells. In the present invention, plant-specific multimerization was induced through specific combination of the above sequences. As a result, not only a large amount of the above-mentioned chimeric antigen was expressed, but also the ratio of the polymeric fragment present in the tailpiece of the chimeric antigen Lt; / RTI > Previously, there have been a number of conventional studies in which antibodies or fragments thereof are mass-produced using IgM tail pieces or IgA tail pieces, but the yield of such multimers has been remarkably increased in plants through a specific combination with an endoplasmic reticulum storage induction sequence, The fact that multimers exhibit considerable high immunogenicity is disclosed for the first time in the present invention and contrasts with previous studies.

The chimeric antigen of step (a) of the present invention may be, for example, a GA-Fc? TpK chimeric antigen represented by SEQ ID NO: 19, a GA-Fc? TpK chimeric antigen represented by SEQ ID NO: 21, a PAP -Fc [alpha] tpK chimeric antigen, or the PAP-Fc [mu] tpK chimeric antigen represented by SEQ ID NO: 25.

The term 'transformation' refers to a modification of the genotype of a host cell by the introduction of a foreign polynucleotide, which means that the foreign polynucleotide has been introduced into the host cell irrespective of the method used for its transformation. The exogenous polynucleotide introduced into the host cell may be maintained integrated or maintained in the genome of the host cell, but the present invention encompasses both.

The term " introduction " refers to an operation of inserting a gene or a gene group into a cell that artificially targets and expressing the gene group, or adding another gene (group) to the genome of the cell. The introduction of these genes can be achieved by indirect methods such as transfection by bacteriophage (bacteria), Agrobacterium spp. Which is a soil bacterium, genegun, electroporation, microinjection, etc. In addition, depending on the characteristics of the target cell and the inserted gene, gene transfection techniques known to those skilled in the art can be selectively used.

Transformation in the step (a) of the present invention means introduction of a polynucleotide encoding the aforementioned chimeric antigen into a plant cell. In step (a) of the present invention, the transgenic plant expressing the chimeric antigen For example, by inserting a desired gene (polynucleotides encoding chimeric antigens) into a vector to transform the recombinant vector into a vector, Transforming the recombinant vector into a strain of the genus Agrobacterium , and then infecting the plant cell with the strain.

The vector is preferably an expression vector comprising at least one of a signal sequence, a replication origin, one or more marker genes, an enhancer element, a promoter and a transcription termination sequence. The expression vector is a form of a vector into which a selected polynucleotide can express. One polynucleotide sequence is "operably linked" to the regulatory sequence when the regulatory sequence affects the expression (e.g., level, timing, or location of expression) of the polynucleotide sequence . The modulatory sequence is a sequence that affects the expression (e.g., level, timing, or location of expression) of the nucleic acid to which it is operatively linked. The modulation sequence can be, for example, a nucleic acid whose effect is directly or indirectly affected by the action of one or more other molecules (e. G., Polypeptides that bind to the regulatory sequence and / . The regulatory sequence includes promoters, enhancers, and other expression control elements.

The field of the molecular biology techniques, standard recombinant DNA and molecular cloning techniques known to those skilled in the are described in the following literature (Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2 nd ed And Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1984, and by Ausubel, LL, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. And Wiley-lnterscience, 1987).

After stable transformation, the transformed plants are then propagated. This breeding means increasing the number of plants. The propagation of the plant is not limited as long as the characteristics of the regenerating plant and the expression of the parent gene transplanting plant remain the same, but may be preferably microproliferation. The microproliferation is a method of growing a second generation plant from a single tissue sample cut from a selected parent plant or cultivar. By this method, it is possible to mass-reproduce plants having desirable tissues and expressing the desired protein. The newly created plant is genetically identical to the original plant and has all of the characteristics of the original plant. Microproliferation allows mass production of excellent plant material in a short period of time and enables rapid propagation of selected crops while preserving the characteristics of the original transgenic or transgenic plant. Advantages of plant cloning methods include the rapidity of plant propagation and the superiority and uniformity of the plant produced.

The term "plant" is not limited as long as the plant is capable of introducing a foreign gene. For example, the plant may be rice, wheat, barley, bamboo shoot, corn, taro, There are hemp and ginger. Examples of dicotyledonous plants include, but are not limited to, Arabidopsis thaliana, eggplant, cigarette, pepper, tomato, burdock, cilantro, lettuce, bellflower, spinach, modern, sweet potato, celery, carrot, parsley, parsley, cabbage, cabbage, Watermelon, melon, cucumber, pak, strawberry, soybean, mung bean, kidney bean, buzz foot trefoil, potato, fowl, perilla, dove bean and pea. Preferably, garlic, broccoli, tobacco, Arabidopsis, cabbage, cabbage, onion, leek, zucchini, radish, carrot. Cucumber, and most preferably a tobacco (Nicotiana tabacum). Tobacco has a high protein content, and has the advantage of easy cultivation methods and high biomass.

In step (b), an immunogenic multimeric antigen protein having a protein component, particularly a macromolecular structure, is obtained from the transgenic plant produced in step (a).

In step (b), 'obtaining a protein from a plant' may be performed by a method of obtaining a known plant cell-derived protein, but is not limited thereto. For example, a crossing plant may be crushed and pulverized, ). ≪ / RTI > The extraction buffer may be a known plant protein extraction buffer, but not limited to, for example, phosphate buffered saline (PBS), or Tris-HCl pH 8, dithiothreitol DTT ), Protease inhibitors (e.g., aprotinin, pepstatin, leupeptine, phenyl methyl sulphonyl fluoride, and [(N- (N- (L 3-trans-carboxyoxirane-2-carbonyl) -Lucyl) -agmantine] and the like.

The time of obtaining the protein component from the transgenic plant prepared in the step (b) in the step (a) may be preferably from the full leaf stage to the mature stage of the plant. It is possible to obtain the target protein (immunogenic multimeric antigen protein) capable of mass production of the immunogenic multimeric antigen protein of the present invention at higher efficiency through the collection of the specific period.

The above-mentioned yielding period is the adult period in which tobacco plants bloom, preferably about 14 weeks.

In the present invention, the protein purification step (step c) may be further included after step (b). The 'protein purification' can be purified in a conventional manner, for example, by salting out (eg, ammonium sulfate precipitation, sodium phosphate precipitation), solvent precipitation (protein fraction precipitation using acetone, ethanol, etc.) Techniques such as filtration, ion exchange, column chromatography such as reversed phase column chromatography and ultrafiltration can be applied alone or in combination (Deutscher, M., Guide to Protein Purification Methods Enzymology, vol. , San Diego, CA (1990)). By the protein purification process, only the immunogenic complex protein of the present invention (i.e., a large quaternary structure antigen-antibody complex) can be obtained at a high concentration.

The present invention provides an immunogenic multimeric antigenic protein prepared by a method comprising the steps (a) and (b) described above. The immunogenic multimeric antigen protein of the present invention has a macromolecular structure.

The immunogenic multimeric antigenic protein molecules of the present invention can be preferably 10 nm to 100 nm in diameter, and most preferably 30 nm to 90 nm in size.

The immunogenic multimeric antigen protein of the present invention has a giant four-dimensional molecular structure and thus has an excellent immune response boosting effect. In particular, the ability to generate antibodies in host animals is excellent, without the use of adjuvant, which has been a problem in the past. In particular, when an Ag-binding Fc fusion is polymerized in an animal cell as in the case of David NA Mekhaiel et al. (2011), immunogenicity of immunoglobulins is lower than that of monomers, Is remarkably improved and exhibits excellent immunostimulating effect.

Accordingly, the present invention provides a vaccine composition comprising the immunogenic multimeric antigen protein.

The term " vaccine " or " vaccine composition " in the present invention refers to a composition that stimulates an immuno response and is used interchangeably herein with the same meaning as an immunogenic composition. The vaccine includes both a prophylactic vaccine and a therapeutic vaccine. A prophylactic vaccine is a vaccine that induces an immune response prior to exposure to a substance comprising an antigen, thereby increasing the ability to resist the substance or cell that carries the antigen, so as to induce a larger immune response when the subject is exposed to the antigen . The therapeutic vaccine is used in a manner that is administered to an individual who already has a disease associated with the antigen of the vaccine, which provides an increased ability to fight the disease or cell that carries the antigen, .

The vaccine composition of the present invention may be administered alone or in combination with a known compound having an effect of preventing or treating a target disease.

The vaccine composition of the present invention may be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration Lt; / RTI >

The vaccine composition of the present invention is characterized by comprising the immunogenic complex protein, and may further contain a pharmaceutically acceptable carrier, excipient or diluent. The term "pharmaceutically acceptable" as used herein means a non-toxic composition which is physiologically acceptable and which, when administered to humans, does not inhibit the action of the active ingredient and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, .

By "carrier" is meant a substance that facilitates the addition of a compound into a cell or tissue. The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, it may contain various drug delivery materials used for oral administration to peptide preparations. In addition, the carrier for parenteral administration may contain water, a suitable oil, a saline solution, an aqueous glucose and a glycol, and may further contain a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, etc. in addition to the above components. Other pharmaceutically acceptable carriers and preparations can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).

In addition, the vaccine composition comprising the immunogenic complex protein of the present invention can be used to immunize an individual in need thereof with an effective amount.

The "subject" may be an animal, preferably a mammal, particularly an animal including a human, and may be an animal derived cell, tissue, organ, or the like. The subject may be a patient in need of treatment.

The 'immunization' refers to the secretion, humoral and / or cellular immune response to the immunogenic complex protein in the individual when the immunogenic complex protein according to the present invention is administered to the individual, These immunizations lead to preventive or therapeutic effects on the target disease.

The target disease is determined by an antigen contained in the immunogenic complex protein according to the present invention, i.e., a substantial immune response domain. The disease is a disease in which a disease-causing antigen is known, that is, a disease- And any of them can be used as long as they can be usefully used for prevention or treatment as an antigen protein. But are not limited to, for example, tumor diseases, autoimmune diseases, metabolic diseases, degenerative diseases, viral or bacterial infections, prion diseases, monorne neuron disease, MND) and the like.

Specifically, the target diseases include melanoma, adenocarcinoma, lung cancer, small cell lung cancer, ovarian cancer, cervical cancer, prostate cancer, bladder cancer, colon cancer, colorectal cancer, testicular cancer, B cell malignant tumor, multiple myeloma, Multiple sclerosis (MS), multiple sclerosis, multiple sclerosis, multiple sclerosis, multiple sclerosis, multiple sclerosis, multiple sclerosis, multiple sclerosis, multiple sclerosis, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, muscle cancer, pancreatic cancer, brain tumor, astroblastoma, glioblastoma, Alzheimer's disease, lewy body disorder (LBD) caused by synuclein protein, Parkinson's disease (PD), multiple nervous system atrophy, diabetes mellitus, rheumatoid arthritis, urinary incontinence, osteoporosis, Alzheimer's disease, (AIDS), hepatitis caused by hepatitis B virus or hepatitis C virus, and human papilloma virus (HPV), which are caused by multiple system atrophy (MSA) And a tumor caused thereby, pneumonia caused by Chlamydia pneumonia, infection caused by Escherichia coli, gastric ulcer induced by Helicobacter pylori, malaria, tuberculosis, Candida albicans, etc. Anthrax, sepsis, variant Creutzfeldt-Jakob disease, vCJD, scrapie, amyotropic lateral sclerosis (ALS), and the like. .

Preferably, the subject disease of the vaccine composition of the present invention may be a tumor disease, more preferably a colon cancer or prostate cancer.

The 'effective amount' refers to an amount of the vaccine composition of the present invention that is effective to prevent or treat the disease of the vaccine composition of the present invention. ≪ / RTI >

The total effective amount of the protein of the present invention may be administered to a subject in a single dose and may be administered by a fractionated treatment protocol in which multiple doses are administered over a prolonged period of time. The content of the active ingredient may be varied depending on the purpose of administration. The effective dose is determined by considering various factors such as age, body weight, health status, sex, disease severity, dietary and excretion rate of the subject in need thereof, as well as the type and severity of the subject disease, route of administration, As an effective dose is determined, a person skilled in the art will be able to determine an appropriate effective dose depending on the purpose of administration. It may also be determined by administering the protein according to the invention and then assaying for the activity of the immune cells or by monitoring the efficacy of the therapy using well-known in vivo assays. The pharmaceutical composition of the present invention is not particularly limited to the formulation, administration route and administration method as long as the effect of the present invention is exhibited.

The administration route of the vaccine composition according to the present invention is as described above. The immunogenic complex protein of the present invention may be administered together with a pharmaceutically acceptable carrier, excipient or diluent. The carrier, excipient or diluent is as described above.

The vaccine composition according to the present invention may be administered alone or in combination with a known compound having an effect of preventing or treating a target disease.

In order to produce the immunogenic multimeric antigen protein of the present invention, a plant produced in step (a) is indispensable. Accordingly, the present invention provides an immune response domain comprising an antigenic protein, an antibody Fc fragment A recombinant vector constructed to express a chimeric antigen comprising a target binding domain comprising a Fc antibody fragment and an IgM or IgA-derived tailpiece, , A method for producing a transgenic plant producing an immunogenic multimeric antigen protein comprising the steps of (a) to (c), and a plant (plant producing the immunogenic multimeric antigen protein) produced by the method. The method for producing the transgenic plants is understood with reference to the description of the step (a).

The method comprising the steps (a) and (b) of the present invention can mass-produce the target protein safely and economically, and the immunogenic polysaccharide antigen protein thus prepared has a large four-dimensional structure, boosting effect, so that it is capable of producing antibodies in host animals without the use of adjuvants.

Figure 1A shows the result of adding an IgA c-terminal tailpiece (alpha tp) to an Fc-fusion protein fused with the colon cancer antigen protein GA733 (denoted as GA) and an IgG Fc region [denoted as GA- , A plant expression vector prepared to express it, and a sugar structure of a protein produced thereby.
Fig. 1B shows the results of adding an IgA c-terminal tailpiece (alpha tp) and an ER retention signal peptide KDEL (indicated by K) to an Fc-fusion protein fused with the colon cancer antigen protein GA733 (denoted as GA) and an IgG Fc region [Collectively referred to as " GA-Fc < RTI ID = 0.0 > atpK "),< / RTI >
Fig. 1C shows the result of adding an IgM c-terminal tailpiece (μtp) to an Fc-fusion protein fused with the colon cancer antigen protein GA733 (denoted as GA) and an IgG Fc region [denoted as 'GA-Fc μtp' , A plant expression vector prepared to express it, and a sugar structure of a protein produced thereby.
Figure 1D shows the addition of an IgM c-tailed tailpiece (μtp) and an ER retention signal peptide KDEL (denoted K) to the Fc-fusion protein fused with the colon cancer antigen protein GA733 (designated GA) and the IgG Fc region [Collectively referred to as " GA-Fc < / RTI > μtpK '], a plant expression vector prepared to express it, and a sugar structure of the protein produced thereby.
FIG. 2A shows the expression of PAP-Fc? Tp by adding an IgA c-terminal tailpiece (? Tp) to an Fc-fusion protein fused with prostate cancer antigen proteins PAP and IgG Fc region A plant expression vector and a sugar structure of a protein produced thereby.
Fig. 2B shows the results of adding an IgA c-terminal tailpiece (alpha tp) and an ER retention signal peptide KDEL (indicated by K) to an Fc-fusion protein fused with prostate cancer antigen proteins PAP and IgG Fc region -Fc [alpha] tpK '], a plant expression vector prepared to express it, and the sugar structure of the protein produced thereby.
FIG. 2C shows the expression of PAP-Fc μtp by adding an IgM c-terminal tailpiece (μtp) to an Fc-fusion protein fused with prostate cancer antigen proteins PAP and IgG Fc region. A plant expression vector and a sugar structure of a protein produced thereby.
FIG. 2D shows the addition of an IgM c-terminal tailpiece (μtp) and an ER retention signal peptide KDEL (denoted by K) to an Fc-fusion protein fused with prostate cancer antigen proteins PAP and IgG Fc region Quot; -Fc < / RTI > μtpK '], a plant expression vector prepared to express it, and the sugar structure of the protein produced thereby.
FIG. 3 is a graph showing the results of immunohistochemical analysis of four kinds of colon cancer vaccine proteins (GA-Fc? Tp, GA-Fc? TpK, GA-Fc? Tp and GA- Ppt-Fc? tpK, PAP-Fc? tpK, PAP-Fc? tpK, PAP-Fc? tpK, PAP-Fc? tpK).
FIG. 4 shows the results of analysis of four types of colon cancer vaccine proteins (GA-Fc? Tp, GA-Fc? TpK, GA-Fc? Tp and GA-Fc? TpK) and four types of prostate vaccine proteins (PAP-Fc αtp, PAP-Fc αtpK, PAP-Fc μtp, and PAP-Fc μtpK) by Western blotting.
FIG. 5 shows the results of analysis of four kinds of colon cancer vaccine proteins (GA-Fc? Tp, GA-Fc? TpK, GA-Fc? Tp and GA-Fc? TpK) and four prostate vaccine proteins (PAP-Fc αtp, PAP-Fc αtpK, PAP-Fc μtp, and PAP-Fc μtpK) were purified and confirmed by SDS-PAGE.
FIG. 6 is a graph showing the results of immunohistochemical analysis for four kinds of colon cancer vaccine proteins (GA-Fc? Tp, GA-Fc? TpK, GA- μtp, and PAP-Fc μtpK) by size-exclusion chromatography-high-performance liquid chromatography (SEC-HPLC).

Hereinafter, the present invention will be described in detail.

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

≪ Example 1 >

Production of plants producing macromolecular multimer protein

Fc fusion fused with colon cancer cell surface specific protein GA733 (SEQ ID NO: 1) and human IgG1 Fc region (SEQ ID NO: 7) was constructed, and a tailpiece having IgA and IgM antibody at the c-terminus of the constructed Fc- [alpha tp (IgA tailpiece, SEQ ID NO: 11) and mu tp (IgM tailpiece, SEQ ID NO: 9)] were added to construct a plant expression vector expressing it. This vector was inserted into a pBI121 binary vector and operatively linked to a Cauliflower Mosaic Virus 35S promoter (CaMV 35S promoter) to prepare a plant expression vector.

In addition, a macromolecular fusion vaccine protein expression system of four types (GA-Fc αtp, GA-Fc αtpK, GA-Fc μtp and GA-Fc μtpK) was established as shown in FIG.

Similarly, Fc fusion fused with prostate cancer antigen PAP protein (prostatic acid phosphatase, SEQ ID NO: 3) and human IgG1 Fc region (SEQ ID NO: 7) was constructed. IgA and IgM antibody (IgA tailpiece, SEQ ID NO: 11) and [mu] tp (IgM tailpiece, SEQ ID NO: 9) were constructed to construct a plant expression vector. In addition, we have established a macromolecular fusion vaccine protein expression system of four types (PAP-Fc αtp, PAP-Fc αtpK, PAP-Fc μtp and PAP-Fc μtpK) as shown in FIG.

Each of the constructed plant expression vectors was digested with Agrobacterium tobacco ( Nicotiana tabacum ) Callus induction in plant leaves. Transgenic plants were obtained from the calli produced, and transformed plants were selected in vitro using plant medium containing kanamycin antibiotics.

≪ Example 2 >

Identification of macromolecular immunogenicity genes

Extracts were prepared from the leaves of the plants selected in Example 1, and gene expression of the above-mentioned eight proteins was confirmed by PCR.

(GA-Fc αtpK, GA-Fc αtpK, GA-Fc μtp and GA-Fc μtpK) and four macromolecular structural prostate vaccine proteins (PAP-Fc αtp and PAP- Genomic DNA was isolated and purified from the leaves of the transgenic plants expressing Fc [alpha] tpK, PAP-Fc [mu] tp, PAP-Fc [mu] tpK using a DNeasy kit (Quiagen, Hilden, Germany). Approximately 50 ~ 100g of plant leaves were harvested, frozen by liquid nitrogen, and DNA was extracted by extraction. PCR was performed using primer and tailpiece primers of the vaccine candidate protein (GA733, PAP) using the genomic DNA as a template. The PCR reaction conditions were 30 sec at 94 ° C, 20 sec at 65 ° C, and 30 sec at 72 ° C, and this was repeated 27 times. Then, electrophoresis was performed to confirm gene insertion.

As a result, as shown in Fig. 3, four types of macromolecule colorectal cancer vaccine proteins (GA-Fc? Tp, GA-Fc? TpK, GA-Fc? Tp and GA- It was confirmed that the genes of four vaccine proteins (PAP-Fc? Tp, PAP-Fc? TpK, PAP-Fc? Tp and PAP-Fc? TpK) were inserted.

≪ Example 3 >

Identification of macromolecular immunogenicity protein expression

Extracts were prepared from the leaves of the plants selected in Example 1 and analyzed by western blot method to confirm the expression of the eight proteins described above.

(GA-Fc αtpK, GA-Fc αtpK, GA-Fc μtp and GA-Fc μtpK) and four macromolecular structural prostate vaccine proteins (PAP-Fc αtp and PAP- Fc αtpK, put in a PAP-Fc μtp, PAP-Fc μtpK) respectively taken 100 mg of leaf from the transgenic plants 1x PBS (NcCl, KCl, Na 2 HPO of 300μl expressing _4, KH ₂ PO ₄₎ And then crushed. The supernatant of the crushed leaves was electrophoresed on a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane, and washed with 5% skim milk (Fluka, Buchs, Switzerland) at 4 ° C For 16 hours. Anti-EpCAM / TROP1 (R & D system, Minneapolis, MN) and anti-PAP were treated with primary antibody and secondary anti-mouse IgG H + L (Bethyl, Montgomery, TX) was diluted at a ratio of 1: 5,000. The membrane was then washed three times for 10 min with 1x PBS (Tween 0.1%) buffer. The buffer in the membrane was removed and treated with Supersignal chemiluminescence substrate (Thermo, Fisher Scientific, Roskilde, Rosilde, Denmark) -ray film.

As shown in FIG. 4, four types of macromolecule-resected colon cancer vaccine proteins (GA-Fc? Tp, GA-Fc? TpK, GA-Fc? Tp and GA-Fc? (PAP-Fc? TpK, PAP-Fc? TpK, PAP-Fc? Tp, and PAP-Fc? TpK) were expressed in the colon and the prostate vaccine proteins of the species were about 68 kDa and 78 kDa As shown in Fig.

<Example 4>

Macromolecular structure immunogenic protein purification

Eight species of proteins expressed from the leaves of the plants identified in Example 3 were purified by SDS-PAGE.

(GA-Fc αtpK, GA-Fc αtpK, GA-Fc μtp, and GA-Fc μtpK) and four macromolecular structural prostate vaccine proteins (PAP-Fc αtp, (37.5 mM Tris-HCl pH 7.5, 50 mM NaCl, 15 mM EDTA, 75 mM sodium citrate, and 0.2 [mu] M) were added to the transformed plant leaves expressing PAP-Fc [alpha] tpK, PAP-Fc [ % Sodium thiosulfate) was added and subjected to homogenization. After centrifugation at 4,800 x g for 30 min at 4 ° C, the supernatant was filtered through Miracloth (Biosciences, La Jolla, CA, USA) and the pH was adjusted to 5.1 with acetic acid. The supernatant was then centrifuged at 10,200 x g for 30 min, and the supernatant was obtained and the pH was adjusted to neutral by the addition of 3 M Tris-HCl. The total soluble protein was then incubated overnight in a cold room, then precipitated with ammonium sulfate and centrifuged at 4 ° C for 30 minutes. After resuspending the pellet to 1/10 of the initial volume of the extraction buffer, the resulting solution was centrifuged at 10,200 x g for 30 minutes at 4 &lt; 0 &gt; C and then the plant extract was diluted with protein A (GE Healthcare, ) column. Protein concentration was measured using Nano-drop (Biotek, Highland, VT, USA) and SDS-PAGE was performed on the purified protein. The results are shown in Fig.

< Example  5>

Identification of macromolecular immunogenic proteins

The eight proteins purified in Example 4 were analyzed by SEC-HPLC (Size exclusion chromatography-High-performance liquid chromatography).

(GA-Fc? TpK, GA-Fc? TpK, GA-Fc? Tp and GA-Fc? TpK) and four macromolecular structural prostate cancer vaccine proteins (PAP- PAP-Fc? TpK, PAP-Fc? Tp, PAP-Fc? TpK). The elution was carried out at pH 7.0 under a mobile phase of 0.1 M sodium phosphate containing 0.3 M sodium chloride at a flow rate of 1 mL / min Respectively.

As a result, as shown in FIG. 6, the 8 kinds of macromolecular structural vaccine proteins were found to be similar in size to IgM (Immunogloblin M), and it was confirmed that they exhibited the same pattern at the same time. Thus, it was confirmed that the macromolecular structural vaccine protein of the present invention has excellent immunogenicity.

&Lt; Example 6 >

Comparison of immunogenicity and evaluation of vaccine performance

In the present invention, a macromolecular structural vaccine protein (immunogenic multimeric antigen protein) produced by inducing plant-specific multimerization in the same manner as in <Example 1> The immunogenicity was compared and evaluated in comparison with the somatic Fc-fusants.

As a result, the macromolecular structural vaccine protein of the present invention showed excellent immunogenicity and cancer growth inhibitory effect, and particularly, it was confirmed that the immunomodulating effect is excellent even without adjuvant.

(A) an immune response domain comprising an antigenic protein, a target binding domain comprising an antibody Fc fragment (Fc antibody fragment), and an IgM or IgA Preparing a transgenic plant expressing a chimeric antigen comprising a polymerisation domain comprising a tailpiece derived from the plant; And (b) obtaining a protein from the transgenic plant, and a method for producing the immunogenic multimeric antigen protein, and a plant therefor.

The method comprising the steps (a) and (b) of the present invention can mass-produce the target protein safely and economically, and the immunogenic polysaccharide antigen protein thus prepared has a large four-dimensional structure, boosting effect and is highly industrially applicable because of its excellent ability to generate antibodies in host animals without the use of adjuvants.

<110> CHUNG ANG University industry Academic Cooperation Foundation <120> Method for preparing polymerized antigen protein with high          Immunogenicity and Plant for the same <130> NP17-0009P <150> KR 10-2017-0046943 <151> 2017-04-11 <160> 26 <170> KoPatentin 3.0 <210> 1 <211> 249 <212> PRT <213> Artificial Sequence <220> <223> GA733 <400> 1 Thr Ala Thr Phe Ala Ala Gln Glu Glu Cys Val Cys Glu Asn Tyr   1 5 10 15 Lys Leu Ala Val Asn Cys Phe Val Asn Asn Asn Arg Gln Cys Gln Cys              20 25 30 Thr Ser Val Gly Ala Gln Asn Thr Val Ile Cys Ser Lys Leu Ala Ala          35 40 45 Lys Cys Leu Val Met Lys Ala Glu Met Asn Gly Ser Lys Leu Gly Arg      50 55 60 Arg Ala Lys Pro Glu Gly Ala Leu Gln Asn Asn Asp Gly Leu Tyr Asp  65 70 75 80 Pro Asp Cys Asp Glu Ser Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly                  85 90 95 Thr Ser Thr Cys Trp Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp             100 105 110 Lys Asp Thr Glu Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile         115 120 125 Ile Ile Glu Leu Lys His Lys Ala Arg Glu Lys Pro Tyr Asp Ser Lys     130 135 140 Ser Leu Arg Thr Ala Leu Gln Lys Glu Ile Thr Thr Arg Tyr Gln Leu 145 150 155 160 Asp Pro Lys Phe Ile Thr Ser Ile Leu Tyr Glu Asn Asn Val Ile Thr                 165 170 175 Ile Asp Leu Val Gln Asn Ser Ser Gln Lys Thr Gln Asn Asp Val Asp             180 185 190 Ile Ala Asp Val Ala Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu Ser         195 200 205 Leu Phe His Ser Lys Lys Met Asp Leu Thr Val Asn Gly Glu Gln Leu     210 215 220 Asp Leu Asp Pro Gly Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys Ala 225 230 235 240 Pro Glu Phe Ser Met Gln Gly Leu Lys                 245 <210> 2 <211> 747 <212> DNA <213> Artificial Sequence <220> <223> GA733 <400> 2 acggcgactt ttgccgcagc tcaggaagaa tgtgtctgtg aaaactacaa gctggccgta 60 aactgctttg tgaataataa tcgtcaatgc cagtgtactt cagttggtgc acaaaatact 120 gtcatttgct caaagctggc tgccaaatgt ttggtgatga aggcagaaat gaatggctca 180 aaacttggga gaagagcaaa acctgaaggg gccctccaga acaatgatgg gctttatgat 240 cctgactgcg atgagagcgg gctctttaag gccaagcagt gcaacggcac ctccacgtgc 300 tggtgtgtga acactgctgg ggtcagaaga acagacaagg acactgaaat aacctgctct 360 gagcgagtga gaacctactg gatcatcatt gaactaaaac acaaagcaag agaaaaacct 420 tatgatagta aaagtttgcg gactgcactt cagaaggaga tcacaacgcg ttatcaactg 480 gatccaaaat ttatcacgag tattttgtat gagaataatg ttatcactat tgatctggtt 540 caaaattctt ctcaaaaaac tcagaatgat gtggacatag ctgatgtggc ttattatttt 600 gaaaaagatg ttaaaggtga atccttgttt cattctaaga aaatggacct gacagtaaat 660 ggggaacaac tggatctgga tcctggtcaa actttaattt attatgttga tgaaaaagca 720 cctgaattct caatgcaggg tctaaaa 747 <210> 3 <211> 384 <212> PRT <213> Artificial Sequence <220> <223> PAP <400> 3 Met Ala Thr Gln Arg Arg Ala Asn Pro Ser Ser Leu His Leu Ile Thr   1 5 10 15 Val Pés Ser Leu Ala Ala Val Val Ser Ala Glu Val Asp Lys Glu              20 25 30 Leu Lys Phe Val Thr Leu Val Phe Arg His Gly Asp Arg Ser Pro Ile          35 40 45 Asp Thr Phe Pro Thr Asp Pro Ile Lys Glu Ser Ser Trp Pro Gln Gly      50 55 60 Phe Gly Gln Leu Thr Gln Leu Gly Met Glu Gln His Tyr Glu Leu Gly  65 70 75 80 Glu Tyr Ile Arg Lys Arg Tyr Arg Lys Phe Leu Asn Glu Ser Tyr Lys                  85 90 95 His Glu Gln Val Thr Ile Arg Ser Thr Asp Val Asp Arg Thr Leu Met             100 105 110 Ser Ala Met Thr Asn Leu Ala Leu Phe Pro Pro Glu Gly Val Ser         115 120 125 Ile Trp Asn Pro Ile Leu Leu Trp Gln Pro Ile Pro Val His Thr Val     130 135 140 Pro Leu Ser Glu Asp Gln Leu Leu Tyr Leu Pro Phe Arg Asn Cys Pro 145 150 155 160 Arg Phe Gln Glu Leu Glu Ser Glu Thr Leu Lys Ser Glu Glu Phe Gln                 165 170 175 Lys Arg Leu His Pro Tyr Lys Asp Phe Ile Ala Thr Leu Gly Lys Leu             180 185 190 Ser Gly Leu His Gly Gln Asp Leu Phe Gly Ile Trp Ser Lys Val Tyr         195 200 205 Asp Pro Leu Tyr Cys Glu Ser Val His Asn Phe Thr Leu Pro Ser Trp     210 215 220 Ala Thr Glu Asp Thr Met Thr Lys Leu Arg Glu Leu Ser Glu Leu Ser 225 230 235 240 Leu Leu Ser Leu Tyr Gly Ile His Lys Gln Lys Glu Lys Ser Arg Leu                 245 250 255 Gln Gly Gly Val Leu Val Asn Glu Ile Leu Asn His Met Lys Arg Ala             260 265 270 Thr Gln Ile Pro Ser Tyr Lys Lys Leu Ile Met Tyr Ser Ala His Hisp         275 280 285 Thr Thr Val Gly Leu Gln Met Ala Leu Asp Val Tyr Asn Gly Leu     290 295 300 Leu Pro Pro Tyr Ala Ser Cys His Leu Thr Glu Leu Tyr Phe Glu Lys 305 310 315 320 Gly Glu Tyr Phe Val Glu Met Tyr Tyr Arg Asn Glu Thr Gln His Glu                 325 330 335 Pro Tyr Pro Leu Met Leu Pro Gly Cys Ser Pro Ser Cys Pro Leu Glu             340 345 350 Arg Phe Ala Glu Leu Val Gly Pro Val Ile Pro Gln Asp Trp Ser Thr         355 360 365 Glu Cys Met Thr Thr Asn Ser His Gln Gly Thr Glu Asp Ser Thr Asp     370 375 380 <210> 4 <211> 1158 <212> DNA <213> Artificial Sequence <220> <223> PAP <400> 4 atggctactc aacgaagggc aaaccctagc tctctccatc taattactgt attctctctg 60 ctagctgctg tcgtctccgc tgaggtcgac aaggagttga agtttgtgac tttggtgttt 120 cggcatggag accgaagtcc cattgacacc tttcccactg accccataaa ggaatcctca 180 tggccacaag gatttggcca actcacccag ctgggcatgg agcagcatta tgaacttgga 240 gagtatataa gaaagagata tagaaaattc ttgaatgagt cctataaaca tgaacaggtt 300 tatattcgaa gcacagacgt tgaccggact ttgatgagtg ctatgacaaa cctggcagcc 360 ctgtttcccc cagaaggtgt cagcatctgg aatcctatcc tactctggca gcccatcccg 420 gtgcacacag ttcctctttc tgaagatcag ttgctatacc tgcctttcag gaactgccct 480 cgttttcaag aacttgagag tgagactttg aaatcagagg aatttcagaa gaggctgcac 540 ccttataagg attttatagc taccttggga aaactttcag gattacatgg ccaggacctt 600 tttggaattt ggagtaaagt ctacgaccct ttatattgtg agagtgttca caatttcact 660 ttaccctcct gggccactga ggacaccatg actaagttga gagaattgtc agaattgtcc 720 ctcctgtccc tctatggtat tcacaagcag aaagagaaat ctaggctcca agggggtgtc 780 ctggtcaatg aaatcctcaa tcacatgaag agagcaactc agataccaag ctacaaaaaa 840 cttatcatgt attctgcgca tgacactact gtgagtggcc tacagatggc gctagatgtt 900 tacaacggac tccttcctcc ctatgcttct tgccacttga cggaattgta ctttgagaag 960 ggggagtact ttgtggagat gtactatcgg aatgagacgc agcacgagcc gtatcccctc 1020 atgctacctg gttgcagccc tagctgtcct ctggagaggt ttgctgagct ggttggccct 1080 gtgatccctc aagactggtc cacggagtgt atgaccacaa acagccatca aggtactgag 1140 gacagtacag atactagt 1158 <210> 5 <211> 192 <212> PRT <213> Artificial Sequence <220> <223> Fc fragment of IgG1 <400> 5 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Ser Glu Asp   1 5 10 15 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn              20 25 30 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val          35 40 45 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu      50 55 60 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys  65 70 75 80 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr                  85 90 95 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr             100 105 110 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu         115 120 125 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu     130 135 140 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 145 150 155 160 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu                 165 170 175 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly             180 185 190 <210> 6 <211> 576 <212> DNA <213> Artificial Sequence <220> <223> Fc fragment of IgG1 <400> 6 cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 60 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 120 cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 180 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 240 accatctcga aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 300 cgggaggaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 360 agcgacatcg ccgtggagtg ggagagcaat gggcagccag agaacaacta caagaccacg 420 cctcccgtgc tggactccga cggctccttc ttcctctata gcaagctcac cgtggacaag 480 agcaggtggc agcaggggaa cgtcttctca tgctctgtga tgcatgaggc tctgcacaac 540 cactacacgc agaagagcct ctccctgtcc ccgggt 576 <210> 7 <211> 237 <212> PRT <213> Artificial Sequence <220> <223> Fc fragment of IgG1 containing hinge region <400> 7 Ala Ala Ala Asp Leu Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr   1 5 10 15 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe              20 25 30 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro          35 40 45 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val      50 55 60 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr  65 70 75 80 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Val Ser                  85 90 95 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys             100 105 110 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser         115 120 125 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro     130 135 140 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 145 150 155 160 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly                 165 170 175 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp             180 185 190 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp         195 200 205 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His     210 215 220 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 225 230 235 <210> 8 <211> 711 <212> DNA <213> Artificial Sequence <220> <223> Fc fragment of IgG1 containing hinge region <400> 8 gcggccgcag atctcgttga gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgc 60 ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 120 accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 180 gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 240 aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 300 caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 360 cccggaccacc accctgcccc catcccggga ggagatgacc aagaaccagg tcagcctgac ctgcctggtc 480 aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccagagaac 540 aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctatagcaag 600 ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc tgtgatgcat 660 gaggctctgc acaaccacta cacgcagaag agcctctccc tgtccccggg t 711 <210> 9 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> IgM tailpiece <400> 9 Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr Ala   1 5 10 15 Gly Thr Cys Tyr              20 <210> 10 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> IgM tailpiece <400> 10 ggtaaaccca ccctgtacaa cgtgtccctg gtcatgtccg acacagctgg cacctgctac 60                                                                           60 <210> 11 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> IgA tailpiece <400> 11 Gly Lys Pro Thr His Val Asn Val Ser Val Met Ala Glu Val Asp Gly   1 5 10 15 Thr Cys Tyr             <210> 12 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> IgA tailpiece <400> 12 ggtaaaccca cccatgtaaa tgtgtctgtt gttatggctg aggttgatgg cacatgttac 60                                                                           60 <210> 13 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> KDEL for ER retention signal <400> 13 Lys Asp Glu Leu   One <210> 14 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> HDEL for ER retention signal <400> 14 His Asp Glu Leu   One <210> 15 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> SEKDEL for ER retention signal <400> 15 Ser Glu Lys Asp Glu Leu   1 5 <210> 16 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> KHDEL for ER retention signal <400> 16 Lys His Asp Glu Leu   1 5 <210> 17 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> KEEL for ER retention signal <400> 17 Lys Glu Glu Leu   One <210> 18 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> SEHDEL for ER retention signal <400> 18 Ser Glu His Asp Glu Leu   1 5 <210> 19 <211> 514 <212> PRT <213> Artificial Sequence <220> <223> GA-Fc ATPK <400> 19 Met Thr Ala Thr Phe Ala Ala Ala Gln Glu Glu Cys Val Cys Glu Asn   1 5 10 15 Tyr Lys Leu Ala Val Asn Cys Phe Val Asn Asn Asn Arg Gln Cys Gln              20 25 30 Cys Thr Ser Val Gly Ala Gln Asn Thr Val Ile Cys Ser Lys Leu Ala          35 40 45 Ala Lys Cys Leu Val Met Lys Ala Glu Met Asn Gly Ser Lys Leu Gly      50 55 60 Arg Arg Ala Lys Pro Glu Gly Ala Leu Gln Asn Asn Asp Gly Leu Tyr  65 70 75 80 Asp Pro Asp Cys Asp Glu Ser Gly Leu Phe Lys Ala Lys Gln Cys Asn                  85 90 95 Gly Thr Ser Thr Cys Trp Cys Val Asn Thr Ala Gly Val Arg Arg Thr             100 105 110 Asp Lys Asp Thr Glu Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp         115 120 125 Ile Ile Ile Glu Leu Lys His Lys Ala Arg Glu Lys Pro Tyr Asp Ser     130 135 140 Lys Ser Leu Arg Thr Ala Leu Gln Lys Glu Ile Thr Thr Arg Tyr Gln 145 150 155 160 Leu Asp Pro Lys Phe Ile Thr Ser Ile Leu Tyr Glu Asn Asn Val Ile                 165 170 175 Thr Ile Asp Leu Val Gln Asn Ser Ser Gln Lys Thr Gln Asn Asp Val             180 185 190 Asp Ile Ala Asp Val Ala Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu         195 200 205 Ser Leu Phe His Ser Lys Lys Met Asp Leu Thr Val Asn Gly Glu Gln     210 215 220 Leu Asp Leu Asp Pro Gly Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys 225 230 235 240 Ala Pro Glu Phe Ser Met Gln Gly Leu Lys Arg Thr Ser Ala Ala Ala                 245 250 255 Asp Leu Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro             260 265 270 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro         275 280 285 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr     290 295 300 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 305 310 315 320 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg                 325 330 335 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val             340 345 350 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser         355 360 365 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys     370 375 380 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 385 390 395 400 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe                 405 410 415 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu             420 425 430 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe         435 440 445 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly     450 455 460 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 465 470 475 480 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Lys Pro Thr His Val                 485 490 495 Asn Val Ser Val Val Met Ala Glu Val Asp Gly Thr Cys Tyr Lys Asp             500 505 510 Glu Leu         <210> 20 <211> 1542 <212> DNA <213> Artificial Sequence <220> <223> GA-Fc ATPK <400> 20 atgacggcga cttttgccgc agctcaggaa gaatgtgtct gtgaaaacta caagctggcc 60 gtaaactgct ttgtgaataa taatcgtcaa tgccagtgta cttcagttgg tgcacaaaat 120 actgtcattt gctcaaagct ggctgccaaa tgtttggtga tgaaggcaga aatgaatggc 180 tcaaaacttg ggagaagagc aaaacctgaa ggggccctcc agaacaatga tgggctttat 240 gatcctgact gcgatgagag cgggctcttt aaggccaagc agtgcaacgg cacctccacg 300 tgctggtgtg tgaacactgc tggggtcaga agaacagaca aggacactga aataacctgc 360 tctgagcgag tgagaaccta ctggatcatc attgaactaa aacacaaagc aagagaaaaa 420 ccttatgata gtaaaagttt gcggactgca cttcagaagg agatcacaac gcgttatcaa 480 ctggatccaa aatttatcac gagtattttg tatgagaata atgttatcac tattgatctg 540 gttcaaaatt cttctcaaaa aactcagaat gatgtggaca tagctgatgt ggcttattat 600 tttgaaaaag atgttaaagg tgaatccttg tttcattcta agaaaatgga cctgacagta 660 aatggggaac aactggatct ggatcctggt caaactttaa tttattatgt tgatgaaaaa 720 gcacctgaat tctcaatgca gggtctaaaa cggactagtg cggccgcaga tctcgttgag 780 cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 840 ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 900 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 960 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 1020 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1080 aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1140 tcgaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggag 1200 gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1260 atcgccgtgg agtgggagag caatgggcag ccagagaaca actacaagac cacgcctccc 1320 gtgctggact ccgacggctc cttcttcctc tatagcaagc tcaccgtgga caagagcagg 1380 tggcagcagg ggaacgtctt ctcatgctct gtgatgcatg aggctctgca caaccactac 1440 acgcagaaga gcctctccct gtccccgggt ggtaaaccca cccatgtaaa tgtgtctgtt 1500 gttatggctg aggttgatgg cacatgttac aaagatgaac tc 1542 <210> 21 <211> 514 <212> PRT <213> Artificial Sequence <220> <223> GA-Fc utPK <400> 21 Met Thr Ala Thr Phe Ala Ala Ala Gln Glu Glu Cys Val Cys Glu Asn   1 5 10 15 Tyr Lys Leu Ala Val Asn Cys Phe Val Asn Asn Asn Arg Gln Cys Gln              20 25 30 Cys Thr Ser Val Gly Ala Gln Asn Thr Val Ile Cys Ser Lys Leu Ala          35 40 45 Ala Lys Cys Leu Val Met Lys Ala Glu Met Asn Gly Ser Lys Leu Gly      50 55 60 Arg Arg Ala Lys Pro Glu Gly Ala Leu Gln Asn Asn Asp Gly Leu Tyr  65 70 75 80 Asp Pro Asp Cys Asp Glu Ser Gly Leu Phe Lys Ala Lys Gln Cys Asn                  85 90 95 Gly Thr Ser Thr Cys Trp Cys Val Asn Thr Ala Gly Val Arg Arg Thr             100 105 110 Asp Lys Asp Thr Glu Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp         115 120 125 Ile Ile Ile Glu Leu Lys His Lys Ala Arg Glu Lys Pro Tyr Asp Ser     130 135 140 Lys Ser Leu Arg Thr Ala Leu Gln Lys Glu Ile Thr Thr Arg Tyr Gln 145 150 155 160 Leu Asp Pro Lys Phe Ile Thr Ser Ile Leu Tyr Glu Asn Asn Val Ile                 165 170 175 Thr Ile Asp Leu Val Gln Asn Ser Ser Gln Lys Thr Gln Asn Asp Val             180 185 190 Asp Ile Ala Asp Val Ala Tyr Tyr Phe Glu Lys Asp Val Lys Gly Glu         195 200 205 Ser Leu Phe His Ser Lys Lys Met Asp Leu Thr Val Asn Gly Glu Gln     210 215 220 Leu Asp Leu Asp Pro Gly Gln Thr Leu Ile Tyr Tyr Val Asp Glu Lys 225 230 235 240 Ala Pro Glu Phe Ser Met Gln Gly Leu Lys Arg Thr Ser Ala Ala Ala                 245 250 255 Asp Leu Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro             260 265 270 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro         275 280 285 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr     290 295 300 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 305 310 315 320 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg                 325 330 335 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val             340 345 350 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser         355 360 365 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys     370 375 380 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 385 390 395 400 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe                 405 410 415 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu             420 425 430 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe         435 440 445 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly     450 455 460 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 465 470 475 480 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Lys Pro Thr Leu Tyr                 485 490 495 Asn Val Ser Leu Val Met Ser Asp Thr Ala Gly Thr Cys Tyr Lys Asp             500 505 510 Glu Leu         <210> 22 <211> 1542 <212> DNA <213> Artificial Sequence <220> <223> GA-Fc utPK <400> 22 atgacggcga cttttgccgc agctcaggaa gaatgtgtct gtgaaaacta caagctggcc 60 gtaaactgct ttgtgaataa taatcgtcaa tgccagtgta cttcagttgg tgcacaaaat 120 actgtcattt gctcaaagct ggctgccaaa tgtttggtga tgaaggcaga aatgaatggc 180 tcaaaacttg ggagaagagc aaaacctgaa ggggccctcc agaacaatga tgggctttat 240 gatcctgact gcgatgagag cgggctcttt aaggccaagc agtgcaacgg cacctccacg 300 tgctggtgtg tgaacactgc tggggtcaga agaacagaca aggacactga aataacctgc 360 tctgagcgag tgagaaccta ctggatcatc attgaactaa aacacaaagc aagagaaaaa 420 ccttatgata gtaaaagttt gcggactgca cttcagaagg agatcacaac gcgttatcaa 480 ctggatccaa aatttatcac gagtattttg tatgagaata atgttatcac tattgatctg 540 gttcaaaatt cttctcaaaa aactcagaat gatgtggaca tagctgatgt ggcttattat 600 tttgaaaaag atgttaaagg tgaatccttg tttcattcta agaaaatgga cctgacagta 660 aatggggaac aactggatct ggatcctggt caaactttaa tttattatgt tgatgaaaaa 720 gcacctgaat tctcaatgca gggtctaaaa cggactagtg cggccgcaga tctcgttgag 780 cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 840 ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 900 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 960 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 1020 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1080 aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1140 tcgaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggag 1200 gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1260 atcgccgtgg agtgggagag caatgggcag ccagagaaca actacaagac cacgcctccc 1320 gtgctggact ccgacggctc cttcttcctc tatagcaagc tcaccgtgga caagagcagg 1380 tggcagcagg ggaacgtctt ctcatgctct gtgatgcatg aggctctgca caaccactac 1440 acgcagaaga gcctctccct gtccccgggt ggtaaaccca ccctgtacaa cgtgtccctg 1500 gtcatgtccg acacagctgg cacctgctac aaagatgaac tc 1542 <210> 23 <211> 645 <212> PRT <213> Artificial Sequence <220> <223> PAP-Fc ATPK <400> 23 Met Ala Thr Gln Arg Arg Ala Asn Pro Ser Ser Leu His Leu Ile Thr   1 5 10 15 Val Pés Ser Leu Ala Ala Val Val Ser Ala Glu Val Asp Lys Glu              20 25 30 Leu Lys Phe Val Thr Leu Val Phe Arg His Gly Asp Arg Ser Pro Ile          35 40 45 Asp Thr Phe Pro Thr Asp Pro Ile Lys Glu Ser Ser Trp Pro Gln Gly      50 55 60 Phe Gly Gln Leu Thr Gln Leu Gly Met Glu Gln His Tyr Glu Leu Gly  65 70 75 80 Glu Tyr Ile Arg Lys Arg Tyr Arg Lys Phe Leu Asn Glu Ser Tyr Lys                  85 90 95 His Glu Gln Val Thr Ile Arg Ser Thr Asp Val Asp Arg Thr Leu Met             100 105 110 Ser Ala Met Thr Asn Leu Ala Leu Phe Pro Pro Glu Gly Val Ser         115 120 125 Ile Trp Asn Pro Ile Leu Leu Trp Gln Pro Ile Pro Val His Thr Val     130 135 140 Pro Leu Ser Glu Asp Gln Leu Leu Tyr Leu Pro Phe Arg Asn Cys Pro 145 150 155 160 Arg Phe Gln Glu Leu Glu Ser Glu Thr Leu Lys Ser Glu Glu Phe Gln                 165 170 175 Lys Arg Leu His Pro Tyr Lys Asp Phe Ile Ala Thr Leu Gly Lys Leu             180 185 190 Ser Gly Leu His Gly Gln Asp Leu Phe Gly Ile Trp Ser Lys Val Tyr         195 200 205 Asp Pro Leu Tyr Cys Glu Ser Val His Asn Phe Thr Leu Pro Ser Trp     210 215 220 Ala Thr Glu Asp Thr Met Thr Lys Leu Arg Glu Leu Ser Glu Leu Ser 225 230 235 240 Leu Leu Ser Leu Tyr Gly Ile His Lys Gln Lys Glu Lys Ser Arg Leu                 245 250 255 Gln Gly Gly Val Leu Val Asn Glu Ile Leu Asn His Met Lys Arg Ala             260 265 270 Thr Gln Ile Pro Ser Tyr Lys Lys Leu Ile Met Tyr Ser Ala His Hisp         275 280 285 Thr Thr Val Gly Leu Gln Met Ala Leu Asp Val Tyr Asn Gly Leu     290 295 300 Leu Pro Pro Tyr Ala Ser Cys His Leu Thr Glu Leu Tyr Phe Glu Lys 305 310 315 320 Gly Glu Tyr Phe Val Glu Met Tyr Tyr Arg Asn Glu Thr Gln His Glu                 325 330 335 Pro Tyr Pro Leu Met Leu Pro Gly Cys Ser Pro Ser Cys Pro Leu Glu             340 345 350 Arg Phe Ala Glu Leu Val Gly Pro Val Ile Pro Gln Asp Trp Ser Thr         355 360 365 Glu Cys Met Thr Thr Asn Ser His Gln Gly Thr Glu Asp Ser Thr Asp     370 375 380 Ala Ala Ala Asp Leu Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 385 390 395 400 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe                 405 410 415 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro             420 425 430 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val         435 440 445 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr     450 455 460 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Val Ser 465 470 475 480 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys                 485 490 495 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser             500 505 510 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro         515 520 525 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val     530 535 540 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 545 550 555 560 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp                 565 570 575 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp             580 585 590 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His         595 600 605 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Lys Pro     610 615 620 Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp Gly Thr Cys 625 630 635 640 Tyr Lys Asp Glu Leu                 645 <210> 24 <211> 1941 <212> DNA <213> Artificial Sequence <220> <223> PAP-Fc ATPK <400> 24 atggctactc aacgaagggc aaaccctagc tctctccatc taattactgt attctctctg 60 ctagctgctg tcgtctccgc tgaggtcgac aaggagttga agtttgtgac tttggtgttt 120 cggcatggag accgaagtcc cattgacacc tttcccactg accccataaa ggaatcctca 180 tggccacaag gatttggcca actcacccag ctgggcatgg agcagcatta tgaacttgga 240 gagtatataa gaaagagata tagaaaattc ttgaatgagt cctataaaca tgaacaggtt 300 tatattcgaa gcacagacgt tgaccggact ttgatgagtg ctatgacaaa cctggcagcc 360 ctgtttcccc cagaaggtgt cagcatctgg aatcctatcc tactctggca gcccatcccg 420 gtgcacacag ttcctctttc tgaagatcag ttgctatacc tgcctttcag gaactgccct 480 cgttttcaag aacttgagag tgagactttg aaatcagagg aatttcagaa gaggctgcac 540 ccttataagg attttatagc taccttggga aaactttcag gattacatgg ccaggacctt 600 tttggaattt ggagtaaagt ctacgaccct ttatattgtg agagtgttca caatttcact 660 ttaccctcct gggccactga ggacaccatg actaagttga gagaattgtc agaattgtcc 720 ctcctgtccc tctatggtat tcacaagcag aaagagaaat ctaggctcca agggggtgtc 780 ctggtcaatg aaatcctcaa tcacatgaag agagcaactc agataccaag ctacaaaaaa 840 cttatcatgt attctgcgca tgacactact gtgagtggcc tacagatggc gctagatgtt 900 tacaacggac tccttcctcc ctatgcttct tgccacttga cggaattgta ctttgagaag 960 ggggagtact ttgtggagat gtactatcgg aatgagacgc agcacgagcc gtatcccctc 1020 atgctacctg gttgcagccc tagctgtcct ctggagaggt ttgctgagct ggttggccct 1080 gtgatccctc aagactggtc cacggagtgt atgaccacaa acagccatca aggtactgag 1140 gacaktacag atactagtgc ggccgcagat ctcgttgagc ccaaatcttg tgacaaaact 1200 cacacatgcc caccgtgccc agcacctgaa ctcctggggg gaccgtcagt cttcctcttc 1260 cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg 1320 gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag 1380 gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc 1440 agcgtcctca ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc 1500 tccaacaaag ccctcccagc ccccatcgag aaaaccatct cgaaagccaa agggcagccc 1560 cgagaaccac aggtgtacac cctgccccca tcccgggagg agatgaccaa gaaccaggtc 1620 agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc 1680 aatgggcagc cagagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc 1740 ttcttcctct atagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc 1800 tcatgctctg tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg 1860 tccccgggtg gtaaacccac ccatgtaaat gtgtctgttg ttatggctga ggttgatggc 1920 acatgttaca aagatgaact c 1941 <210> 25 <211> 645 <212> PRT <213> Artificial Sequence <220> <223> PAP-Fc utPK <400> 25 Met Ala Thr Gln Arg Arg Ala Asn Pro Ser Ser Leu His Leu Ile Thr   1 5 10 15 Val Pés Ser Leu Ala Ala Val Val Ser Ala Glu Val Asp Lys Glu              20 25 30 Leu Lys Phe Val Thr Leu Val Phe Arg His Gly Asp Arg Ser Pro Ile          35 40 45 Asp Thr Phe Pro Thr Asp Pro Ile Lys Glu Ser Ser Trp Pro Gln Gly      50 55 60 Phe Gly Gln Leu Thr Gln Leu Gly Met Glu Gln His Tyr Glu Leu Gly  65 70 75 80 Glu Tyr Ile Arg Lys Arg Tyr Arg Lys Phe Leu Asn Glu Ser Tyr Lys                  85 90 95 His Glu Gln Val Thr Ile Arg Ser Thr Asp Val Asp Arg Thr Leu Met             100 105 110 Ser Ala Met Thr Asn Leu Ala Leu Phe Pro Pro Glu Gly Val Ser         115 120 125 Ile Trp Asn Pro Ile Leu Leu Trp Gln Pro Ile Pro Val His Thr Val     130 135 140 Pro Leu Ser Glu Asp Gln Leu Leu Tyr Leu Pro Phe Arg Asn Cys Pro 145 150 155 160 Arg Phe Gln Glu Leu Glu Ser Glu Thr Leu Lys Ser Glu Glu Phe Gln                 165 170 175 Lys Arg Leu His Pro Tyr Lys Asp Phe Ile Ala Thr Leu Gly Lys Leu             180 185 190 Ser Gly Leu His Gly Gln Asp Leu Phe Gly Ile Trp Ser Lys Val Tyr         195 200 205 Asp Pro Leu Tyr Cys Glu Ser Val His Asn Phe Thr Leu Pro Ser Trp     210 215 220 Ala Thr Glu Asp Thr Met Thr Lys Leu Arg Glu Leu Ser Glu Leu Ser 225 230 235 240 Leu Leu Ser Leu Tyr Gly Ile His Lys Gln Lys Glu Lys Ser Arg Leu                 245 250 255 Gln Gly Gly Val Leu Val Asn Glu Ile Leu Asn His Met Lys Arg Ala             260 265 270 Thr Gln Ile Pro Ser Tyr Lys Lys Leu Ile Met Tyr Ser Ala His Hisp         275 280 285 Thr Thr Val Gly Leu Gln Met Ala Leu Asp Val Tyr Asn Gly Leu     290 295 300 Leu Pro Pro Tyr Ala Ser Cys His Leu Thr Glu Leu Tyr Phe Glu Lys 305 310 315 320 Gly Glu Tyr Phe Val Glu Met Tyr Tyr Arg Asn Glu Thr Gln His Glu                 325 330 335 Pro Tyr Pro Leu Met Leu Pro Gly Cys Ser Pro Ser Cys Pro Leu Glu             340 345 350 Arg Phe Ala Glu Leu Val Gly Pro Val Ile Pro Gln Asp Trp Ser Thr         355 360 365 Glu Cys Met Thr Thr Asn Ser His Gln Gly Thr Glu Asp Ser Thr Asp     370 375 380 Ala Ala Ala Asp Leu Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 385 390 395 400 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe                 405 410 415 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro             420 425 430 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val         435 440 445 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr     450 455 460 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Val Ser 465 470 475 480 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys                 485 490 495 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser             500 505 510 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro         515 520 525 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val     530 535 540 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 545 550 555 560 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp                 565 570 575 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp             580 585 590 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His         595 600 605 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Lys Pro     610 615 620 Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr Ala Gly Thr Cys 625 630 635 640 Tyr Lys Asp Glu Leu                 645 <210> 26 <211> 1941 <212> DNA <213> Artificial Sequence <220> <223> PAP-Fc utPK <400> 26 atggctactc aacgaagggc aaaccctagc tctctccatc taattactgt attctctctg 60 ctagctgctg tcgtctccgc tgaggtcgac aaggagttga agtttgtgac tttggtgttt 120 cggcatggag accgaagtcc cattgacacc tttcccactg accccataaa ggaatcctca 180 tggccacaag gatttggcca actcacccag ctgggcatgg agcagcatta tgaacttgga 240 gagtatataa gaaagagata tagaaaattc ttgaatgagt cctataaaca tgaacaggtt 300 tatattcgaa gcacagacgt tgaccggact ttgatgagtg ctatgacaaa cctggcagcc 360 ctgtttcccc cagaaggtgt cagcatctgg aatcctatcc tactctggca gcccatcccg 420 gtgcacacag ttcctctttc tgaagatcag ttgctatacc tgcctttcag gaactgccct 480 cgttttcaag aacttgagag tgagactttg aaatcagagg aatttcagaa gaggctgcac 540 ccttataagg attttatagc taccttggga aaactttcag gattacatgg ccaggacctt 600 tttggaattt ggagtaaagt ctacgaccct ttatattgtg agagtgttca caatttcact 660 ttaccctcct gggccactga ggacaccatg actaagttga gagaattgtc agaattgtcc 720 ctcctgtccc tctatggtat tcacaagcag aaagagaaat ctaggctcca agggggtgtc 780 ctggtcaatg aaatcctcaa tcacatgaag agagcaactc agataccaag ctacaaaaaa 840 cttatcatgt attctgcgca tgacactact gtgagtggcc tacagatggc gctagatgtt 900 tacaacggac tccttcctcc ctatgcttct tgccacttga cggaattgta ctttgagaag 960 ggggagtact ttgtggagat gtactatcgg aatgagacgc agcacgagcc gtatcccctc 1020 atgctacctg gttgcagccc tagctgtcct ctggagaggt ttgctgagct ggttggccct 1080 gtgatccctc aagactggtc cacggagtgt atgaccacaa acagccatca aggtactgag 1140 gacaktacag atactagtgc ggccgcagat ctcgttgagc ccaaatcttg tgacaaaact 1200 cacacatgcc caccgtgccc agcacctgaa ctcctggggg gaccgtcagt cttcctcttc 1260 cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg 1320 gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag 1380 gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc 1440 agcgtcctca ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc 1500 tccaacaaag ccctcccagc ccccatcgag aaaaccatct cgaaagccaa agggcagccc 1560 cgagaaccac aggtgtacac cctgccccca tcccgggagg agatgaccaa gaaccaggtc 1620 agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc 1680 aatgggcagc cagagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc 1740 ttcttcctct atagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc 1800 tcatgctctg tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg 1860 tccccgggtg gtaaacccac cctgtacaac gtgtccctgg tcatgtccga cacagctggc 1920 acctgctaca aagatgaact c 1941

Claims (11)

(a) an immune response domain comprising an antigenic protein, a target binding domain comprising an Fc antibody fragment, and an IgM or IgA-derived tailpiece Producing a transgenic plant expressing a chimeric antigen comprising a polymerisation domain which is capable of producing a chimeric antigen; And
(b) obtaining a protein from said transgenic plant.
2. The method of claim 1, wherein the chimeric antigen comprises an endoplasmic reticulum storage inducible sequence.
2. The method of claim 1, wherein the target binding domain of step (a) further comprises an immunoglobulin hinge region, a heavy chain CH1 domain or a linker.
The method according to claim 1, wherein the antigenic protein of step (a) is a colorectal cancer antigen GA733 or prostate acid phosphatase (PAP).
The method of claim 1, wherein the antibody Fc fragment of step (a) comprises a hinge region of IgG specific for GA733 or PAP (prostatic acid phosphatase), a CH2 domain and a CH3 domain.
4. The plant according to claim 1, wherein the plant is selected from the group consisting of tobacco ( Nicotiana tabacum ). &lt; / RTI &gt;
6. An immunogenic multimeric antigenic protein produced by the method of claim 1.
A vaccine composition comprising the immunogenic multimeric antigenic protein of claim 7 and a pharmaceutically acceptable carrier or diluent.
A chimeric antibody comprising an immune response domain comprising an antigenic protein, a target binding domain comprising an antibody Fc fragment (Fc antibody fragment), and a tailpiece derived from IgM or IgA Transforming a plant cell with a recombinant vector constructed to express a chimeric antigen
&Lt; / RTI &gt; wherein the method comprises the steps of:
10. The method of claim 9, wherein the chimeric antigen comprises an endoplasmic reticulum storage inducible sequence.
10. A plant produced by the method of claim 9 producing an immunogenic multimeric antigen protein.

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