KR20100097526A - Peptide antigen with enhanced immunogenicity and preparation method thereof - Google Patents

Abstract

PURPOSE: A peptide antigen with enhanced immunogenicity and a method for producing the same are provided to enhance immunogenicity and to increase antibody production efficiency. CONSTITUTION: A peptide antigen with enhanced immunogenicity comprises an amino acid sequence with two or more tandem repeat amino acid sequence unit. The peptide antigen additionally contains proline between amino acid sequence units. A vaccine composition contains the peptide antigen, nucleic acid encoding the same, or antibody to the peptide antigen. A method for producing the peptide antigen with immunogenicity comprises: a step of determining amino acid sequence corresponding to an epitope of a target antigen; and a step of preparing a polypeptide comprising two or more tandem repeat amino acid sequence units; or a step of preparing amino acid sequence which additionally contains proline.

Description

Peptide Antigen with enhanced immunogenicity and preparation method

The present invention relates to a method for producing a peptide antigen excellent in immunogenicity, a peptide antigen prepared by this method, a vaccine composition comprising the antigen, and a method for producing an antibody using the antigen.

The research for the present invention is the basic research project of the Korea Research Foundation, Ministry of Education, Science and Technology. [Task ID: KRF-2008-314-C00231 Title: Biological role of microsomal monooxygenase (MMO) and mitochondria and endoplasmic reticulum Cross-talk identification] and the Korea Research Foundation's key research institute support project [Project No.: KRF-2006-005-J03003 Project Name: Study on Regulatory Mechanism of Hormonal Metabolic Diseases].

Natural or recombinant proteins have been used to produce antibodies. However, native protein antigens are difficult to obtain in pure form, and recombinant proteins are limited to be used as ideal antigens due to expression in host cells, folding related problems, purification procedures, and the like.

Peptide antigens, on the other hand, can target specific region (s) of the entire protein, minimize the number of epitopes (antigenic determinants) and reduce cross-reactivity with antibodies produced by other antigens, while synthetic peptides are highly pure It has the advantage that it can be obtained in the form and can produce antibodies against proteins identified only by cDNA or EST clones without purification (1). However, it is not easy to identify epitope regions from the entire protein sequence. There has been a limitation in using it widely.

A vaccine is a medicine that injects an antigen into the human body to form an antibody necessary for an immune response, thereby having resistance to disease and immunity. Vaccines also refer to the introduction of harmless antigens with epitopes found in pathogens. Vaccination is also called an active immune process because it stimulates the immune system to develop its immunity to pathogens. Passive immunity, on the other hand, is a case of immediate but temporary protection obtained by injection of an antibody made by another animal.

As vaccines, killed or attenuated microorganisms, substances purified therefrom, subunits of proteins of microorganisms causing immune responses, and the like are used. Recently, synthetic vaccines composed of synthetic peptides have been developed. Synthetic vaccines can be synthesized by combining peptides with the same amino acid sequence as the epitope of the pathogen, or by combining them with other proteins, and genetic engineering techniques of genetic engineering are also applied.

The use of epitope-based vaccines has several advantages over the use of conventional vaccines. With the use of epitope-based vaccines, it is possible to avoid immunosuppressive epitopes that may be present in the full antigen, to selectively bind the necessary epitopes, and to modify the epitope composition to achieve improved immunogenicity, thereby targeting It can regulate the immune response to disease. Another major advantage of epitope-based immune stimulating vaccines is safety. Eliminates pathological side effects that are likely to be caused by infectious agents or whole protein antigens that may possess inherent biological activity.

In addition, DNA vaccines have been developed that administer DNA encoding the antigen. Usually DNA vaccines are injected into muscles in the form of plasmids.

In general, vaccines are used by adding an immunoactive agent such as alum and Freund's adjuvant in addition to the antigen to increase the formation of antibodies.

The contents of the prior art cited in the specification of the present invention are all incorporated herein. In addition, the information described herein is only intended to help the understanding of the technical background of the present invention, it is natural that it cannot act as a prior art for the present invention.

Peptide antigens and vaccines comprising them have many advantages, but have relatively low immunogenicity due to peptide length. Therefore, it is a problem to be solved in the present invention to develop a method for producing a peptide antigen with improved immunogenicity.

In order to solve the above problems, the present invention provides a polypeptide in which a proline is introduced between a polypeptide having a tandem repeat of an epitope region of a target antigen and a sequence having a sequence of repeating the epitope region, and a method of using the same.

Polypeptides having a repeating epitope region of the present invention have a higher immunogenicity than mono-epitope peptides, and when proline is introduced, the immunogenicity is higher, resulting in more antibody production efficiency. Therefore, the polypeptide according to the present invention enables more efficient production of antibodies to target antigens, and can be used to prepare more effective vaccine compositions.

The inventors of the present invention have shown that polypeptides having a series of repeated epitope regions in the peptide antigen have higher immunogenicity than mono-epitope peptides, and have higher immunogenicity when proline is introduced, thereby increasing antibody production efficiency. More specifically, the inventors of the present invention compared the effects of antibody production when using mono-epitope peptides and mono-epitope peptides in the epitope region from CYP1A2 or CYP 3A4. As a result, the single-repeat peptides in the epitope region were found to be 2.2-2.6 fold more efficient in producing polyclonal antibodies compared to mono-epitope peptides (FIG. 1A). Antibodies prepared from tandem peptides of CYP1A2 and 3A4 specifically recognized the corresponding peptides of human liver microsomes (FIG. 1B). These results indicate that repeated epitope peptides promote increased production of polyclonal antibodies as compared to conventional epitope sequences.

The inventors also introduced amino acid proline between repeating sequences and compared antibody production with no proline to investigate the relationship between the distance between epitope regions and antigenicity. The introduction of proline promoted antibody production, so that the absorbance value of the ELISA assay was about 2.0-2.5 times when the proline was not introduced at 100% (FIG. 2). This result suggests that the introduction of proline improves the antigenicity of CYP1A2 and 3A4 epitope repeat sequences. Introducing two prolines produced more antibodies.

In order to examine the effect on the antibody production when introducing another amino acid as a spacer between the sequence sequences, alanine (Ala) or glycine (Gly) instead of proline was introduced between the repeat sequences of CYP1A2, and the same assay was performed. Using alanine did not show a specific antibody production increase effect. On the other hand, when glycine was introduced, the absorbance was significantly decreased in the ELISA assay, indicating that the antigenicity of the peptide was reduced (FIG. 3).

Based on the experimental results, the present invention provides a peptide antigen with increased immunogenicity and a method of producing the same. The present invention is to determine the amino acid sequence corresponding to the epitope of the target antigen (s), and to prepare a polypeptide such that the unit of the amino acid is composed of two or more tandem repeat amino acid sequence, or the repeated amino acid sequence unit It includes a method of producing a peptide antigen having a high immunogenicity by producing a polypeptide consisting of an amino acid sequence further comprises a proline further in between.

A polypeptide according to the present invention is a polypeptide consisting of an amino acid sequence of two or more tandem repeat units of an amino acid sequence corresponding to an epitope of a target antigen (s). In addition, the polypeptide provided by the present invention is composed of an amino acid sequence of two or more tandem repeats of amino acid sequence units corresponding to epitopes of the target antigen (s), and between 0-3 repeating amino acid sequence units. A polypeptide consisting of an amino acid sequence further comprising dog, preferably 0-2 prolines.

2 to 10 epitope regions each, and more Can be repeated.

Peptides according to the invention may be prepared synthetically by recombinant DNA techniques or chemical synthesis or from natural sources such as natural tumors or pathogenic organisms. While the peptide is preferably substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments, the peptides can be synthetically conjugated to natural fragments or particles. Peptides according to the invention may vary in length and may be in their neutral (non-charged) form or in salt form.

Peptides of the invention can be prepared in a wide variety of ways. For relatively short sizes, the peptides can be synthesized in solution or on a solid support according to conventional techniques. Various automated synthesizers are commercially available and can be used according to known protocols (see, eg, Stewart amp: Young, SOLID PHASE PEPTIDE SYNTHESIS, 2D. ED .. Pierce Chemical Co .. 1984). Alternatively, recombinant DNA is inserted into an expression vector that encodes the immunogenic peptide to be prepared, transfected into a suitable host cell, such as a bacterium, yeast, insect, plant or mammalian cell, and cultured under conditions suitable for expression. Technology can be used. In general, such procedures are known in the art as described in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Press. Cold Spring Harbor.New York (1989). Nucleotide coding sequences for the peptide epitopes of the present invention can be synthesized by chemical techniques such as the phosphoester ester method of Matteucci, et al., J. Am. Chem. Soc. 103: 3185 (1981).

The peptide antigen with improved immunogenicity of the present invention can be used in combination with a polymer or other protein. The polymer that can be used may include, but is not limited to, polystyrene, polylactic-glycolic acid, and the like. In addition, the protein that can be bound to the peptide antigen of the present invention may include, but is not limited to, keyhole limpet hemocyanin, bovine plasma protein, obvalmin, tetanus toxin, and the like.

In addition, the present invention provides a peptide antigen having improved immunogenicity provided by the present invention, that is, a polypeptide comprising two or more tandem repeats of amino acid sequences of selected epitope regions of a target antigen or a sequence in which the epitope regions are in line repeat Provided is a vaccine composition comprising a polypeptide introduced with proline between and a method of producing the same. The present invention also provides a vaccine composition comprising a nucleic acid encoding a peptide antigen with improved immunogenicity and a method of producing the same. The present invention also provides a vaccine composition comprising an antibody against the polypeptide having improved immunogenicity provided by the present invention and a method of producing the same. The vaccine composition of the present invention can be used to prevent or treat infectious diseases, malignant diseases or autoimmune diseases.

Carriers that can be included in the vaccine composition of the present invention are well known in the art and include, for example, tyroglobulin, albumin such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, Hepatitis B virus core protein. The vaccine may contain physiologically tolerant (ie acceptable) diluents such as water or saline, preferably phosphate complete saline. Typically, the vaccine also contains an adjuvant. Adjuvant such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide or alum are examples of materials well known in the art.

In addition, the vaccines of the invention may comprise antigen presenting cells (APCs), such as dendritic cells (DCs), as vehicles for providing peptides of the invention. After the vaccine composition is produced in vitro, loading of the dendritic cells takes place in vitro by immobilizing and harvesting the dendritic cells. For example, dendritic cells are transfected with, for example, the minigene according to the invention, or pulsed with peptides. Dendritic cells can then be administered to the patient to elicit an immune response in vivo.

The vaccine composition of the present invention may comprise DNA or RNA encoding the peptide antigen of the present invention. Methods for this are described, for example, in Woff et al., Science 247: 1465 (1990), as well as US Pat. Nos. 5,580,859, 5,589,466, 5,804,566, 5,739,118, 5,736,524, 5,679,647, WO 98 / 04720, incorporated herein by this reference.

Peptides according to the invention can be immunized with a host by injection, aerosol, oral, transdermal, dural, pleural, intravesical or other routes. The peptide of the present invention and / or nucleic acid encoding the peptide can also be administered using liposomes.

The vaccine composition of the present invention can also be used as a prophylactic agent. Typically, the initial vaccination dose will generally be in the range of about 1, 5, 50, 500 or 1000 μg at low doses and about 10,000, 20,000, 30,000 or 50,000 μg at high doses. Dosages for administration to humans typically range from about 500 μg to about 50,000 μg for a 70 kg patient. Following the initial vaccine administration, a boosting dosage of about 1.0 μg to about 50,000 μg of peptide is administered at prescribed intervals of about 4 weeks to 6 months.

The peptide and nucleic acid compositions of the invention can be provided in kit form with instructions for vaccine administration. Typically, the kit will include the desired peptide composition in a container, preferably in unit dosage form, and instructions for administration. Other kit components may also include, for example, sterile syringes, booster doses and other excipients, and the like.

The present invention also provides a method for more efficiently producing an antibody against a target antigen. For this purpose, a polypeptide prepared by repeating two or more tandem repeats of the amino acid sequence of the selected epitope region of the target antigen or a polypeptide in which proline is introduced between the sequences in which the epitope region is in series repeat. Antibodies are prepared using conventional methods known in the art.

The invention also provides antibodies that selectively bind to the polypeptides of the invention. Antibodies of the invention include polyclonal antibodies, monoclonal antibodies, human antibodies and humanized antibodies. Examples of antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments; Diabody; Linear antibodies (Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995)); Single chain antibody molecules; And multispecific antibodies formed from antibody fragments and the like. Digestion of the antibody with papain results in two identical antigen binding fragments, each "Fab" fragment with a single antigen binding site, and the remainder "Fc" fragment. Treatment of pepsin results in an F (ab ') 2 fragment that has two antigen binding sites and still can crosslink antigen. Fv is a minimal antibody fragment comprising a complete antigen recognition and binding site. This site consists of a dimer of one heavy chain and one light chain variable region and is tightly coupled non-covalently.

Methods of making polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies can be prepared by injecting the mammal with one or more immunizing agents, if necessary, with an adjuvant. Typically, an immunizing agent and / or adjuvant is injected into the mammal several times by subcutaneous injection or intraperitoneal injection. The immunizing agent may be a protein of the invention or a fusion protein thereof. Injecting an immunizing agent with a protein known to be immunogenic in the mammal being immunized can be effective. Adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose decolinomycolate). Immunization methods can be chosen by one skilled in the art without undue experimentation.

Monoclonal antibodies according to the invention can be prepared by the hybridoma method described in Kohler et al., Nature, 256: 495 (1975), or by recombinant DNA methods (e.g., US Pat. No. 4,816,576). Reference). Monoclonal antibodies are also described, for example, in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991). It can be isolated from phage antibody libraries using the techniques described.

Monoclonal antibodies herein are specifically identical to or different from the corresponding sequences of antibodies from a particular species or antibodies belonging to a particular antibody class or subclass, provided that a portion of the heavy and / or light chains exhibits the desired activity. Although homologous, the remainder of the chain (s) includes "chimeric" antibodies that are identical or homologous to antibodies from other species or to antibodies belonging to different antibody classes or subclasses or fragments of such antibodies (Morrison et al. , Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

A “humanized” form of a non-human (eg murine) antibody is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof comprising a minimum sequence derived from a non-human immunoglobulin (eg, Fv, Fab, Fab ', F (ab') 2 or other antigen binding sequence of the antibody. In most cases humanized antibodies are human immunoglobulins in which residues of the complementarity determining regions (CDRs) of the recipient are replaced with CDR residues of species other than the human (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and ability. (Receptor antibody). In some cases, Fv framework residues of human immunoglobulins are replaced by corresponding non-human residues. Humanized antibodies may also include residues that are not found in the recipient antibody or in the CDR or framework sequences to be introduced. In general, humanized antibodies comprise substantially all of one or more, generally two or more variable domains, wherein all or substantially all CDR regions correspond to regions of non-human immunoglobulins, and all or substantially all FR regions Corresponds to the region of human immunoglobulin sequence. Humanized antibodies also include at least a portion of an immunoglobulin constant region (Fc), generally a portion of a human immunoglobulin region (Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992)).

"Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody present in a single polypeptide chain. Preferably, the Fv polypeptide also comprises a polypeptide linker between the VH and VL domains that allows the sFv to form the structure necessary for antigen binding. "Diabody" refers to a small antibody fragment (VH-VL) with two antigen binding sites, comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) within the same polypeptide chain. By using a linker that is too short to join two domains in the same chain, the domain is forcibly bound to the complementary domains of the other chain to create two antigen binding sites.

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

Example

BALB / c mice (5-7 weeks old) used in the present invention were purchased from Samtaco (Osan, Korea). Peptide antigens listed in Table 1 were synthesized in peptron (Daejeon, Korea). Keyhole Limpet Hemocyanin (KLH) was purchased from Pierce Biotechnology (Rockford, IL) and human human microsomes from BD Biosciences (San Jose, Calif.).

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Peptides and KLH Combination of

Peptides were bound to KLH using glutaraldehyde as a crosslinker (8). The molar ratio of KLH: peptide antigen was 1: 100. KLH and peptide were dissolved in 2 mL of 0.01 M phosphate buffer (pH 7.4). Glutalaldehyde (20 mM, 1 mL) was added to the reaction sample for peptide-KLH binding. Unreacted glutaraldehyde was removed by dialysis with an excess of phosphate buffered saline. Peptide concentration was determined by fluorescamine assay (9).

immune

Peptide-KLH complexes were mixed with complete or incomplete Freund's adjuvant 1: 1 (v / v) in a mullion to prepare injection samples. The injection volume for immunization of each mouse was about 0.4 mL. Antigen was intracutaneously administered (100 μl / injection) four times using a 28-gauge standard disposable gauge to the skin of mice (5 mice per group) and the like. Two weeks later the animals were boosted in the same manner. Two weeks after the last injection, a total of about 300 μl of blood sample was taken from the tail vein of each mouse.

Weston Blot Assay

Western blotting analysis was performed using human liver microsomes to detect cytochrome P450 1A2 (CYP1A2) and 3A4 (CYP3A4) proteins.

ELISA Assay

Levels of anti-peptide antibodies were determined using conventional ELISA methods. Each peptide is first combined with activated bovine serum albumin (Imject Maleimide Activated BSA, Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions, and the peptide-serum albumin (BSA) complex as an antigen of ELISA Used.

Statistical analysis

The results of five independent experiments were expressed as mean ± SE, and Student's t testes were performed.

< Example 1 > Repeated Epitope  Antibody Production Effects of Antigens Containing

Cytochrome Peptide antigens of 1A2 (CYP1A2) and 3A4 (Table 1) were established as epitope regions (2,3). The inventors of the present invention have reported that polyclonal antibodies can be produced in rabbits when the peptide is bound to a polymer carrier (4). In the present invention, the effects of antibody production when tandem repeats of epitope regions were studied.

Blood was collected from immunized mice by injecting antigen and serum samples diluted 2000-fold were analyzed by conventional ELISA methods using the peptide-BSA conjugate described above as antigen. Anti-mouse IgG peroxidase conjugate and 2,2'-azino-di- (3 ethylbenzthiazoline sulfonic acid) were used as substrates of secondary antibodies and peroxidases, respectively. Values due to antigens that specifically bind to the wells were subtracted (FIG. 1A). Western blot analysis of human liver microsomes using anti-sera prepared from CYP1A2 or 3A4 single repeat peptides. Blot results were developed with ECL ^™ by conventional methods. Two microsome protein samples (80 μg for CYP1A2 detection and 40 μg for CYP3A4 detection) were used for immunoblotting (FIG. 1 B).

As a result of antibody analysis after antigen injection, the sequence repeating peptides (1A2-TR and 3A4-TR in Table 1, respectively) of epitope regions from CYP1A2 or 3A4 were poly-unsaturated compared to mono-epitope peptides (1A2-M and 3A4-M). 2.2-2.6 fold more efficient in clonal antibody production (FIG. 1A). Antibodies prepared from tandem peptides of CYP1A2 and 3A4 specifically recognized the corresponding peptides of human liver microsomes (FIG. 1B). These results indicate that repeated epitope peptides promote increased production of polyclonal antibodies as compared to conventional epitope sequences.

< Example  2> Repeated Epitope  Effect of the introduction of amino acid proline between regions

To investigate the relationship between the distance between epitope regions and antigenicity, amino acid proline was introduced between repeating sequences and antibody production was compared with no proline introduction.

Peptide antigens were synthesized to contain one or two prolines between repeated epitopes, and the amount of antibody produced was compared to that of epitope line repeats without control of proline (control: 1A2-TR and 3A4-TR). . All experimental conditions were the same as in Example 1.

The introduction of proline promoted antibody production, so that the absorbance value of the ELISA assay was about 2.0-2.5 times when the proline was not introduced (using the 1A2-TR peptide control) at 100% (FIG. 2). This result suggests that when proline is introduced (1A2-TR / P, 3A4-TR / P), the antigenicity of CYP1A2 and 3A4 epitope repeat sequences is improved. Introducing two prolines (1A2-TR / PP, 3A4-TR / PP) produced more antibodies.

Since proline is known as a helix-breaking amino acid in the alpha-helix region of the polypeptide, this effect of proline was thought to be derived from the secondary structure of the peptide. For example, the network protein sequence analysis (5) method predicted that the 3A4-TR peptide had a high alpha-helix content, while the 3A4-TR / P and 3A4-TR / PP peptides had almost random structures. This may be due to the presence of proline in the peptide sequence. Thus, the results of this experiment contradict the previous reports (6,7) that peptide antigenicity is enhanced by stabilizing helix at the C-terminus of chicken riboflavin carrier protein.

< Example  3> Repeated Epitope  Between amino acid alanine or glycine Degree Mouth effect

To investigate the effect on the antibody production when introducing other amino acids as spacers between the sequence of epitopes, alanine (Ala) or glycine (Gly) was introduced instead of proline between the repeats of CYP1A2 and the same assay was introduced. It was. The experiment was performed under the same conditions as described in Example 1.

When alanine was used as a spacer (1A2-TR / A, 1A2-TR / AA), it did not show an increase in specific antibody production compared to the sequence of repeating epitopes without a spacer (1A2-TR). On the other hand, when glycine (1A2-TR / G, 1A2-TR / GG), which is also known as helix-breaking amino acid, was introduced, the ELISA assay compared to the sequence of repeating epitopes without a spacer (1A2-TR). The absorbance was markedly reduced, showing that the antigenicity of the peptide was reduced (FIG. 3).

references

Angeletti, RH (1999) Design of useful peptide antigens. J. Biomol . Tech. 10 , 2-10.

Edwards, RJ, Singleton, AM, Murray, BP, Davies, DS, and Boobis, AR (1995) Short synthetic peptides exploited for reliable and specific targeting of antibodies to the C-termini of cytochrome P450 enzymes. Biochem . Pharmacol . 49 , 39-47.

Wang, RW, Newton, DJ, Liu, NY, Shou, M., Rushmore, T., and Lu, AY (1999) Inhibitory anti-CYP3A4 peptide antibody: mapping of inhibitory epitope and specificity toward other CYP3A isoforms. Drug Metab . Dispos . 27 , 167-172.

4.Kim, M., Yun, CH., Park, SK, Seo, JH, and Ahn, T. (2007) Production of polyclonal antibodies against peptide antigens using polystyrene beads as a carrier. Biotechnol . Lett . 29 , 1735-1740.

5. Combet, C., Blanchet, C., Geourjon, C., and Deleage, G. (2000) NPS @: network protein sequence analysis. Trends Biochem . Sci . 25 , 147-150.

6. Gurunath, R., Beena, TK, Adiga, PR, and Balaram, P. (1995) Enhancing peptide antigenicity by helix stabilization. FEBS Lett . 361 , 176-178.

7. Subramanian, S., Karande, AA, and Adiga, PR (2001) Helix stabilization in the C-terminal peptide of chicken riboflavin carrier protein enhances immunogenicity and prolongs contraceptive potential as an epitope-based vaccine in female rats. Biochem . Biophys . Res . Commun. 14 , 236-243.

8. Reichlin, M. (1980) Use of glutaraldehyde as a coupling agent for proteins and peptides. Methods Enzymol . 70 , 159-165.

9. Castell, JV, Cervera, M., and Marco, R. (1979) A convenient micromethod for the assay of primary amines and proteins with fluorescamine. A re-examination of the conditions of reaction. Anal . Biochem . 99 , 379-391.

Figure 1 shows the effect (A) on the antibody production of the repeating peptide sequence of the epitope and Western blotting results (B) using the antibody against the repeating peptide.

Figure 2 shows the effect on antibody production when proline is introduced into the CYP1A2 or 3A4 epitope peptide.

3 is This is a result of comparing the antibody produced by the peptide antigen with the case where no spacer was introduced when alanine or glycine was introduced between the sequence sequences of epitopes of CYP1A2.

Claims (9)

A polypeptide consisting of an amino acid sequence of two or more tandem repeat units of an amino acid sequence corresponding to an epitope of a target antigen (s). Polypeptide consisting of an amino acid sequence of two or more tandem repeats of amino acid sequence units corresponding to epitopes of the target antigen (s), and an amino acid sequence further comprising proline between repeated amino acid sequence units . The polypeptide of claim 2, wherein 1-2 prolines are added. The polypeptide of claim 1, wherein the polypeptide is linked to a polymer or other protein. A nucleic acid encoding the polypeptide of claim 1. An antibody against the polypeptide of claim 1. A vaccine composition comprising one or more selected from the group consisting of the polypeptide of claim 1, the nucleic acid of claim 5, and the antibody of claim 6. A method of producing an antibody using the polypeptide of claim 1. Determine an amino acid sequence corresponding to the epitope of the target antigen (s) and prepare a polypeptide such that the unit of amino acid consists of two or more tandem repeat amino acid sequences, or between the repeated amino acid sequence units A method of producing a peptide antigen having high immunogenicity by producing a polypeptide consisting of an amino acid sequence further comprising proline.

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