KR102508883B1 - A method for producing immunogenic composition - Google Patents

Abstract

The present invention relates to a method for manufacturing an immunogenic composition. The manufacturing method includes a step of forming multimeric protein assemblies self-assembled by mixing: (i) a fusion protein in which SEQ ID NO: 4 or a protein having at least 75% sequence identity therewith and SEQ ID NO: 5 or a protein having at least 75% sequence identity therewith are linked via a linker; and (ii) a protein having SEQ ID NO: 1 or at least 75% sequence identity therewith. The present invention has an effect of achieving excellent yield even in terms of scale-up.

Description

Method for producing an immunogenic composition {A method for producing immunogenic composition}

The present invention relates to methods of preparing immunogenic compositions.

The new coronavirus (2019-nCoV) that occurred in Wuhan, China in 2019 was named COVID-19 in the sense that human infection was first confirmed in the second half of 2019, and it is a virus that causes respiratory diseases in humans and animals. It is known that various symptoms appear after an incubation period of up to two weeks at work. The main symptoms include lethargy, high fever of 37.5 degrees or higher, cough, sore throat, phlegm, muscle pain, headache, shortness of breath and pneumonia, and respiratory failure due to lung damage. Similar diseases include MERS and SARS, which have a lower mortality rate than MERS or SARS, but are known to be much more contagious. There is an increasing demand in the medical industry for the production of efficient vaccines for the prevention of COVID-19 and the production of vaccine antigen proteins.

Accordingly, the present inventors developed a method for producing a protein used in a coronavirus vaccine and completed the present invention.

US 2021-0338807 A1

An object of the present invention is to provide a method for preparing an immunogenic composition.

One aspect of the present invention is a method for preparing an immunogenic composition.

In one embodiment, the preparation method comprises (i) SEQ ID NO: 4 or a protein having at least 75% sequence identity therewith and SEQ ID NO: 5 or a protein having at least 75% sequence identity therewith linked through a linker, a fusion protein; and (ii) mixing SEQ ID NO: 1 or a protein having at least 75% sequence identity thereto to form a self-assembled multimeric protein assembly.

As the manufacturing method according to the foregoing embodiment, the molar ratio of (i) and (ii) is 1:10 to 2:1.

As the manufacturing method according to any one of the preceding embodiments, the mixing reaction time exceeds 1 hour.

As the manufacturing method according to any one of the preceding embodiments, the mixing step is performed in the presence of a buffer containing a stabilizer.

As the manufacturing method according to any one of the preceding embodiments, the stabilizer is polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85 , Poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleate , arginine, sucrose or combinations thereof.

As the manufacturing method according to any one of the preceding embodiments, the content of the stabilizer in the buffer is greater than 0 and less than 0.050 v / v%.

A manufacturing method according to any one of the preceding embodiments, wherein the manufacturing method includes filtering the composition after the mixing step.

As the manufacturing method according to any one of the preceding embodiments, the filtration step includes filtering with a 0.01 μm to 0.5 μm filter.

As a manufacturing method according to any one of the preceding embodiments, the linker of (i) is a peptide linker.

In the preparation method according to any one of the preceding embodiments, the linker of (i) includes a Gly-Ser linker.

In the preparation method according to any one of the preceding embodiments, the Gly-Ser linker is (GGGGS) n, (GSSGSS) n, (GSSSSSS) n, (SSSSSSS) n, (GSGGSGSGSGGSGSG) n, (GGSGGSGS) n or (GSGGSGSG) )n is the linker.

In the preparation method according to any one of the preceding embodiments, the linker of (i) includes a helical linker sequence.

In the preparation method according to any one of the preceding embodiments, the helix linker sequence is represented by EKAAKAEEAAR.

In the preparation method according to any one of the above embodiments, the fusion protein of (i) has SEQ ID NO: 6 or 75% or more identity therewith.

As the production method according to any one of the preceding embodiments, the multimeric protein assembly includes (i) a trimer of a fusion protein and (ii) a pentamer of a protein.

As the production method according to any one of the preceding embodiments, the multimeric protein assembly is composed of (i) 20 trimers of fusion proteins and (ii) 12 pentamers of proteins.

As a manufacturing method according to any one of the preceding embodiments, the multimeric protein assembly has an average diameter of 20 nm to 80 nm.

As a manufacturing method according to any one of the preceding embodiments, the multimeric protein assembly has an icosahedral structure.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below.

In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Also, such equivalents are intended to be included in this invention.

As used herein, the term "about" may be preceded by a specific numerical value. As used in this application, the term "about" includes not only the exact number that follows the term, but also a range that is or is close to that number. It can be determined whether the number is close to or nearly the specific number mentioned, given the context in which it is presented. As an example, the term “about” can refer to a range of -10% to +10% of a numerical value. As another example, the term "about" can refer to a range of -5% to +5% of a given numerical value. However, it is not limited thereto.

As used herein, the term “and/or” when used herein is to be understood as specifically disclosing each of the two specified features or elements, either with or without the other. Thus, the term "and/or" as used herein in phrases such as "A and/or B" includes "A and B", "A or B", "A" (alone), and "B" (alone). It is intended to include Likewise, the term "and/or" as used in phrases such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

As used herein, the term "consisting essentially of" means that a non-specified ingredient may be present if the characteristics of the claimed subject matter are not substantially affected by the presence of the non-specified ingredient.

As used herein, the term “consisting of” means that the proportions of the specified component(s) total 100%. Ingredients or features that come after the term “consisting of” may be essential or mandatory. In some embodiments, in addition to the ingredients or features that come under "consisting of", any other ingredients or non-essential ingredients may be excluded.

As used herein, the term "comprising" refers to the presence of any of the features, steps or components described below the term, and does not exclude the presence or addition of one or more features, steps or components. Components or features described herein under "comprising" may be required or mandatory, but in some embodiments may further include other optional or non-essential components or features.

As used herein, the term “comprising” may, in some embodiments, be modified to refer to “consisting essentially of” or “consisting of”.

With reference to amino acid sequences herein, a polypeptide "comprising" the amino acid sequence set forth in a particular SEQ ID NO, a polypeptide "consisting of' the amino acid sequence set forth in a particular SEQ ID NO, or a polypeptide or protein that "has" the amino acid sequence set forth in a particular SEQ ID NO. Even if it is described, as long as it has the same or equivalent activity as the polypeptide consisting of the amino acid sequence of the corresponding sequence number, proteins having amino acid sequences in which some sequences have been deleted, modified, substituted, conservatively substituted or added can also be used in this application. It is self-evident that it can For example, if the amino acid sequence has an addition of a sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation thereof, or a conservative substitution to the N-terminus and/or C-terminus of the amino acid sequence. It may, but is not limited thereto.

One aspect of the present invention is (i) a fusion protein in which SEQ ID NO: 4 or a protein having at least 75% sequence identity therewith and SEQ ID NO: 5 or a protein having at least 75% sequence identity therewith are linked via a linker; and (ii) mixing SEQ ID NO: 1 or a protein having 75% or more sequence identity thereto to form a self-assembled nanostructure. The multimeric protein assembly of the present invention may also be referred to as "nanostructure (sieve)" or "Nanoparticle".

The fusion protein of (i), which is one component of the multimeric protein assembly of the present invention, can be used to prepare a multimeric protein assembly displaying an antigen or antigen fragment used in the preparation of an immunogenic composition. The fusion protein of (i) may include proteins disclosed in WO2019-169120, US 9630994 B2, WO2021-163481, WO2021-163438, and PCT/US2021/037341.

Specifically, in the fusion protein, the protein of SEQ ID NO: 4 is a fragment of the SARS-CoV-2 Spike protein, and may exhibit immunogenicity. As used herein, the term "immunogenicity" refers to the ability of a specific protein or a specific region thereof to elicit an immune response against a specific protein or a protein comprising an amino acid sequence having a high degree of identity with the specific protein. do. Thus, the immunogenic protein that may be included in the multimeric protein assembly is SEQ ID NO: 4 or at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, It may have sequences with greater than 97%, 98% or 99% identity.

The protein of SEQ ID NO: 5 of the present invention is linked to an immunogenic protein to form the structure of a multimeric protein assembly and to display an antigen. The protein of SEQ ID NO: 5 may include any modification that does not disrupt assembly into a multimeric protein assembly, and may include, for example, amino acid addition, deletion, insertion, substitution, or modification. In one embodiment, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of SEQ ID NO: 5 or thereto. It can have sequences with identity. In any one of the embodiments described above, the protein that forms the structure of the multimeric protein assembly and displays the antigen linked to the immunogenic protein has the amino acid sequence of SEQ ID NO: 5, 17, 18, 19, 20 or 21 It may include, consist essentially of, or consist of the sequence.

In any one of the above-described embodiments, in the range of proteins linked to immunogenic proteins to form the structure of multimeric protein assemblies and displaying antigens, SEQ ID NO: 5 and at least 1, 2, 3, 4 or 5 contain identical amino acid sequences at identified interface positions. In the protein, the interface residues identified as being present at the interface to be assembled to form the nanostructure may be 25, 29, 33, 54, and 57 from the N-terminus of SEQ ID NO: 5.

The linker may be of any suitable length. For example, the linker may be a peptide linker, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 It may be composed of one or more amino acids. In one embodiment, it may be a Gly-Ser linker containing glycine and/or serine residues. In any one of the foregoing embodiments, the linker is a (GGGGS)n, (GSSGSS)n, (GSSSSSS)n, (SSSSSSS)n, (GSGGSGSGSGGSGSG)n, (GGSGGSGS)n or (GSGGSGSG)n linker. can be In any one of the foregoing embodiments, the linker may comprise a helical linker sequence. Specifically, the helix linker may be represented by an EKAAKAEEAAR sequence. As a specific example of the linker, the sequence of amino acids 237 to 263 from the N-terminus of SEQ ID NO: 6 may be cited, but is not limited thereto.

Connection of the antigenic protein and the nanostructure may be achieved by expressing a fusion protein fused to the N- or C-terminus, such as, for example, the fusion protein of SEQ ID NO: 6, but is not limited thereto, and post-translational covalent attachment. or can be achieved by non-covalent attachment. Also, depending on how the antigenic protein is attached, the antigenic protein can be displayed in any orientation.

In any one of the foregoing embodiments, the fusion protein of (i) is SEQ ID NO: 6 or at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, It may have sequences with at least 95%, 96%, 97%, 98% or 99% identity. The protein of SEQ ID NO: 6 herein may be referred to as "Component A". A plurality of isolated protein monomers of SEQ ID NO: 6 may self-assemble through interactions to form a trimer. This assembly can be achieved through non-covalent protein-protein interaction reactions.

In the multimeric protein assembly of the present invention, the protein of (ii) may be used to prepare a multimeric protein assembly displaying an antigen or antigen fragment used in the preparation of an immunogenic composition. The protein of (ii) may be a protein disclosed in WO2019-169120, US 9630994 B2, WO2021-163481, WO2021-163438, PCT/US2021/037341. Specifically, the protein of (ii) may be the protein represented by SEQ ID NO: 1, but those having the same function are included without limitation.

In one embodiment, the target protein of the present invention is SEQ ID NO: 1 or at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, It may have sequences with greater than 98% or 99% identity. In any one of the foregoing embodiments, the protein of (ii) comprises, or consists essentially of, the amino acid sequence of SEQ ID NO: 1, 9, 10, 11, 12, 13, 14, 15 or 16 It may consist essentially of or consist of. In any one of the foregoing embodiments, the protein of interest of the invention has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 identified interfaces ) contains the same amino acid sequence at the position. In the target protein, the interface residues identified as being present at the interface to be assembled to form the nanostructure are 24, 28, 36, 124, 125, 127, 128, 129, 131 from the N-terminus of SEQ ID NO: 1. , 132, 133, 135, and 139. The protein of SEQ ID NO: 1 herein may be referred to as "Component B". A plurality of isolated protein monomers of SEQ ID NO: 1 may self-assemble through interactions to form a pentamer. This assembly can be achieved through non-covalent protein-protein interaction reactions.

Such Component B and Component A may self-assemble to form an immunogenic composition, and for the purpose of the present application, may be one component of a vaccine against coronavirus. In one aspect, the multimeric protein assembly prepared by self-assembly of Component A and Component B is a nanoparticle disclosed in WO2019-169120, US 9630994 B2, WO2021-163481, WO2021-163438, PCT / US2021 / 037341 can

The proteins (i) and (ii) of the present invention may contain any modifications that do not perturb assembly into multimeric protein assemblies, for example including amino acid additions, deletions, insertions, substitutions or modifications. can Examples include cases involving conservative sequence modifications.

In the present invention, "conservative sequence modifications" may refer to amino acid modifications that do not significantly affect or change the structural properties of a protein of interest. Such conservative modifications include amino acid substitutions, additions and deletions. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Such substitutions are generally based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Families of amino acid residues with similar side chains have been defined in the art. This family includes basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), non-charged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. For example, tyrosine, phenylalanine, tryptophan, histidine).

The multimeric protein assembly of the present invention may be formed by self-assembly of a trimer of Component A and a pentamer of Component B. The multimeric protein assembly formed by self-assembly of Component A and Component B may also be referred to as "Nanoparticle", "Nanoparticle", or "Nanostructure".

Specifically, the multimeric protein assembly of the present invention may have a structure including (i) 20 trimers of the fusion protein (Component A) and (ii) 12 pentamers of the protein (Component B). there is. Thus, the multimeric protein assembly of the present invention contains 60 copies of Component A and 60 copies of Component B. Meanwhile, the multimeric protein assembly may be formed by non-covalent interactions between (i) and (ii). That is, it may be that no covalent bond is formed between (i) and (ii).

The multimeric protein assembly of the present invention may have an icosahedral symmetrical structure. The multimeric protein assembly of the present invention has an average diameter of about 20 nm to about 80 nm, wherein the lumen may be about 15 nm to about 32 nm horizontally.

In one embodiment, the method for preparing a multimeric protein assembly of the present invention may include mixing the fusion protein of (i) and the protein of (ii) at a molar ratio of 1:10 to 2:1.

For example, in the mixing step, the molar ratio of the fusion protein of (i) to the protein of (ii) is about 1:9, 1:8, 1:7, 1:6, 1:5.5, 1:5, 1: 4.5, 1:4, 1:3.5, 1:3, 1:2.5, 1:2.4, 1:2.3, 1:2.2, 1:2.1, 1:2, 1:1.9, 1:1.8, 1:1.7, 1:1.6, 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1, 1:1, 1.05:1, 1.1:1, 1.15:1, 1.2:1, 1.25:1 or about 1.3 : can be 1. In another aspect, the molar ratio between the fusion protein of (i) and the protein of (ii) may be about 1:0.9 to 1.1, or 1:1.0 to 1.1.

The time for performing the mixing reaction is not particularly limited, but may be performed in excess of 1 hour. For example, it may be performed for about 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours or about 4 hours. For example, greater than about 0.5 hours and less than 4 hours, greater than about 0.5 hours and less than 3.5 hours, greater than about 1 hour and less than 3.5 hours, greater than about 1.5 hours and less than 3 hours, greater than 1.5 hours and less than 2.5 hours, greater than 1.8 hours and less than 2.2 hours, or 1.9 hours. It can be performed for more than 2.1 hours or less.

In any one of the foregoing embodiments, the mixing step may be performed in the presence of a buffer containing a stabilizer.

The stabilizer is polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer-188, sorbitan monolaurate, selected from sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleate, arginine, sucrose or combinations thereof can For example, it may be polysorbate-80.

The content of the stabilizer may be greater than 0 and less than 0.050 v/v%. Specifically, about 0.001% or more and less than 0.050%, 0.005% or more and less than 0.050%, 0.01% or more and less than 0.050%, 0.001% or more and 0.045% or less, 0.005% or more and 0.045% or less, 0.01% or more and 0.045% or less, 0.001% or more 0.040 % or less, 0.005% or more 0.040% or less, 0.01% or more 0.040% or less, 0.001% or more 0.030% or less, 0.005% or more 0.030% or less, 0.01% or more 0.030% or less, 0.001% or more 0.035% or less, 0.005% or more 0.035% 0.01% or more and 0.035% or less, 0.01% or more and 0.025% or less, 0.015% or more and less than 0.050%, 0.02% or more and less than 0.050%, 0.015% or more and 0.045% or less, 0.02% or more and 0.045% or less, 0.015% or more and 0.040% or less . In one aspect, the content of the stabilizer may be about 0.01% or more and 0.025% or less.

In any one of the above-described embodiments, after the mixing step, the step of filtering the composition comprising the multimeric protein assembly of the present invention may be further included. The filtration step may include passing the permeate through a filter having a pore size of about 0.01 μm to about 0.5 μm, for example about 0.1 μm to about 0.3 μm, specifically about 0.2 μm. However, it is not limited thereto.

A solution containing the fusion protein of (i) and the protein of (ii), which are components of the multimeric protein assembly of the present invention, can be provided as described below.

The target protein of the present invention can be recovered from prokaryotic or eukaryotic cells. For example, it can be produced in animal cells, Escherichia coli, yeast, insect cells, plant cells, and living animals through genetic recombination. For example, prokaryotic and eukaryotic cells may be transformed or transfected with an expression vector containing a nucleic acid encoding the target protein of the present invention. Such transformation or transfection of expression vectors includes standard bacterial transformation, calcium phosphate co-precipitation, electroporation, or liposome mediated transfection, DEAE dextran mediated transfection, polycationic mediated transfection or viral mediated transfection. This may be achieved through any technique known in the art, but is not limited thereto. (See, e.g., Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press); Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY])).

In one embodiment, the fusion protein of (i) may be recovered from a eukaryotic cell expressing the protein. Animal cells may be used as examples of the eukaryotic cells, examples of which may include mammalian cells, insect cells, fish cells, and the like, and for example, hamster ovary cells (CHO cells) may be used, but are not limited thereto.

In one embodiment, the protein of (ii) may be recovered from a strain (prokaryotic cell) expressing the protein. In any one of the above embodiments, the strain may be a microorganism of the genus Escherichia, for example, Escherichia coli ( E. coli ), but is not limited thereto.

The prokaryotic or eukaryotic cell may include a polynucleotide encoding a component of the multimeric protein assembly of the present invention, or a vector containing the polynucleotide.

The polynucleotide encoding the target protein provided in the present invention can be used within a range that does not change the amino acid sequence of the protein due to codon degeneracy or considering codons preferred in organisms in which the protein is to be expressed. Various modifications may be made to the coding region.

In addition, the polynucleotide of the present invention hybridizes with a probe that can be prepared from a known gene sequence, for example, a complementary sequence for all or part of the nucleotide sequence under stringent conditions to encode the target protein of the present invention. Any sequence may be included without limitation.

The "stringent condition" means a condition that allows specific hybridization between polynucleotides. These conditions are described in the literature (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology , John Wiley & Sons, Inc., New York).

A multimeric protein assembly comprising the protein of interest of the present invention can be used as a component of an immunogenic composition. Accordingly, another aspect of the present invention provides an immunogenic composition comprising the above multimeric protein assembly.

The immunogenic composition can be used to raise antibodies in mammals.

An immunogenic composition of the present invention may include an immunologically effective amount of a multimeric protein assembly. The term "immunologically effective amount" is an amount effective to elicit an antibody response to an antigen when administered to a subject. The immunologically effective amount depends on the health and physical condition of the individual being treated, the age of the individual, the ability of the individual's immune system to synthesize antibodies, the level of protection required, the formulation of the composition, the treating physician's assessment of the medical situation, and other relevant factors. may vary depending on An immunologically effective amount of an immunological composition of the present invention can be expressed as a multimeric protein assembly or antigen content within the assembly, and can be expressed in terms of mass of protein per dose. For example, based on the antigen content in the assembly, it may be a dose of 1 μg / dose to 100 μg / dose, for example, 5 μg to 50 μg.

An immunogenic composition of the present invention can be administered to an individual to prevent infection in the individual. Thus, an immunogenic composition of the present invention may be a vaccine composition.

The vaccine composition of the present invention can be used to prevent diseases caused by infection with SARS-CoV-2. The term "prevention" of the present invention refers to any action that inhibits or delays the onset of a disease. The term "SARS-CoV-2" of the present invention refers to a virus that causes respiratory disease coronavirus infection-19 (COVID-19).

In the method for producing an immunogenic composition provided in one aspect of the present invention, when inducing self-assembly of components A and B defined herein, the mixing molar ratio, reaction time, and conditions for adding a specific concentration of a stabilizer are specified. Protein assemblies can be produced with a particle size and polydispersity index (PDI) value that are desirable for use as vaccine antigens, and an excellent yield can be achieved in terms of scale-up.

Figure 1 confirms the particle size and polydispersity index according to the mixing molar ratio of components A and B.
Figure 2 confirms the particle size and polydispersity index according to the reaction time when mixing components A and B.

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these Examples and Experimental Examples.

Manufacturing Example 1. Manufacturing of Component A

RBD-Component A cDNA (NT-SARS-CoV-2RBD-I53-50A-16GS-he synthesized by Genscript) for the production of RBD-Component A (SEQ ID NO: 6), an antigen of SARS-CoV-2 recombinant nanoparticle vaccine ) was inserted into the donor plasmid (pJV145) using the SalI/NotI restriction enzyme reaction to construct a recombinant expression vector plasmid (M-2560). As a result of sequencing analysis of the RBD-Component A cDNA (NT-SARS-CoV-2RBD-I53-50A-16GS-he) inserted into the final expression vector plasmid (M-2560), the nucleotide sequence of the inserted gene is the purpose It was confirmed that the gene was 100% identical to the nucleotide sequence (SEQ ID NO: 7). The vector sequence is shown in SEQ ID NO: 8.

The prepared vector was transfected into the HD-BIOP3 host cell line (HORIZON Discovery) by electroporation. Cell lines with high productivity were selected by performing fed-batch, and single cell-derived clones were obtained from them to construct a cell bank.

Cells were thawed, seed culture in a flask and main culture were performed in a bioreactor, and the main culture was centrifuged to collect the supernatant, which was then filtered through a 0.5/0.2 μm filter. In order to remove cell line-derived DNA, the filtered main culture medium was treated with benzonase, a DNA cutting enzyme, and reacted.

The recovered culture medium was first subjected to HIC (hydrophobic interaction chromatography) chromatography, treated with 5 M HCl to a pH of 3.5 to inactivate viruses while stirring, and first ultrafiltration was performed using a 100 kDa filter. In the second chromatography, remaining impure proteins were removed using mixed mode resin and Component A was purified. In the third purification process, Component A was purified using a cation exchange column and impurity proteins and nucleic acids were removed. Viruses were removed by performing a virus filtration process using the column flow-through solution of the third purification process. Next, concentration and buffer exchange were performed by performing secondary ultrafiltration. The ultrafiltration-completed sample was filtered to obtain Component A.

Manufacturing Example 2. Manufacturing of Component B

Component B (SEQ ID NO: 1) serving as an antigen carrier for the SARS-CoV-2 recombinant nanoparticle vaccine was prepared as follows.

BL21 competent cells distributed by Thermofisher were transformed with the vector into which the Component B protein expression gene was introduced. As a vector for gene introduction, pET29b+ was used. The prepared plasmid represented by SEQ ID NO: 3 was transformed into E. coli BL21 strain, and an E. coli strain having kanamycin resistance was selected, and the selected E. coli BL21 strain was inoculated into a soybean-based medium containing yeast extract and heated to 200 °C at 37 ° C. Incubation was performed with shaking until the absorbance was 1.8 or more at rpm. When the culture was completed, sterilized glycerol was added to a final concentration of 25% to construct a cell bank for research.

Cells were thawed and seed culture in flasks and main cultures in bioreactors were performed. The seed culture was cultured with shaking at 37 ° C. at 200 rpm so that the final absorbance exceeded 1.5, and then the entire amount of the seed culture was inoculated and cultured at 37 ° C. at 200 rpm until the absorbance was 25 (Growth phase). When the absorbance reached 25, IPTG was added to a final concentration of 0.5 mM (induction phase), and the main culture was terminated after culturing for 10 hours.

Cells were recovered by centrifugation of the main culture medium, suspended in a buffer solution, and disrupted with a high-pressure disruptor. In order to remove cell line-derived DNA, benzonase, a DNA cleavage enzyme, was treated and reacted with cell homogenate to a final concentration of 5 Unit/mL. The cell lysate after treatment with Benzonase was centrifuged, the supernatant was discarded, and inclusion bodies were recovered. The recovered inclusion bodies were washed using a sodium phosphate buffer solution, dissolved using a sodium phosphate buffer solution containing 2 M urea, and filtered through a 0.2 μm filter.

Purification was performed by first chromatography (negative mode) and second chromatography (positive mode) using a column filled with diethylaminoethyl (DEAE) resin. The recovered solution was filtered through 0.2 μm.

The secondary chromatography recovered solution was concentrated (Ultrafiltration) to 5 kg using a tangential flow filtration system equipped with a 100 kDa MWCO (Molecular weight cut-off) membrane with an area of 0.7 m ² , and It was recovered by performing buffer exchange (diafiltration) using 50 mM Tris buffer (500 mM NaCl, including 0.75 w/v% CHAPS). At this time, the transmembrane pressure (TMP) was maintained at 1 bar or less. The recovered solution was filtered through 0.2 μm.

Example 1. Assembly of Component A and Component B

Component A obtained in Preparation Example 1 and Component B obtained in Preparation Example 2 were assembled to prepare a multimeric protein assembly Nanoparticle used in a vaccine.

A reaction of mixing the Component A stock solution and the Component B stock solution at room temperature, 2 hours, and a stirring speed of 80 rpm was performed. The mixing ratio of Component A: Component B was 1: 1.1 (molar concentration), and the reaction time was 2 hours. At this time, the buffer solution in which self-assembly is induced by mixing the stock solutions of Component A and B is the buffer solution in the above preparation example. Corresponds to the 50 mM Tris buffer (500 mM NaCl, containing 0.75 w/v% CHAPS) used when performing the exchange. Polysorbate-80 (PS80) was added to the buffer as an additional stabilizer to adjust the PS80 content in the buffer to 0.025 v/v%. Upon completion of the reaction, the reaction solution was filtered through a 0.2 μm filter.

Meanwhile, in order to optimize the assembly reaction, the optimal mixing ratio and buffer composition were changed.

Example 1-1. Setting the optimal mixing ratio

A reaction of mixing the Component A stock solution and the Component B stock solution at room temperature, 2 hours, and a stirring speed of 80 rpm was performed. Upon completion of the reaction, the reaction solution was filtered through a 0.2 μm filter.

Meanwhile, in order to confirm the optimal mixing molar ratio, the particle size and polydispersity index were measured while changing the molar concentration ratio of Component A and Component B, and the results are shown in the table below and FIG. 1.

Group Reaction ratio
(A:B) Particle size
(d, nm) Polydipersity Index (PDI) One 1:1.1 54.6 0.210 2 1:2 68.9 0.310 3 1:3 84.3 0.432

Group Reaction ratio
(A:B) Particle size
(d, nm) Polydipersity Index (PDI) One 1.5:1 72.7 0.496 2 1.1:1 56.8 0.275 3 1:1.5 69.4 0.306

As shown in Tables 1 and 2, it can be seen that the particle size and polydispersity index also change as the molar concentration ratio of Component A and Component B is changed. From this, it was confirmed that a 1:1.1 molar concentration ratio that secures the most desirable particle size and PDI value for vaccine antigen use is the optimal mixing molar ratio.

Example 1-2. Optimal mixing time setting

The self-assembly of Component A and Component B was induced at the optimal molar concentration ratio identified above, but only the reaction time was set differently to evaluate the final prepared particle size and yield, and the results are shown in the table below and FIG. 2.

Group Reaction time (hr) Particle size
(nm) Process yield (%) One 0.5 60.2 28 2 One 67.8 26 3 2 53.4 49 4 4 58.3 47

As shown in Table 3, it can be seen that the particle size and yield change as the self-assembly induction reaction time of Component A and Component B is changed. From this, it was confirmed that the most desirable particle size for vaccine antigen use was secured and the reaction time with the highest yield was 2 hours.

Example 1-3. Optimization of buffer composition

As the component A and B stock solutions are mixed, the buffer in which self-assembly is induced corresponds to the 50 mM Tris buffer (including 500 mM NaCl and 0.75 w/v% CHAPS) used for buffer exchange in the above preparation example. Polysorbate-80 (PS80) was added as an additional stabilizer to the buffer, and at this time, the optimal concentration of the stabilizer in the buffer to induce self-assembly was sought. Yield, particle size, and polydispersity index were confirmed in the table below while polysorbate-80 (PS80) was not added or the content was changed from 0.05 v/v% to 0.01 v/v%.

Test group PS80 concentration (v/v %) Process yield
(%) Particle size (DLS) Z-average PDI Group 1 0.050 67.3 82.57 0.556 Group 2 0.025 62.0 41.92 0.263 Group 3 0.010 61.1 40.24 0.270 Group 4 0.000 56.6 62.09 0.635

As shown in the table above, Group 4, which does not contain PS80, has a relatively low yield and high PDI value, and in the case of Group 1, which has a PS80 content of 0.05 v/v%, the average particle size value is too large, making it unsuitable for use as an antigen in a vaccine. confirmed that it is not. In addition, when PS80 was not included in the self-assembly induction buffer or was included at a concentration of 0.05 v/v%, intensity and mass peaks of various sizes were observed.

On the other hand, in the case of Groups 2 and 3 in which PS80 was included in the self-assembly induction buffer at a concentration of 0.025 v / v% or 0.010 v / v%, considering the scale-up process, a yield higher than 60% was achieved, and Z- It was confirmed that the average and PDI values were secured within the preferred ranges for use as antigens in vaccines. The polydispersity index PDI means the value (Mw/Mn) divided by the weight average molecular weight (Mw) by the number average molecular weight (Mn), and a high polydispersity index means that the size, shape or mass distribution of the manufactured particles is not constant. Non-uniform means that the quality is not uniform and the workability is lowered.

From this, it was confirmed that the preferred concentration range of the stabilizer in the self-assembly induction buffer was in the range of 0.010-0.025 v/v%. Finally, considering the possibility of loss during the process, 0.025% v/v% was set as the most preferred concentration of the stabilizer in the self-assembly induction buffer.

Example 2. UF/DF (Ultrafiltration/diafiltration) process

The assemble reaction liquid filtered in Example 1 was concentrated to 4 kg using a tangential flow filtration system equipped with a 300 kDa MWCO (Molecular weight cut-off) membrane with an area of 0.7 m ² (Ultrafiltration), It was recovered by performing buffer exchange (diafiltration) using 50 mM Tris buffer in a volume 10 times the concentrated solution. At this time, the transmembrane pressure (TMP) was maintained at 1 bar or less, and a PES (Polyethersulfone) filter was used. As a result of the UF/DF process, it was confirmed that Component A, which did not assemble, was removed.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

<110> SK bioscience Co., Ltd. <120> A method for producing immunogenic composition <130> KPA211656-KR-P4 <150> KR 10-2021-0137835 <151> 2021-10-15 <160> 21 <170> KoPatentIn 3.0 <210> 1 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> Component B <400> 1 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asn Gly Met Met Asn Val Gln Leu Asn Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Asn Tyr Asp Lys Ser Lys Ala His Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 2 <211> 474 <212> DNA <213> Artificial Sequence <220> <223> Component B <400> 2 atgaaccagc acagtcacaa ggaccacgaa acggtaagaa tagcggtagt gcgtgcgcgt 60 tggcatgcgg aaattgtgga cgcctgtgtg agtgcgtttg aagccgcgat gcgtgatatt 120 ggcggcgatc gttttgccgt ggatgtgttt gatgtgccgg gtgcgtacga aattccactg 180 catgcgcgta ccctggcgga aaccggccgt tatggcgcgg tgttaggcac cgcctttgtg 240 gtgaatggtg gcatttatcg tcacgaattt gtggcgagcg cggttattaa cggcatgatg 300 aatgtgcagc tgaacacggg cgtgccagtg ttaagtgccg tgctgacccc acacaactat 360 gataaaagca aagcccatac cctgctgttc ttagcgctgt ttgcggtgaa aggcatggaa 420 gcggcgcgtg cctgcgtgga gattttagcg gcccgtgaaa agattgcggc gtga 474 <210> 3 <211> 5712 <212> DNA <213> Artificial Sequence <220> <223> plasmid <400> 3 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgcc acgttcgccg gctttccc cg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccag cgca tcaacaatat 1020 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg a acgacctac 1860 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctg ttt 2700 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 35 40 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780 atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380 ggg ccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc 4980 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa 5040 ataattttgt ttaactttaa gaaggagata tacatatgaa ccagcacagt cacaaggacc 5100 acgaaacggt aagaatagcg gtagtgcgtg cgcgttggca tgcggaaatt gtggacgcct 5160 gtgtgagtgc gtttgaagcc gcgatgcgtg atattggcgg cgatcgtttt gccgtggatg 5220 tgtttgatg t gccgggtgcg tacgaaattc cactgcatgc gcgtaccctg gcggaaaccg 5280 gccgttatgg cgcggtgtta ggcaccgcct ttgtggtgaa tggtggcatt tatcgtcacg 5340 aatttgtggc gagcgcggtt attaacggca tgatgaatgt gcagctgaac acgggcgtgc 5400 cagtgttaag tgccgtgctg accccacaca actatgataa aagcaaagcc cataccctgc 5460 tgttcttagc gctgtttgcg gtgaaaggca tggaagcggc gcgtgcctgc gtggagattt 5520 tagcggcccg tgaaaagatt gcggcgtgac tcgagcacca ccaccaccac cactgagatc 5580 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac 5640 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa 5700 ctatatccgg at 5712 <210> 4 <211> 204 <212> PRT <213> Artificial Sequence <220> <223> SARS-CoV-2 RBD <400> 4 Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn 1 5 10 15 Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser 20 25 30 Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser 35 40 45 Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys 50 55 60 Phe Thr Asn Val Tyr Ala As p Ser Phe Val Ile Arg Gly Asp Glu Val 65 70 75 80 Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr 85 90 95 Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn 100 105 110 Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu 115 120 125 Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu 130 135 140 Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn 145 150 155 160 Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val 165 170 175 Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His 180 185 190 Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr 195 200 <210> 5 <211> 204 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 5 Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu Arg 1 5 10 15 Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe Ala 20 25 30 Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala Asp 35 40 45 Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile Ile 50 55 60 Gly Ala Gly Thr Val Thr Ser Val Glu Gln Ala Arg Lys Ala Val Glu 65 70 75 80 Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile Ser 85 90 95 Gln Phe Ala Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met Thr 100 105 110 Pro Thr Glu Leu Leu Val Lys Ala Met Lys Leu Gly His Thr Ile Leu Lys 115 120 125 Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met Lys 130 135 140 Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn Leu 145 150 155 160 Asp Asn Val Ala Glu Trp Phe Lys Ala Gly Val Leu Ala Val Gly Val 165 170 175 Gly Ser Ala Leu Val Lys Gly Thr Pro Asp Glu Val Arg Glu Lys Ala 180 185 190 Lys Ala Phe Val Glu Lys Ile Arg Gly Ala Thr Glu 195 200 <210> 6 <211> 467 <212> PRT <213> Artificial Sequence <220> <223> Component A <400> 6 Met Gly Ile Leu Pro Ser Pro Gly Met Pro Ala Leu Leu Ser Leu Val 1 5 10 15 Ser Leu Leu Ser Val Leu Leu Met Gly Cys Val Ala Glu Thr Gly Thr 20 25 30 Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn 35 40 45 Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser 50 55 60 Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser 65 70 75 80 Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys 85 90 95 Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val 100 105 110 Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr 115 120 125 Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn S er Asn 130 135 140 Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu 145 150 155 160 Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu 165 170 175 Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn 180 185 190 Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val 195 200 205 Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His 210 215 220 Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Gly Gly Ser Gly 225 230 235 240 Gly Ser Gly Ser Gly Gly Ser Gly Gly Ser Gly Ser Glu Lys Ala Ala 245 250 255 Lys Ala Glu Glu Ala Ala Arg Lys Met Glu Glu Leu Phe Lys Lys His 260 265 270 Lys Ile Val Ala Val Leu Arg Ala Asn Ser Val G lu Glu Ala Ile Glu 275 280 285 Lys Ala Val Ala Val Phe Ala Gly Gly Val His Leu Ile Glu Ile Thr 290 295 300 Phe Thr Val Pro Asp Ala Asp Thr Val Ile Lys Ala Leu Ser Val Leu 305 310 315 320 Lys Glu Lys Gly Ala Ile Ile Gly Ala Gly Thr Val Thr Ser Val Glu 325 330 335 Gln Ala Arg Lys Ala Val Glu Ser Gly Ala Glu Phe Ile Val Ser Pro 340 345 350 His Leu Asp Glu Glu Ile Ser Gln Phe Ala Lys Glu Lys Gly Val Phe 355 360 365 Tyr Met Pro Gly Val Met Thr Pro Thr Glu Leu Val Lys Ala Met Lys 370 375 380 Leu Gly His Thr Ile Leu Lys Leu Phe Pro Gly Glu Val Val Gly Pro 385 390 395 400 Gln Phe Val Lys Ala Met Lys Gly Pro Phe Pro Asn Val Lys Phe Val 405 410 415 Pro Thr Gly Gly Val Asn Leu Asp A sn Val Ala Glu Trp Phe Lys Ala 420 425 430 Gly Val Leu Ala Val Gly Val Gly Ser Ala Leu Val Lys Gly Thr Pro 435 440 445 Asp Glu Val Arg Glu Lys Ala Lys Ala Phe Val Glu Lys Ile Arg Gly 450 455 460 Ala Thr Glu 465 <210> 7 <211> 1404 <212> DNA <213> Artificial Sequence <220> <223> Component A <400> 7 atgggaatcc tgccaagccc tggaatgcca gccctgctgt ccctggtgtc tctgctgagc 60 gtgctgctga tgggatgcgt ggcagagacc ggaacaaggt tccctaacat caccaacctg 120 tgcccattcg gcgaggtgtt taacgccaca cgctttgcct ccgtgtatgc ctggaaccgg 180 aagagaatct ctaattgcgt ggccgactat agcgtgctgt acaatagcgc ctccttctct 240 acctttaagt gctatggcgt gtctcccacc aagctgaacg acctgtgctt cacaaacgtg 300 tacgccgaca gctttgtgat ccggggcgat gaggtgagac agatcgcacc aggacagacc 360 ggcaagatcg cagactacaa ctataagctg cctgacgatt tcacaggctg cgtgatcgcc 420 tggaatagca acaatctgga ttccaaagtg ggcggcaact acaattatct gtacaggctg 480 ttccgcaaga gcaacctgaa gccatttgag cgggacatca gca ccgagat ctaccaggca 540 ggctccacac catgcaacgg agtggagggc ttcaattgtt attttcccct gcagagctac 600 ggcttccagc ctaccaatgg cgtgggctat cagccataca gagtggtggt gctgtccttt 660 gagctgctgc acgcaccagc aaccgtgtgc ggacctaaga agtccacagg cggctctgga 720 ggaagcggat ccggaggatc cggaggatct ggaagcgaga aggcagcaaa ggcagaggag 780 gcagcaagga agatggagga gctgttcaag aagcacaaga tcgtggccgt gctgagagcc 840 aactctgtgg aggaggccat cgagaaggca gtggccgtgt tcgcaggagg agtgcacctg 900 atcgagatca cctttacagt gcccgacgcc gataccgtga tcaaggccct gtccgtgctg 960 aaggagaagg gagcaatcat cggagcagga accgtgacat ctgtggagca ggcaaggaag 1020 gcagtggagt ccggagccga gtttatcgtg tctcctcacc tggatgagga gatctcccag 1080 ttcgccaagg agaagggcgt gttttacatg cctggcgtga tgaccccaac agagctggtg 1140 aaggccatga agctgggcca caccatcctg aagctgttcc caggagaggt ggtgggacca 1200 cagtttgtga aggccatgaa gggcccattc cccaatgtga agtttgtgcc tacaggcggc 1260 gtgaacctgg acaatgtggc agagtggttc aaggcaggcg tgctggcagt gggagtggga 1320 tctgccctgg tgaagggaac cccagatgag gtgagggaaa aggccaaggc ctttgt ggag 1380 aagatcaggg gagcaacaga gtga 1404 <210> 8 <211> 10704 <212> DNA <213> Artificial Sequence <220> <223> Plasmid (M-2560) <400> 8 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ggagatcggt acttcgcgaa 420 tgcgtcgaga tgtttaaact cccgccccta actccgccca gttccgccca ttctccgccc 480 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta 540 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctagctggt 600 tctttccgcc tcagaaggta cctaaccaag ttcctctttc agaggttatt tcaggccacc 660 ttccaccatg gccacctcag caagttccca cttgaacaaa aacatcaagc aaatgtactt 720 gtgcctgccc cagggtgaga aagtccaagc catgtatatc tgggttgatg gtactggaga 780 aggactgcgc tgcaaaaccc gcaccctgga ctgtgagccc aagtgtgtag aagagttacc 840 tgagtggaat tttgatggct ctagtacctt tcagtctgag ggctccaaca gtgacatgta 900 tctcagccct gttgccatg t ttcgggaccc cttccgcaga gatcccaaca agctggtgtt 960 ctgtgaagtt ttcaagtaca accggaagcc tgcagagacc aatttaaggc actcgtgtaa 1020 acggataatg gacatggtga gcaaccagca cccctggttt ggaatggaac aggagtatac 1080 tctgatggga acagatgggc acccttttgg ttggccttcc aatggctttc ctgggcccca 1140 aggtccgtat tactgtggtg tgggcgcaga caaagcctat ggcagggata tcgtggaggc 1200 tcactaccgc gcctgcttgt atgctggggt caagattaca ggaacaaatg ctgaggtcat 1260 gcctgcccag tgggaatttc aaataggacc ctgtgaagga atccgcatgg gagatcatct 1320 ctgggtggcc cgtttcatct tgcatcgagt atgtgaagac tttggggtaa tagcaacctt 1380 tgaccccaag cccattcctg ggaactggaa tggtgcaggc tgccatacca actttagcac 1440 caaggccatg cgggaggaga atggtctgaa gcacatcgag gaggccatcg agaaactaag 1500 caagcggcac cggtaccaca ttcgagccta cgatcccaag gggggcctgg acaatgcccg 1560 tcgtctgact gggttccacg aaacgtccaa catcaacgac ttttctgctg gtgtcgccaa 1620 tcgcagtgcc agcatccgca ttccccggac tgtcggccag gagaagaaag gttactttga 1680 agaccgccgc ccctctgcca attgtgaccc ctttgcagtg acagaagcca tcgtccgcac 1740 atgccttctc aatgagactg gcgac gagcc cttccaatac aaaaactaac gcccgcccca 1800 cgacccgcag cgcccgaccg aaaggagcgc acgaccccat gcatcgcaca catcataaga 1860 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt 1920 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac 1980 aacaacaatt gcattcattt tatgtttcag gttcaggggg agatgtggga ggttttttaa 2040 agcaagtaaa acctctacaa atgtggtaga attctacgta gataaaagtt ttgttacttt 2100 atagaagaaa ttttgagttt ttgttttttt taataaataa ataaacataa ataaattgtt 2160 tgttgaattt attattagta tgtaagtgta aatataataa aacttaatat ctattcaaat 2220 taataaataa acctcgatat acagaccgat aaaacacatg cgtcaatttt acacatgatt 2280 atctttaacg tacgtcacaa tatgattatc tttctagggt taatctagct gcgtgttctg 2340 cagcgtgtcg agcatcttca tctgctccat cacgctgtaa aacacatttg caccgcgagt 2400 ctgcccgtcc tccacgggtt caaaaacgtg aatgaacgag gcgcgctcat atcatgatta 2460 cgccaagcgc gcccgccggg taactcacgg ggtatccatg tccatttctg cggcatccag 2520 ccaggatacc cgtcctcgct gacgtaatat cccagcgccg caccgctgtc attaatctgc 2580 acaccggcac ggcagttcca tttaaatggc tgtcgccggt attgttcggg ttgctgatgc 2640 gcttcgggct gaccatccgg aactgtgtcc ggaaaagccg cgacgaactg gtatcccagg 2700 tggcctgaac gaacagttca ccgttaaagg cgtgcatggc cacaccttcc cgaatcatca 2760 tggtaaacgt gcgttttcgc tcaacgtcaa tgcagcagca gtcatcctcg gcaaactctt 2820 tccatgccgc ttcaacctcg cgggaaaagg cacgggcttc ttcctccccg atgcccagat 2880 agcgccagct tgggcgatga ctgagccgga aaaaagaccc gacgatatga tcctgatgca 2940 gctagattaa ccctagaaag atagtctgcg taaaattgac gcatgcattc ttgaaatatt 3000 gctctctctt tctaaatagc gcgaatccgt cgctgtgcat ttaggacatc tcagtcgccg 3060 cttggagctc ccgtgaggcg tgcttgtcaa tgcggtaagt gtcactgatt ttgaactata 3120 acgaccgcgt gagtcaaaat gacgcatgat tatcttttac gtgactttta agatttaact 3180 catacgataa ttatattgtt atttcatgtt ctacttacgt gataacttat tatatatata 3240 ttttcttgtt atagatatca agcttataga tctggggaca gccccccccc aaagccccca 3300 gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct 3360 ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg 3420 gggaaggtgg cacgggatcg ctttcctctg aacgct tctc gctgctcttt gagcctgcag 3480 acacctgggg ggatacgggg aaaaagcttt aggctgaaag agagatttag aatgacagaa 3540 tcatagaacg gcctgggttg caaaggagca cagtgctcat ccagatccaa ccccctgcta 3600 tgtgcagggt catcaaccag cagcccaggc tgcccagagc cacatccagc ctggccttga 3660 atgcctgcag ggatggggca tccacagcct ccttgggcaa cctgttcagt gcgtcaccac 3720 cctctggggg aaaaactgcc tcctcatatc caacccaaac ctcccctgtc tcagtgtaaa 3780 gccattcccc cttgtcctat caagggggag tttgctgtga cattgttggt ctggggtgac 3840 acatgtttgc caattcagtg catcacggag aggcagattt ggggataagg aagtgcagga 3900 cagcatggac gtgggacatg caggtgttga gggctctggg acactctcca agtcacagcg 3960 ttcagaacag ccttaaggat aagaagatag gatagaagga caaagagcaa gttaaaaccc 4020 agcatggaga ggagcacaaa aaggccacag acactgctgg tccctgtgtc tgagcctgca 4080 tgtttgatgg tgtctggatg caagcagaag gggtggaaga gcttgcctgg agagatacag 4140 ctgggtcagt aggactggga caggcagctg gagaattgcc atgtagatgt tcatacaatc 4200 gtcaaatcat gaaggctgga aaagccctcc aagatcccca agaccaaccc caacccaccc 4260 accgtgccca ctggccatgt ccctcagtgc cacatcccca c agttcttca tcacctccag 4320 ggacggtgac ccccccacct ccgtgggcag ctgtgccact gcagcaccgc tctttggaga 4380 aggtaaatct tgctaaatcc agcccgaccc tcccctggca caacgtaagg ccattatctc 4440 tcatccaact ccaggacgga gtcagtgagg atggggctct agagtcaaca ggaaagttcc 4500 attggagcca agtacattga gtcaataggg actttccaat gggttttgcc cagtacataa 4560 ggtcaatggg aggtaagcca atgggttttt cccattactg gcacgtatac tgagtcatta 4620 gggactttcc aatgggtttt gcccagtaca taaggtcaat aggggtgaat caacaggaaa 4680 gtcccattgg agccaagtac actgagtcaa tagggacttt ccattgggtt ttgcccagta 4740 caaaaggtca atagggggtg agtcaatggg tttttcccat tattggcacg tacataaggt 4800 caataggggt gagtcattgg gtttttccag ccaatttaat taaaacgcca tgtactttcc 4860 caccattgac gtcaatgggc tattgaaact aatgcaacgt gacctttaaa cggtactttc 4920 ccatagctga ttaatgggaa agtaccgttc tcgagccaat acacgtcaat gggaagtgaa 4980 agggcagcca aaacgtaaca ccgccccggt tttcccctgg aaattccata ttggcacgca 5040 ttctattggc tgagctgcgt tctacgtggg tatataagca gagctctccc tatcagtgat 5100 agagatctcc ctatcagtga tagagatcga gctcagcgtc ggtaccg tac ctcttccgca 5160 tcgctgtctg cgagggccag ctgttggggt gagtggcggg tgtggcttcc gcgggccccg 5220 gagctggagc cctgctctga gcgggccggg ctgatatgcg agtgtcgtcc gcagggttta 5280 gctgtgagca ttcccacttc gagtggcggg cggtgcgggg gtgagagtgc gaggcctagc 5340 ggcaaccccg tagcctcgcc tcgtgtccgg cttgaggcct agcgtggtgt ccgccgccgc 5400 gtgccactcc ggccgcacta tgcgtttttt gtccttgctg ccctcgattg ccttccagca 5460 gcatgggcta acaaagggag ggtgtggggc tcactcttaa ggagcccatg aagcttacgt 5520 tggataggaa tggaagggca ggaggggcga ctggggcccg cccgccttcg gagcacatgt 5580 ccgacgccac ctggatgggg cgaggcctgt ggctttccga agcaatcggg cgtgagttta 5640 gcctacctgg gccatgtggc cctagcactg ggcacggtct ggcctggcgg tgccgcgttc 5700 ccttgcctcc caacaagggt gaggccgtcc cgcccggcac cagttgcttg cgcggaaaga 5760 tggccgctcc cggggccctg ttgcaaggag ctcaaaatgg aggacgcggc agcccggtgg 5820 agcgggcggg tgagtcaccc acacaaagga agagggcctt gcccctcgcc ggccgctgct 5880 tcctgtgacc ccgtggtcta tcggccgcat agtcacctcg ggcttctctt gagcaccgct 5940 cgtcgcggcg gggggagggg atctaatggc gttggagttt gttcacattt gg tgggtgga 6000 gactagtcag gccagcctgg cgctggaagt cattcttgga atttgcccct ttgagtttgg 6060 agcgaggcta attctcaagc ctcttagcgg ttcaaaggta ttttctaaac ccgtttccag 6120 ctcgcggttg aggacaaact cttcgcggtc tttccagtac tcttggatcg gaaacccgtc 6180 ggcctccgaa cggtactccg ccaccgaggg acctgagcga gtccgcatcg accggatcgg 6240 aaaacctcgt cgacgccgcc accatgggaa tcctgccaag ccctggaatg ccagccctgc 6300 tgtccctggt gtctctgctg agcgtgctgc tgatgggatg cgtggcagag accggaacaa 6360 ggttccctaa catcaccaac ctgtgcccat tcggcgaggt gtttaacgcc acacgctttg 6420 cctccgtgta tgcctggaac cggaagagaa tctctaattg cgtggccgac tatagcgtgc 6480 tgtacaatag cgcctccttc tctaccttta agtgctatgg cgtgtctccc accaagctga 6540 acgacctgtg cttcacaaac gtgtacgccg acagctttgt gatccggggc gatgaggtga 6600 gacagatcgc accaggacag accggcaaga tcgcagacta caactataag ctgcctgacg 6660 atttcacagg ctgcgtgatc gcctggaata gcaacaatct ggattccaaa gtgggcggca 6720 actacaatta tctgtacagg ctgttccgca agagcaacct gaagccattt gagcgggaca 6780 tcagcaccga gatctaccag gcaggctcca caccatgcaa cggagtggag ggcttcaa tt 6840 gttattttcc cctgcagagc tacggcttcc agcctaccaa tggcgtgggc tatcagccat 6900 acagagtggt ggtgctgtcc tttgagctgc tgcacgcacc agcaaccgtg tgcggaccta 6960 agaagtccac aggcggctct ggaggaagcg gatccggagg atccggagga tctggaagcg 7020 agaaggcagc aaaggcagag gaggcagcaa ggaagatgga ggagctgttc aagaagcaca 7080 agatcgtggc cgtgctgaga gccaactctg tggaggaggc catcgagaag gcagtggccg 7140 tgttcgcagg aggagtgcac ctgatcgaga tcacctttac agtgcccgac gccgataccg 7200 tgatcaaggc cctgtccgtg ctgaaggaga agggagcaat catcggagca ggaaccgtga 7260 catctgtgga gcaggcaagg aaggcagtgg agtccggagc cgagtttatc gtgtctcctc 7320 acctggatga ggagatctcc cagttcgcca aggagaaggg cgtgttttac atgcctggcg 7380 tgatgacccc aacagagctg gtgaaggcca tgaagctggg ccacaccatc ctgaagctgt 7440 tcccaggaga ggtggtggga ccacagtttg tgaaggccat gaagggccca ttccccaatg 7500 tgaagtttgt gcctacaggc ggcgtgaacc tggacaatgt ggcagagtgg ttcaaggcag 7560 gcgtgctggc agtgggagtg ggatctgccc tggtgaaggg aaccccagat gaggtgaggg 7620 aaaaggccaa ggcctttgtg gagaagatca ggggagcaac agagtgagcg gccgccacac 768 0 atcataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc 7740 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa 7800 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga gatgtgggag 7860 gttttttaaa gcaagtaaaa cctctacaaa tgtggtacac aaagtgggga gctctagagg 7920 gacagccccc ccccaaagcc cccagggatg taattacgtc cctcccccgc tagggggcag 7980 cagcgagccg cccggggctc cgctccggtc cggcgctccc cccgcatccc cgagccggca 8040 gcgtgcgggg acagcccggg cacggggaag gtggcacggg atcgctttcc tctgaacgct 8100 tctcgctgct ctttgagcct gcagacacct ggggggatac ggggaaaaag gggagctcta 8160 gagggacagc ccccccccaa agcccccagg gatgtaatta cgtccctccc ccgctagggg 8220 gcagcagcga gccgcccggg gctccgctcc ggtccggcgc tccccccgca tccccgagcc 8280 ggcagcgtgc ggggacagcc cgggcacggg gaaggtggca cgggatcgct ttcctctgaa 8340 cgcttctcgc tgctctttga gcctgcagac acctgggggg atacggggaa aaaggatcca 8400 tggccggcca tctgcagatc atatgatcgg atgccgggac cgacgagtgc agaggcgtgc 8460 aagcgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 8520 caat tccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 8580 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 8640 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 8700 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 8760 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 8820 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 8880 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 8940 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 9000 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 9060 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 9120 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 9180 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 9240 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 9300 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 9360 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 9420 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 9480 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 9540 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 9600 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 9660 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 9720 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 9780 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 9840 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 9900 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 9960 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 10020 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 10080 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 10140 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 10200 caaccaagtc a ttctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 10260 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 10320 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 10380 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 10440 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 10500 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 10560 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 10620 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata 10680 ggcgtatcac gaggcccttt cgtc 10704 <210> 9 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 9 Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala 20 25 30 Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Ala Glu His Arg 115 120 125 Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala 130 135 140 Cys Ile Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 10 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 10 Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Ala Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Ala His Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 11 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400 > 11 Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Gln Cys Val Arg Ala 20 25 30 Phe Glu Glu Ala Met Ala Asp Ala Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Ser Ser Arg Glu His Glu 115 120 125 Phe Phe Arg Glu His Phe Met Val Lys Gly Val Glu Ala Ala Ala Ala 130 135 140 Cys Ile Thr Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 12 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide < 400 > 12 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala 20 25 30 Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Glu His Arg 115 120 125 Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala 130 135 140 Cys Ile Glu Ile Leu Asn Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 13 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 13 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala 20 25 30 Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asp Gly Gly Ile Tyr Asp His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Glu Tyr Glu Asp Ser Asp Glu Asp His Glu 115 120 125 Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala 130 135 140 Cys Ile Glu Ile Leu Asn Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 14 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 14 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Ala His Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 15 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 15 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asp Gly Gly Ile Tyr Asp His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Glu Tyr Glu Asp Ser Asp Ala Asp Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 16 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <220> <221> MISC_FEATURE <222> (9) <223> Xaa is Tyr or His <220> <221> MISC_FEATURE <222> (82) <223> Xaa is Asn or Asp <220> <221> MISC_FEATURE <222> (87) <223> Xaa is Arg or Asp <220> <221 > MISC_FEATURE <222> (105) <223> Xaa is Ser or Asp <220> <221> MISC_FEATURE <222> (119) <223> Xaa is Arg or Glu <220> <221> MISC_FEATURE <222> (121) <223> Xaa is Arg or Glu <220> <221> MISC_FEATURE <222> (124) <223> Xaa is Ala or Asp <220> <221> MISC_FEATURE <222> (126) <223> Xaa is His or Asp <220> <221> MISC_FEATURE <222> (128) <223> Xaa is Arg or Glu <220> <221> MISC_FEATURE <222> (150) <223> Xaa is Ala or Asn <400> 16 Met Asn Gln His Ser His Lys Asp Xaa Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala 20 25 30 Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Xaa Gly Gly Ile Tyr Xaa His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Xaa Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Xaa Tyr Xaa Asp Ser Xaa Glu Xaa His Xaa 115 120 125 Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala 130 135 140 Cys Ile Glu Ile Leu Xaa Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 17 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 17 Met Lys Met Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu 1 5 10 15 Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile 85 90 95 Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Thr Ile Leu 115 120 125 Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met 130 135 140 Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn 145 150 155 160 Leu Asp Asn Val Cys Glu Trp Ph e Lys Ala Gly Val Leu Ala Val Gly 165 170 175 Val Gly Ser Ala Leu Val Lys Gly Thr Pro Asp Glu Val Arg Glu Lys 180 185 190 Ala Lys Ala Phe Val Glu Lys Ile Arg Gly Cys Thr Glu 195 200 205 <210> 18 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 18 Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu 1 5 10 15 Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile 85 90 95 Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 Thr Pro Thr Glu Leu Val Ly s Ala Met Lys Leu Gly His Asp Ile Leu 115 120 125 Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met 130 135 140 Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn 145 150 155 160 Leu Asp Asn Val Cys Glu Trp Phe Lys Ala Gly Val Leu Ala Val Gly 165 170 175 Val Gly Asp Ala Leu Val Lys Gly Asp Pro Asp Glu Val Arg Glu Lys 180 185 190 Ala Lys Lys Phe Val Glu Lys Ile Arg Gly Cys Thr Glu 195 200 205 <210> 19 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 19 Met Lys Met Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu 1 5 10 15 Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile 85 90 95 Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Asp Ile Leu 115 120 125 Lys Leu Phe Pro Gly Glu Val Val Gly Pro Glu Phe Val Glu Ala Met 130 135 140 Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asp 145 150 155 160 Leu Asp Asp Val Cys Glu Trp Phe Asp Ala Gly Val Leu Ala Val Gly 165 170 175 Val Gly Asp Ala Leu Val Glu Gly Asp Pro Asp Glu Val Arg Glu Asp 180 185 190 Ala Lys Glu Phe Val Glu Glu Ile Arg Gly Cys Thr Glu 195 200 205 <210> 20 <211> 205 <21 2> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 20 Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu 1 5 10 15 Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile 85 90 95 Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Asp Ile Leu 115 120 125 Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met 130 135 140 Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn 145 150 155 160 Leu Asp Asn Val Cys Lys Trp Phe Lys Ala Gly Val Leu Ala Val Gly 165 170 175 Val Gly Lys Ala Leu Val Lys Gly Lys Pro Asp Glu Val Arg Glu Lys 180 185 190 Ala Lys Lys Phe Val Lys Lys Ile Arg Gly Cys Thr Glu 195 200 205 <210> 21 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <220> <221> MISC_FEATURE <222> (126) <223> Xaa is Thr or Asp <220> <221> MISC_FEATURE <222> (139) <223> Xaa is Gln or Glu <220> <221> MISC_FEATURE <222> (142) <223> Xaa is Lys or Glu <220> <221 > MISC_FEATURE <222> (160) <223> Xaa is Asn or Asp <220> <221> MISC_FEATURE <222> (163) <223> Xaa is Asn or Asp <220> <221> MISC_FEATURE <222> (166) <223> Xaa is Glu or Lys <220> <221> MISC_FEATURE <222> (169) <223> Xaa is Lys or Asp <220> <221> MISC_FEATURE <222> (179) <223> Xaa is Ser, Lys or Asp <220> <221> MISC_FEATURE <222> (183) <223> Xaa is Lys or Glu < 220> <221> MISC_FEATURE <222> (185) <223> Xaa is Thr, Asp or Lys <220> <221> MISC_FEATURE <222> (192) <223> Xaa is Lys or Asp <220> <221> MISC_FEATURE <222> (195) <223> Xaa is Ala, Glu or Lys <220> <221> MISC_FEATURE <222> (198) <223> Xaa is Glu or Lys <220> <221> MISC_FEATURE <222> (199) <223> Xaa is Lys or Glu <400> 21 Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu 1 5 10 15 Arg Ala Asn Ser Val Glu Ala Ile Glu Lys Ala Val Ala Val Phe 20 25 30 Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala 35 40 45 Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile 50 55 60 Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val 65 70 75 80 Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile 85 90 95 Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met 100 105 110 Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Xaa Ile Leu 115 120 125 Lys L eu Phe Pro Gly Glu Val Val Gly Pro Xaa Phe Val Xaa Ala Met 130 135 140 Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Xaa 145 150 155 160 Leu Asp Xaa Val Cys Xaa Trp Phe Xaa Ala Gly Val Leu Ala Val Gly 165 170 175 Val Gly Xaa Ala Leu Val Xaa Gly Xaa Pro Asp Glu Val Arg Glu Xaa 180 185 190 Ala Lys Xaa Phe Val Xaa Xaa Ile Arg Gly Cys Thr Glu 195 200 205

Claims (16)

(i) the fusion protein of SEQ ID NO: 6; and
(ii) a method for producing an immunogenic composition comprising the step of mixing the protein of SEQ ID NO: 1 to form a self-assembled multimeric protein assembly,
The molar ratio of (i) and (ii) is 1.1:1 to 1:1.1,
The mixing step is performed in the presence of a buffer solution containing polysorbate-80 in an amount of 0.01 v / v% or more and 0.025 v / v% or less,
The buffer solution contains Tris, NaCl and CHAPS, and the mixing reaction time is more than 1 hour and 4 hours or less, the method for producing an immunogenic composition.
delete delete According to claim 1, wherein the buffer is a 50mM Tris buffer, 500mM NaCl and 0.75% (w / v) CHAPS containing a method for preparing an immunogenic composition.
delete The method of claim 1, wherein the method comprises filtering the composition after the mixing step.
The method of claim 6, wherein the filtration step comprises filtering with a 0.01 μm to 0.5 μm filter.
delete The method of claim 1, wherein the molar ratio of (i) and (ii) is 1:1.1.
The method for preparing an immunogenic composition according to claim 1, wherein the mixing reaction is performed for 2 hours.
The method of claim 6, wherein the filtration step comprises filtering with a 0.1 μm to 0.3 μm filter.
The method of claim 1, wherein the manufacturing method
(i) the fusion protein of SEQ ID NO: 6; and
(ii) a method for producing an immunogenic composition comprising the step of mixing the protein of SEQ ID NO: 1 to form a self-assembled multimeric protein assembly,
The molar ratio of (i) and (ii) is 1:1.1,
The mixing reaction execution time is 2 hours,
The buffer solution contains polysorbate-80 in an amount of 0.025 v / v%, the buffer solution is a 50 mM Tris buffer, 500 mM NaCl and 0.75% (w / v) CHAPS, Method for producing an immunogenic composition.
13. The method according to any one of claims 1, 4, 6, 7, 9, 10, 11 and 12, wherein the multimeric protein assembly comprises (i) a fusion protein A method for preparing an immunogenic composition comprising a trimer and (ii) a pentamer of a protein.
13. The method according to any one of claims 1, 4, 6, 7, 9, 10, 11 and 12, wherein the multimeric protein assembly comprises (i) a fusion protein A method for preparing an immunogenic composition comprising 20 trimers and (ii) 12 pentamers of a protein.
The method according to any one of claims 1, 4, 6, 7, 9, 10, 11 and 12, wherein the multimeric protein assembly has a size of 20 nm to 80 nm. A method for producing an immunogenic composition having an average diameter.
The method according to any one of claims 1, 4, 6, 7, 9, 10, 11 and 12, wherein the multimeric protein assembly has an icosahedral structure, A method for preparing an immunogenic composition.

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