CN102816216A - Polypeptide capable of inducing granulation and application thereof - Google Patents

Abstract

The invention relates to polypeptide (Multimerization-Inducing Peptide, MIP) capable of inducing target protein to gather and granulate, particularly provides the sequence of the polypeptide, relative code sequences and application of the polypeptide for inducing the target protein to gather and granulate. The invention further provides a method for inducing the target protein to gather and granulate, protein granules prepared from the method, and application of the protein granules for vaccine, diagnosis or nanotechnology.

Description

A kind of polypeptide that can induce granulation and its use

technical field

The invention relates to a polypeptide (Multimerization-Inducing Peptide, MIP) that can aggregate or granulate a target protein. The aggregate or particle formed by the polypeptide can be used in vaccine development, diagnostic signal amplification, nanoparticle production etc. Specifically, the present invention provides a polypeptide capable of aggregating or granulating a target protein, its sequence and related coding sequences, and its method and use for aggregating or granulating a target protein. According to the method, protein particles, and the use of these particles for vaccines, diagnostics or nanotechnology. The invention also provides the in vitro assembly mechanism and method of such particles, and their preparation application for vaccines, particle antigens or nanoparticles.

Background technique

In the life activities of biological organisms, protein-protein interactions are ubiquitous, such as monomer-monomer, particle-particle in the virus assembly process, antigen-antibody in the immune system, enzyme-substrate in biochemical reactions, Effector molecules-receptors in signal transduction, virus-cell receptors in virus infection, etc., all exert their unique biological functions through protein-protein interactions. Biochemistry and structural biology have revealed that protein-protein interactions can be carried out through hydrophobic interactions, van der Waals forces, electrostatic interactions, etc., generally with high specificity, which may also induce allosteric phenomena and cascade effects. Interaction processes are also thermodynamic and kinetic processes, accompanied by changes in entropy, enthalpy and free energy.

Among the many forms of protein interaction, the virus capsid is uniquely arranged and aggregated with a small number of capsid proteins or envelope proteins, which can form particles of tens of nanometers or even hundreds of nanometers. The assembly process is strictly controlled. Therefore, it performs biological functions such as encapsulating genomic DNA/RNA, recognizing virus receptors on host cells, undergoing conformational changes, and uncapsiding. These capsids have different morphological structures, such as spherical, rod-shaped, elastic, brick-shaped, crown-shaped, cup-shaped, ring-shaped, wheel-shaped, kidney-shaped, and sand-embedded. However, the symmetry of the basic form can be divided into four types, namely spiral symmetry, three-dimensional symmetry (mainly icosahedral symmetry), compound symmetry and complex symmetry. Among them, the icosahedral symmetry is the most common.

People use various expression systems through genetic engineering, such as Escherichia coli, yeast, insect cells, CHO cells, and mammalian cells, to express all or part of the capsid protein of the virus exogenously, which can simulate the assembly of the virus capsid, Obtain artificial capsids similar to virus morphology and structure, also known as virus-like particles (Virus Like Particle, VLP). VLP has similar structural characteristics and antigenicity to the virus it is derived from, and does not contain viral genome nucleic acid, and has good safety, so it is an ideal form of vaccine, such as the genetically engineered recombinant hepatitis B vaccine, human papilla tumor virus vaccines, etc. VLPs have also been shown to insert or fuse heterologous antigen sequences, resulting in chimeric particles and displaying foreign antigens on the surface of the particles. VLP can also be linked to exogenous antigens via chemical coupling methods, including protein components and non-protein components (such as sugar groups), and VLP is also called a carrier at this time. Compared with general monomeric or oligomeric antigens, granulated antigens are more likely to be recognized by the body's immune system and induce corresponding humoral or cellular immunity (Grgacic EV, Anderson DA. Methods. 2006.40: 60-65). The size of VLP belongs to the nanometer level, and it has been proved that it can be combined with metal atoms to form nanoparticles, which may have the same properties as chemically prepared nanoparticles and have the characteristics of viral antigens.

VLPs are an ordered form of aggregation, therefore, there may be key core aggregation elements. The present invention is based on such a hypothesis, and invented a general-purpose polypeptide that can induce aggregation and granulation of target proteins. The structure of a protein includes multiple domains that perform different functions. For example, the capsid protein of HEV includes S shell domain, P1 protruding domain and P2 outer protruding domain (Yamashita T, et al.PNAS.2009.106 (31): 12986-12991.Guu TS, et al.PNAS.2009.106 (31): 12992-12997). It is generally believed that the S domain constitutes the basic structure of the capsid, while the P domain protrudes from the surface of the particle and forms the domain of the antigenic epitope.

When the protein is expressed exogenously, the introduction of certain N-terminal amino acid sequences or N-terminal fusion polypeptides is often beneficial to increase the expression of the protein and improve the folding properties of the protein. Some polypeptides, such as GST (glutathione S-transferase), Trx (thioredoxin), MBP (maltose binding protein), His Tag (tandem histidine tag), etc., are often used as fusion proteins and The target polypeptide is expressed in fusion, and the obtained protein often has a higher yield, is better folded, and is conducive to affinity chromatography. The corresponding commercially available pGEX, pTrxFUS, pMAL, and pET plasmid vectors of these fusion proteins use fusion proteins for high-efficiency expression and purification. The MIP polypeptide in the expression vector constructed by the present invention can assist high-yield protein expression and correct folding, and the induced particles can be distinguished from general proteins in terms of molecular size, and can be easily achieved by purification methods based on molecular size separation. The purpose of purification, such as using molecular sieve chromatography, non-denaturing polyacrylamide/agarose electrophoresis, ultracentrifugation and other methods for separation and purification.

Contents of the invention

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the laboratory operation steps of cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are all routine steps widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

According to the present invention, the term "Escherichia coli expression system" refers to an expression system composed of Escherichia coli (bacterial strain) and vector, wherein Escherichia coli (bacterial strain) is derived from commercially available bacterial strains, such as but not limited to: GI698, ER2566, BL21(DE 3), B834(DE3), BLR(DE3).

According to the present invention, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector is capable of achieving expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; bacteriophages; cosmids and the like.

According to the present invention, the term "HEV ORF2" refers to the ORF2 of human hepatitis E virus (HEV), the sequence of which is known in the art (see, for example, DDBJ Data Accession No.: D11092). In the present invention, when referring to the sequence of HEV ORF2, it is described using the sequence shown in DDBJ data accession number: D11092. For example, the 370-435 amino acid residues in the expression "the 370-435 amino acid residues of the polypeptide encoded by HEV ORF2" refer to the 370-435 amino acid residues of the polypeptide encoded by D11092. However, those skilled in the art understand that in HEV ORF2 or its encoded polypeptide, mutations or variations (including but not limited to, substitutions, deletions and/or additions) can be naturally produced or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "HEV ORF2" shall include all such sequences, including for example the sequence shown in D11092 and its natural or artificial variants. And, when describing the sequence fragments of HEV ORF2 (or its encoded polypeptides), it not only includes the sequence fragments of D11092 (or its encoded polypeptides), but also includes the natural or artificial variants of D11092 (or its encoded polypeptides). corresponding sequence fragments. For example, the expression "amino acid residues 370-435 of the polypeptide encoded by HEV ORF2" includes amino acid residues 370-435 of the polypeptide encoded by D11092, and the variants (natural or artificial) of the polypeptide encoded by D11092 corresponding fragment. According to the present invention, the expression "corresponding sequence fragment" or "corresponding fragment" means, when the sequences are optimally aligned, i.e. when the sequences are aligned for the highest percentage identity, the equivalent positions in the compared sequences fragments.

According to the present invention, when used in the context of a protein/polypeptide, the term "variant" refers to a protein whose amino acid sequence has one or more differences from the amino acid sequence of a reference protein/polypeptide (e.g., a MIP polypeptide of the invention). (e.g. 1-10 or 1-5 or 1-3) amino acid differences (e.g. conservative amino acid substitutions), or have at least 60%, 80%, 85%, 90%, 95%, 96%, 97% %, 98%, or 99% identity, and it retains the essential properties of the reference protein/polypeptide. In the present invention, the essential property of the protein/polypeptide (eg, the MIP polypeptide of the present invention) may refer to the ability to aggregate or granulate the protein fused or conjugated thereto.

According to the present invention, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by an adenine, or both a position in each of the polypeptides is occupied by lysine), then the molecules are identical at that position. "Percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions being compared x 100. For example, two sequences are 60% identical if 6 out of 10 positions match. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, comparisons are made when two sequences are aligned for maximum identity. Such alignments can be achieved using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453 which can be conveniently performed by computer programs such as the Align program (DNAstar, Inc.). The algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), can also be used, using the PAM120 weight residue table , a gap length penalty of 12, and a gap penalty of 4 to determine the percent identity between two amino acid sequences. In addition, the algorithm of Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) in the GAP program that has been incorporated into the GCG software package (available at www.gcg.com) can be used, using the Blossum 62 matrix or PAM250 matrix with a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine percent identity between two amino acid sequences .

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the biological activity of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions for amino acid residues with amino acid residues that have similar side chains, e.g., are physically or functionally similar (e.g., have similar size, shape, charge, chemical properties, including Substitution of residues with the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art. These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine) , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acid, proline, phenylalanine, methionine), beta branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine) amino acids. Therefore, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying amino acid conservative substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999) and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

According to the present invention, linking of MIP peptides to target proteins may include, for example, fusion and conjugation. In particular, a MIP polypeptide can be linked to a protein of interest via a linker. According to the invention, the term "linker" refers to a short peptide used to link two molecules, such as proteins. Usually, the fusion protein is obtained by introducing (for example, by PCR amplification or ligase) the polynucleotide sequence encoding the short peptide between two DNA fragments respectively encoding the two target proteins to be linked, and performing protein expression , such as target protein 1-linker-target protein 2. As known to those skilled in the art, linkers include, but are not limited to, flexible linker peptides such as Gly-Gly-Gly-Gly, Gly-Gly-Gly-Gly-Ser, Gly-Gly-Ser-Ser and (Gly-Gly-Gly -Gly-Ser)3.

In particular, as used in the present invention, the term "conjugation" means that two or more molecules (such as protein and protein, protein and polysaccharide, protein and label, etc.) are covalently (such as chemical coupling) or Non-covalent means (such as adsorption) for attachment. Thus, in the present invention, the term "conjugate" refers to two or more molecules linked together by covalent means (eg chemical coupling) or non-covalent means (eg adsorption). For example, the conjugate of the present invention may refer to, for example, a MIP peptide and a target protein linked together by covalent means or non-covalent means.

According to the present invention, the term "protein denaturation (denaturation)" means that the protein is affected by certain physical or chemical factors, and the spatial conformation of the molecule is destroyed, resulting in changes in its physical and chemical properties and loss of its original biological activity. Phenomenon. Denaturation does not cause the destruction of the primary structure of the protein, but the destruction of the higher structure above the secondary structure. The denatured protein is called denatured protein.

Denaturation of proteins is irreversible if denaturing conditions are severe and prolonged. If the denaturation conditions are not severe, this denaturation is reversible, and the internal structure of the protein molecule does not change much. At this time, if the denaturing factors are removed, the denatured protein can recover its natural conformation and biological activity under appropriate conditions. This phenomenon is called protein renaturation.

According to the invention, the term "hydrophobic interaction" refers to a weak, non-covalent interaction between non-polar molecules. These non-polar molecules (such as some neutral amino acid residues, also known as hydrophobic residues) have a tendency to avoid water and aggregate with each other in an aqueous environment. The hydrophobic interaction occurs through the mutual repulsion between the hydrophobic group of the hydrophobe and water, and the hydrophobic group is generally a non-polar group. This action brings the hydrophobic groups closer to each other while allowing the water to concentrate and structure to a greater extent. Through hydrophobic interactions, clathrate hydrates, which are clathrates, can sometimes be produced. Hydrophobic interactions are critical to the structure and properties of most proteins.

According to the present invention, in addition to the meaning of particle assembly in general structural biology and virology known in the art, particle assembly in the present invention also refers to the assembly of foreign proteins or other substances by using the aggregation of MIP polypeptides as the structural basis of particles. (such as sugars, lipids, compounds, metal ions, etc.) are presented as particles based on this. This particle assembly mainly occurs through the hydrophobic interactions of MIPs, and may also be accompanied by other forces (such as hydrogen bonds, van der Waals forces, etc.). Therefore, factors that induce hydrophobic interactions can induce the assembly of particles, such as adding salts with a certain ionic strength to the solution, initiating the occurrence of hydrophobic interactions on the hydrophobic surface, using hydrophobic interaction chromatography for binding, and so on. Therefore, according to the present invention, the term "particle" refers to a complex of proteins or derivatives thereof formed by aggregation of at least two protein monomers or derivatives thereof through various interaction methods, including but not limited to virus-like particles. According to the present invention, the term "virus-like particle" generally refers to a particle similar to a natural virus structure formed by the capsid protein of a virus in vivo or in vitro by virtue of protein interaction. For example, virus-like particles with icosahedral symmetry will be aggregated in an approximately equal area (quasi-equivalent) according to the Platonic solid geometry principle of 5:3:2, and the particles may contain 60T (T=h ² +k ² + hk, wherein h and k are non-negative integers) protein monomers, such as 60-mer, 180-mer, 420-mer, etc.

According to the invention, the term "aggregate" refers to a plurality of proteins associated together by non-covalent bonds. When proteins in the form of particles or aggregates are used as antigens, they will be more easily recognized by the body's immune system to induce corresponding humoral or cellular immunity. Thus, particularly advantageously, the particles or aggregates according to the invention can be used as antigens for the preparation of vaccines. Such antigens in the form of particles or aggregates are also referred to as "particulate antigens" or "particulated antigens". Furthermore, when the particle or aggregate (eg, particulate antigen) of the present invention is nanoscale in size, it may have the same properties as a chemically prepared nanoparticle, and thus may also be referred to and used as a "nanoparticle".

According to the present invention, the term "immunogenicity" refers to the ability to stimulate the body to form specific antibodies or sensitize lymphocytes. It not only refers to the characteristics that antigens can stimulate specific immune cells to activate, proliferate, and differentiate immune cells, and finally produce immune effect substances such as antibodies and sensitized lymphocytes, but also means that after antigens stimulate the body, the immune system of the body can form antibodies or Specific immune response of sensitized T lymphocytes.

In the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

In the present invention, the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. In particular, in the present invention, the target protein/polypeptide can be any protein, such as viral protein, such as AIV protein, HIV protein, HBV protein and the like.

The present invention is based at least in part on the inventors' discovery that when the MIP peptides of the invention are linked (e.g., fused or conjugated) to the polypeptide/protein of interest, the MIP peptides of the invention induce aggregation or particle formation of the polypeptide/protein of interest , forming aggregates or particles, and the obtained aggregates or particles have a variety of excellent properties, so they can be widely used in the fields of biology and medicine.

Therefore, in one aspect, the present invention provides a peptide capable of aggregating or granulating a target protein (also referred to as a MIP polypeptide or MIP peptide in this application), comprising

(1) an amino acid sequence, the amino acid sequence being a fragment of a polypeptide encoded by HEV ORF2, and at least comprising amino acid residues 370-435 of the polypeptide encoded by HEV ORF2;

(2) A variant of the amino acid sequence defined in (1), the difference between the variant and the amino acid sequence defined in (1) lies in the deletion, substitution, and/or addition of 1-20 (such as 1- 15, 1-10, 1-5, 1, 2, 3, or 4) amino acid residues; or

(3) A variant of the amino acid sequence defined in (1), which has at least 80% (such as at least 85%, 90%, 91%, 92%, 93%) of the amino acid sequence defined in (1). %, 94%, 95%, 96%, 97%, 98%, or 99%) identity.

In a preferred embodiment, the N-terminal of the amino acid sequence defined in (1) is located between the 345-370 (such as 345-368) amino acid residues of the polypeptide encoded by HEV ORF2, and/or its C The end is located between amino acid residues 435-460 (eg, 451-460) of the polypeptide encoded by HEV ORF2.

In another preferred embodiment, the amino acid sequence defined in (1) comprises amino acid residues 368-451 or 368-460 of the polypeptide encoded by HEVORF2, or amino acid residues 368-451 of the polypeptide encoded by HEV ORF2 or 368-460 amino acid residues.

In another preferred embodiment, the variant as defined in (2) differs from the amino acid sequence as defined in (1) in that the 12A10 epitope is deleted or mutated. In another preferred embodiment, the variant as defined in (2) is a peptide comprising amino acid residues 368-460 of the polypeptide encoded by HEV ORF2 and whose 12A10 epitope is deleted or mutated. The 12A10 epitope is known in the art and consists of amino acid residues 423-438 of the polypeptide encoded by HEV ORF2, see, eg, He et al. JGV, 2008, which is incorporated herein by reference.

In another preferred embodiment, the variant as defined in (2) or (3) differs from the amino acid sequence as defined in (1) by one or more conservative amino acid substitutions, for example 1-20 (such as 1 -15, 1-10, 1-5, 1, 2, 3, or 4) conservative amino acid substitutions.

In another preferred embodiment, the MIP peptide of the present invention consists of amino acid residues 368-451 or 368-460 of the polypeptide encoded by HEV ORF2.

In another aspect, the present invention provides a fusion protein comprising the above-defined MIP peptide and a target polypeptide (or called target protein). Optionally, in the fusion protein of the present invention, the MIP peptide according to the present invention is connected to the target polypeptide through a linker.

In a preferred embodiment, the polypeptide of interest is linked to the N-terminus of the MIP peptide according to the invention. In another preferred embodiment, the polypeptide of interest is linked to the C-terminus of the MIP peptide according to the invention. In yet another preferred embodiment, the polypeptide of interest is linked to the N- and C-terminus of the MIP peptide according to the invention.

In another preferred embodiment, the linker is a flexible linker peptide, such as Gly-Gly-Gly-Gly, Gly-Gly-Ser-Ser, (Gly-Gly-Gly-Gly-Ser)3 and the like. Such linkers are well known in the art and their selection is well within the purview of those skilled in the art.

In another aspect, the present invention provides a conjugate comprising a MIP peptide according to the present invention and a target polypeptide (or target protein), wherein the MIP peptide according to the present invention is covalently linked to the target polypeptide (optionally , via a linker) or non-covalently.

The benefits of the present invention can be achieved by linking the MIP peptide according to the present invention to the target polypeptide in various ways, such as granulating the target polypeptide. These linking means are well known in the art, including but not limited to fusion and conjugation and the like. Therefore, the MIP peptide according to the invention and the target polypeptide linked together by these means constitute a further aspect of the invention.

In another aspect, the invention provides a polynucleotide encoding a peptide or fusion protein as defined above. Also provided are constructs comprising such polynucleotides.

In another aspect, the invention provides a vector comprising a polynucleotide encoding a peptide or fusion protein as defined above or a construct comprising such a polynucleotide. The vector of the present invention can be a cloning vector or an expression vector.

In a preferred embodiment, the vectors of the invention are eg plasmids, cosmids, phages, cosmids and the like.

In another aspect, there is also provided a host cell or organism comprising a polynucleotide or construct or vector of the invention. Such host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The cells of the invention may also be cell lines, such as 293T cells. In one embodiment, the organism is a plant or an animal.

In another aspect, the present invention provides an aggregate or particle comprising, or consisting of, at least 2 fusion proteins or conjugates of the present invention. Preferably, the particles of the invention are nanoparticles.

The particles and aggregates of the present invention have various advantages, for example, have higher immunoreactivity and/or immunogenicity; have higher enzymatic action; are easier to be resolved, etc., and thus can be widely used in biology and the medical field. For example, in the particles and aggregates of the present invention, the number of protein monomer molecules is greatly increased, and the active sites or binding sites are correspondingly increased and aggregated. Therefore, in the particles and aggregates of the present invention, if the aggregated target protein is an enzyme molecule, its local action concentration will be greatly increased to have a higher enzymatic effect; if the target protein is a photoprotein or labeled with a luminescent substance , it will be easier to distinguish; if the target protein is an antigenic molecule, it may have higher immunoreactivity and/or immunogenicity, so it can be used as particle antigen and for preparing vaccines. Furthermore, the particles and aggregates of the invention can also be used as nanoparticles, provided they have the same size and properties as chemically prepared nanoparticles.

Therefore, in another aspect, the use of the MIP peptide of the present invention is also provided, which can be used to improve the activity of the target protein/polypeptide, or improve the immunogenicity of the target protein/polypeptide, or produce aggregates of the target protein/polypeptide or Particles, or granulate or form aggregates of the target protein/polypeptide.

In another aspect, the present invention provides methods for improving the activity of the target protein/polypeptide, or improving the immunogenicity of the target protein/polypeptide, or producing aggregates or particles of the target protein/polypeptide, or granulating the target protein/polypeptide Or a method of forming an aggregate comprising linking said protein/polypeptide of interest and a MIP peptide of the invention together, such as conjugation or fusion (optionally using a linker, such as a flexible linker peptide).

In another aspect, the present invention provides a method for preparing a particulate antigen or vaccine comprising linking, e.g. conjugating or fusing, a polypeptide as an antigen and a MIP peptide of the present invention (optionally using a linker, e.g. a flexible linking peptide), followed by the formation of particulate antigens or vaccines.

In another aspect, the invention provides a method for preparing nanoparticles comprising linking, e.g. conjugating or fusing (optionally using a linker, e.g. a flexible linker peptide), a polypeptide of interest and a MIP peptide of the invention, Nanoparticles are then formed.

In the present invention, the target polypeptide can be any polypeptide of interest, including but not limited to antigens, enzymes, and fluorescent proteins. For example, the polypeptide of interest can be an antigen such as a viral protein (e.g., capsid protein, envelope protein, and surface protein), a marker protein such as a fluorescent protein (e.g., green fluorescent protein, red fluorescent protein, and blue fluorescent protein), or a protein known in the art. various enzymes such as luciferase. Viral proteins can be derived from, for example, but not limited to, influenza viruses (eg, H5 subtype avian influenza virus), human immunodeficiency virus (HIV, eg, HIV-1), hepatitis B virus (HBV), human papillomavirus (HPV), and the like. Furthermore, in the present invention, the target polypeptide/protein may be of any origin, such as virus origin. Those skilled in the art will understand that the MIP peptides and methods of the present invention can be applied to any protein/polypeptide and obtain the benefits of the present invention.

In particular, in the present invention, the protein/polypeptide of interest may be, for example, viral proteins, such as influenza virus proteins, HIV proteins, HBV proteins and HPV proteins.

In another aspect, the present invention also provides protein particles obtained by the above method. Preferably, such protein particles are nanoparticles.

Beneficial Effects of the Invention

Currently commonly used granulation carriers include: HBV core granules, HPV, etc. These granulated vectors are all inserted into small short peptide fragments at specific positions of the complete virus-like particle, and are usually only suitable for displaying polypeptides. If longer polypeptide fragments or complete protein domains are inserted into these vectors, the assembly of the particles is often affected, and the target peptide fragments are difficult to fold correctly due to the need to use the structure of the particles themselves.

The MIP polypeptide of the present invention can form particles by itself, and its N or C terminal is exposed on the periphery of the particles for fusion with the target protein, and the fused protein can still fold freely without being affected by the particles. In addition, the present invention provides a choice of flexible extension arms, and two different lengths of flexible connections are tried. Those skilled in the art understand that in practical applications, the flexible link can also be properly adjusted to obtain a better supporting distance of the target protein, so as to ensure the formation of particles and the correct folding and reactivity of the protein. Appropriate flexible stretching arms may also have the function of maintaining particle stability and preventing enzymatic degradation. Due to the existence of flexible extension arms, the target protein can still maintain the natural polymer form of monomer, dimer or multimer after being granulated, thereby maintaining the natural epitope conformation.

The theory of antigen presentation in immunology shows that granulated antigens are easily presented by presenting cells to stimulate the body to produce high-titer antibodies. If there are immunodominant epitopes in the antigen, the body will be stimulated to produce immunodominant antibodies; if these immunodominant epitopes are also neutralizing epitopes for pathogens such as viruses and bacteria, then the granulated antigens can stimulate the body to produce High titers of neutralizing antibodies. These neutralizing antibodies can resist the infection of pathogens, so that the granulated antigen constructed in the present invention has a better vaccine effect.

In addition, granulated protein has a variety of uses.

For example, the number of molecules of the granulated protein is greatly increased, and the active or binding sites are correspondingly increased and aggregated. Therefore, if the target protein is an enzyme molecule, its local concentration will be greatly increased to have a higher enzymatic effect; if the target protein is a photoprotein or labeled with a luminescent substance, it will be easier to be distinguished; if the target protein If it is an antigen molecule, the granulated antigen can be combined with multiple and/or multiple antibodies at the same time, so that the monomeric protein can be used for antibody or antigen detection methods such as immunosandwich method or immunoprecipitation method or colloidal gold method, and combined with markers Antibody molecules to enzymes can also enhance the signal amplification of enzymes.

Granulated proteins can also be used for structure elucidation, especially single particle structure elucidation by cryo-EM. In particular, after the protein is aggregated by the method of the present invention, the number of protein molecules used for structural reconstruction can be stacked orderly and effectively. Structurally, the MIP polypeptide can be used as a backbone for the construction of virus-like particles. The granulated protein can also be made into nanoparticles for other related industrial fields.

MIP polypeptide can also be used as a guide peptide to construct a protein expression vector, like the existing GST, MPL maltose binding protein, pTrx, His tag, signal peptide, positioning peptide, etc., which is conducive to the expression, folding and purification of the target protein.

In addition, if the target molecule is a polysaccharide molecule, the polysaccharide molecule can be conjugated to the surface of the particle formed by the MIP polypeptide by chemical conjugation, or the glycosylation site can be fused on the MIP. Based on the prokaryotic expression system, the expression of polysaccharide-conjugated MIP conjugates is carried out, and the particle assembly is carried out by using the inducing granulation property of MIP, so as to develop polysaccharide-conjugated vaccines.

In conclusion, the present invention provides a peptide and method that can effectively aggregate proteins or other molecules into particles, which can realize the application intention of spatially arranging target proteins or other molecules in the form of particles to perform their functions.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention, rather than limiting the scope of the present invention. Various objects and advantages of this invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiment.

Description of drawings

Figure 1: Cloning design of various target polypeptides fused to MIP1 or MIP2. I272N, aa272 (aa represents an amino acid, when it is placed before the number n, it means the nth amino acid (for example, aa272 means the 272nd amino acid), when it is placed after the number, it means that the length of the polypeptide is n amino acids, the same below.) Ile is mutated into Gln; N63, 63 amino acids at the N-terminal of the protein are deleted (the number behind N indicates the number of N-terminal amino acids deleted, the same below); C46, 46 amino acids at the C-terminal of the protein are deleted (behind C The numbers in the numbers indicate the number of deleted C-terminal amino acids, the same below); Linker, the flexible connecting peptide, its amino acid sequence is GGGGSGGGGSGGGGS; MIP1h, aa423-438 on MIP1 (corresponding to the 12A10 epitope) is mutated into 16 concatenated groups MIP1Δ, aa423-438 on MIP1 (corresponding to the 12A10 epitope) was deleted; MIP1n, the target polypeptide was fused to the N-terminus of MIP1; MIP1nL, the target polypeptide was fused to the N-terminus of MIP1 through a flexible linker peptide; MIP1nLc, the target The polypeptide is fused to the N-terminus of MIP1 through a flexible connecting peptide, and is also fused to the C-terminus of MIP1.

Figure 2: SDS-PAGE of HA1 protein fused to MIP1 or MIP2. A: Swimming lanes 1 and 11, protein molecular weight markers; Swimming lane 2, MIP1-HA1-I272N bacterial cell ultrasonic lysate; Swimming lane 3, 4M urea dissolving supernatant of MIP1-HA1-I272N; Swimming lane 4, MIP1-HA1-I272N renaturation Lane 5, ultrasonic lysate of MIP1-HA1-N63C51 cells; lane 6, 4M urea dissolving supernatant of MIP1-HA1-N63C51; lane 7, refolding solution of MIP1-HA1-N63C51; lane 8, MIP2-HA1 -Sonic lysate of N57C67 cells; lane 9, 4M urea dissolving supernatant of MIP2-HA1-N57C67; lane 10, refolding solution of MIP2-HA1-N57C67; lane 12, ultrasonic lysate of MIP1Δ-HA1-N63C51 cells; lane 13, 4M urea dissolving supernatant of MIP1Δ-HA1-N63C51; lane 14, refolding solution of MIP1Δ-HA1-N63C51; lane 15, ultrasonic lysate of MIP1n-HA1-N63C51 cells; lane 16, 4M of MIP1n-HA1-N63C51 Urea dissolved supernatant; lane 17, MIP1n-HA1-N63C51 refolding solution. B: Lane 1, ultrasonic lysate of MIP1-HA1 cells; lane 2, ultrasonic supernatant of MIP1-HA1; lane 3, supernatant of MIP1-HA1 dissolved in 4M urea; lane 4, ultrasonic lysis of MIP1nL-HA1-N63C51 cells Lane 5, ultrasonic supernatant of MIP1nL-HA1-N63C51; lane 6, 4M urea-dissolved supernatant of MIP1nL-HA1-N63C51; lane 7, ultrasonic lysate of MIP1nLc-HA1-N63C51 cells; lane 8, MIP1nLc-HA1 -N63C51 ultrasonic supernatant; lane 9, 4M urea dissolving supernatant of MIP1nLc-HA1-N63C51; lane 10, ultrasonic lysate of MIP1-HA1-N63C46 cells; 12, 4M urea dissolving supernatant of MIP1-HA1-N63C46; lane 13, ultrasonic lysate of MIP1-HA1-N57C51 cells; lane 14, ultrasonic supernatant of MIP1-HA1-N57C51; lane 15, of MIP1-HA1-N57C51 4M urea dissolving supernatant; lane 16, MIP1-HA1-N52C51 bacterial cell ultrasonic lysate; swimming lane 17, MIP1-HA1-N52C51 ultrasonic supernatant; swimming lane 18, 4M urea dissolving supernatant of MIP1-HA1-N52C51; Lane 19, ultrasonic lysate of MIP1-HA1-N63 cells; lane 20, ultrasonic supernatant of MIP1-HA1-N63; lane 21, 4M urea-dissolved supernatant of MIP1-HA1-N63; lane 22, MIP1-HA1-N57C67 Sonic lysate of bacteria; lane 23, ultrasonic supernatant of MIP1-HA1-N57C67; lane 24, 4M urea dissolving supernatant of MIP1-HA1-N57C67; lane 25, ultrasonic lysate of MIP2-HA1-N57C51 bacteria; lane 26 , sonicated supernatant of MIP2-HA1-N57C51; lane 27, 4M urea-dissolved supernatant of MIP2-HA1-N57C51; lane 28, sonicated lysate of MIP1-HA1-N15C51 cells; Supernatant; lane 30, 4M urea dissolving supernatant of MIP1-HA1-N15C51; lane 31, ultrasonic lysate of MIP1h-HA1-N63C51 cells; lane 32, ultrasonic supernatant of MIP1h-HA1-N63C51; lane 33, MIP1h- The supernatant of HA1-N63C51 was dissolved in 4M urea. C: Lanes 1, 6, 15 and 24, protein molecular weight markers; lane 2, ultrasonic lysate of MIP1(C45)n-HA1-N63C51 cells; lane 3, ultrasonic supernatant of MIP1(C45)n-HA1-N63C51; Lane 4, 4M urea dissolving supernatant of MIP1(C45)n-HA1-N63C51; lane 5, refolding solution of MIP1(C45)n-HA1-N63C51; lane 7, sonication of MIP1(C35)n-HA1-N63C51 cells Lysate; lane 8, sonicated supernatant of MIP1(C35)n-HA1-N63C51; lane 9, 4M urea-dissolved supernatant of MIP1(C35)n-HA1-N63C51; lane 10, MIP1(C35)n-HA1- N63C51 refolding solution; lane 11, ultrasonic lysate of MIP1(C25)n-HA1-N63C51 bacteria; lane 12, ultrasonic supernatant of MIP1(C25)n-HA1-N63C51; lane 13, MIP1(C25)n-HA1 -N63C51 4M urea dissolving supernatant; lane 14, MIP1(C25)n-HA1-N63C51 refolding solution; lane 16, MIP1(C20)n-HA1-N63C51 cell ultrasonic lysate; lane 17, MIP1(C20) Ultrasonic supernatant of n-HA1-N63C51; lane 18, 4M urea dissolving supernatant of MIP1(C20)n-HA1-N63C51; lane 19, refolding solution of MIP1(C20)n-HA1-N63C51; 20, MIP1(C15 )n-HA1-N63C51 cell ultrasonic lysate; lane 21, MIP1(C15)n-HA1-N63C51 ultrasonic supernatant; lane 22, 4M urea dissolving supernatant of MIP1(C15)n-HA1-N63C51; lane 23 , MIP1(C15)n-HA1-N63C51 refolding solution; lane 25, MIP1(C10)n-HA1-N63C51 cell ultrasonic lysate; lane 26, MIP1(C10)n-HA1-N63C51 ultrasonic supernatant; lane 27, 4M urea dissolving supernatant of MIP1(C10)n-HA1-N63C51; lane 28, MIP1(C10)n-HA1-N63C51 refolding solution; lane 29, ultrasonic lysis of MIP1(C5)n-HA1-N63C51 cells Lane 30, MIP1(C5)n-HA1-N63C51 ultrasonic supernatant; Swimming lane 31, 4M urea-dissolved supernatant of MIP1(C5)n-HA1-N63C51; Swimming lane 32, MIP1(C5)n-HA1-N63C51 Refolding solution. D: Lane 1, protein molecular weight marker; lane 2, HA1-N63C51 bacterial cell ultrasonic lysate; lane 3, ultrasonic supernatant of HA1-N63C51; lane 4, 4M urea dissolved supernatant of HA1-N63C51; lane 5, HA1- N63C51 refolding solution.

Figure 3: HPLC molecular sieve analysis of HA1 protein fused to MIP1/MIP2. min: minutes, the same below.

Figure 4: Electron microscope observation of HA1 protein particles fused with MIP1/MIP2. Bar, 100 nm.

Figure 5: Western blot analysis of HA1 protein fused to MIP1/MIP2 with H5-specific mAb. Lane 1, MIP1-HA1-N63C51; Lane 2, MIP1-HA1-N63C46; Lane 3, MIP1-HA1-N57C51; Lane 4, MIP1-HA1-N52C51; Lane 5, MIP2-HA1-N57C51; Lane 6, MIP1nLc- HA1-N63C51; lane 7, HA1-N63C51.

Figure 6: The immunoblot reaction of HA1 protein MIP1-HA1-N63C51 fused with MIP1/MIP2 and H5-specific broad-spectrum neutralizing monoclonal antibody. pl-cHA1, MIP1-HA1-N63C51; HA1, yeast expressed HA1.

Figure 7: ELISA immunoreactivity of HA1 protein fused to MIP1/MIP2 with anti-H5 mAb. HA1 (derived from yeast), the full-length HA1 protein expressed by yeast, used as a control for ELISA reaction; A, clustering with each protein antigen, showing the changes in the reaction spectrum of protein particles and various antibodies after different fusion methods; B, Clustering with each monoclonal antibody shows the change in reactivity of different monoclonal antibodies with protein particles of various fusion modes.

Figure 8: Immunogenicity of HA1 protein particles fused with MIP1/MIP2 in mice.

Figure 9: Expression and purification of HIV-1-env-gp41-26 and gp120-222 proteins fused with MIP1/MIP2. A: Lane M, standard molecular weight marker; lane 1, sonic lysate of MIP2-gp41-26 cells; lane 2, sonic lysate of MIP1-gp41-26 cells; lane 3, 8M urea solution of MIP2-gp41-26 4, 8M urea dissolving supernatant of MIP1-gp41-26; 5, refolding supernatant of MIP2-gp41-26; 6, refolding supernatant of MIP1-gp41-26. B: Lane M, standard molecular weight marker; Lane 1, MIP2-gp120-222 bacterial cell ultrasonic lysate; Swimming lane 2, MIP1-gp120-222 bacterial cell ultrasonic lysate; Swimming lane 3, MIP2-gp120-222 dissolved in 8M urea Lane 4, 8M urea-dissolved supernatant of MIP1-gp120-222; lane 5, refolded supernatant of MIP2-gp120-222; lane 6, refolded supernatant of MIP1-gp120-222.

Figure 10: Molecular sieve chromatography analysis of HIV-1-env-gp41-26 and gp120-222 proteins fused with MIP1/MIP2. A: 239, p239 particle control; H0-HIV-1-env-gp41-26, MIP1-gp41-26 protein; H09-HIV-1-env-gp41-26, MIP2-gp41-26 protein. B: 239, p239 particle control; H0-HIV-1-env-gp120-222, MIP1-gp120-222 protein; H09-HIV-1-env-gp120-222, MIP2-gp120-222 protein.

Figure 11: Dynamic light scattering analysis of HIV-1-env-gp41-26, gp120-222 proteins fused to MIP1/MIP2. A: MIP2-gp120-222 protein; B: MIP1-gp120-222 protein; C: MIP2-env-gp41-26 protein; D: MIP1-gp41-26 protein.

Figure 12: Electron microscope analysis of HIV-1 env protein fused with MIP1/MIP2. H09-env-gp41-26, MIP2-gp41-26 protein; H0-env-gp41-26, MIP1-gp41-26 protein; H09-HIV-1-env-gp120-222, MIP2-gp120-222 protein; H0 -env-gp120-222, MIP1-gp120-222 protein. Bars, 100 nm.

Figure 13: Immunogenicity of HIV-1 gp41-26 protein fused to MIP1/MIP2.

Figure 14: Immunogenicity of HIV-1 gp120-222 protein fused to MIP1/MIP2.

Figure 15: Expression and purification of HIV-1 P24 protein fused with MIP1/MIP2. Lane M, standard molecular weight markers; lane 1, ultrasonic lysate of MIP2-P24 cells; lane 2, 4M urea-dissolved supernatant of MIP2-P24 protein; lane 3, refolding supernatant of MIP2-P24; lane 4, MIP1- Ultrasonic lysate of P24 bacteria; lane 5, supernatant of MIP1-P24 protein dissolved in 4M urea; lane 6, supernatant of refolding of MIP1-P24.

Figure 16: Molecular sieve chromatography analysis of MIP1/MIP2 fused HIV-1 P24 protein. 239, p239 particle control; H0-HIV-1-P24, MIP1-P24 protein; H09-HIV-1-P24, MIP2-P24 protein.

Figure 17: Dynamic light scattering analysis of HIV-1 P24 protein fused to MIP1/MIP2. A: MIP1-P24; B: MIP2-P24.

Figure 18: Electron microscope observation of HIV-1 P24 protein fused with MIP1/MIP2. HEV-H0-HIV-1-P24, MIP1-P24 protein; HEV-H09-HIV-1-P24, MIP2-P24 protein. Bars, 100 nm.

Figure 19: Immunogenicity of HIV-1 P24 protein fused to MIP1/MIP2.

Figure 20: Purification of HBsAg-1021 protein fused to MIP2. Swimming lane M, standard molecular weight markers; H 09-HBsAg-1021, the renatured supernatant of MIP2-HBsAg-1021.

Figure 21 : Dynamic light scattering analysis of MIP2-fused HBsAg-1021 protein.

Figure 22: Electron microscope observation of HBsAg-1021 protein fused with MIP2. HEV-H09-HBVSAg-1021, MIP2-HbsAg-1021; bars, 100 nm.

Figure 23: Expression and purification of HPV16L2C50 protein fused with MIP1. Lane M, standard molecular weight marker; lane 1, supernatant of MIP1-L2C50 cell lysate; lane 2, pellet of MIP1-L2C50 cell lysate; 4M urea dissolved precipitate; Lane 5, MIP1-L2C50 in 4M urea, 20mM phosphate buffer pH7.5; Lane 6, Q-FF flow-through fraction of MIP1-L2C50; Lane 7, Q-FF of MIP1-L2C50 80mM NaCl elution fraction of FF chromatography; lane 8, 150mM NaCl elution fraction of Q-FF chromatography of MIP1-L2C50; lane 9, 500mM NaCl elution fraction of Q-FF chromatography of MIP1-L2C50.

Figure 24: Molecular sieve chromatography analysis of HPV16 L2C50 protein fused to MIP1. 239, p239 particle control; HEV-H0-L2C50, MIP1-L2C50 protein.

Figure 25: Dynamic light scattering analysis of HPV16 L2C50 protein fused to MIP1.

Figure 26: Electron microscope observation of HPV16 L2C50 protein fused with MIP1. Bars, 100 nm.

Detailed ways

Below in conjunction with embodiment the present invention is further described by way of example.

Unless otherwise specified, the molecular biology experiment methods and immunoassay methods used in the present invention are basically with reference to J.Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M.Ausubel et al., Compiled Molecular Biology Experimental Guide, 3rd Edition, John Wiley & Sons, Inc., 1995 by the method described; restriction endonucleases were used in accordance with the conditions recommended by the product manufacturer. Those skilled in the art understand that the examples describe the present invention by way of example and are not intended to limit the scope of the claimed invention.

Example 1: Construction of an expression vector containing a polypeptide that can induce aggregation or granulation

In order to facilitate the fusion of other proteins, an expression vector containing a polypeptide (Multimerization-Inducing Peptide, MIP) that can induce aggregation or granulation of the target protein was constructed first. According to the inventor's design, the polypeptide at least comprises the amino acid sequence located in the aa368-451 region of HEV ORF2 (DDBJ data accession number: D11092). Combined with the previous granulation research results (see the results of Example 18 of the authorized patent [China Invention Patent No.: ZL02822218.0, International Invention Patent Application No. PCT/CN02/00797]), the N-terminal position of the polypeptide can be located at HEV ORF2aa345 to between aa370. In order to meet the needs of fusion of different target proteins, the C-terminal position of the polypeptide can be at least located between aa451 and aa460 of HEV ORF2. Of course, the C-terminal of the polypeptide is used to connect with the target protein, so the short C-terminal peptide (HEV ORF2aa451-460) can also be replaced by other reported flexible linkers, such as Gly-Gly-Gly-Gly, Gly-Gly -Ser-Ser, Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser, etc. The main function of the flexible link is to enable the target protein to be folded relatively independently after being linked with the MIP polypeptide of the present invention, so as to obtain high biological activity. In this example, two expression vectors were exemplarily constructed, which respectively contained two polypeptides (MIPs) that could induce aggregation or granulation, namely MIP1 (HEV ORF2 aa368-460) and MIP2 (HEV ORF2 aa368-45l). MIP1 vectors with longer linker chains are generally tried preferentially during application. The present invention further proves that the C-terminus of MIP can be located at least at HEV ORF2 aa435-460 (see Example 4 below).

Using the pTO-T7-p239 plasmid (Li et al, J. Biol. Chem. 2005, 28(5): 3400-3406) containing HEV ORF2 aa368-606 as a template, 368F (SEQ ID NO: 63): 5' -CGG GAA TTC CAT ATG ATA GCG CTT ACC CTG TT-3' is the forward primer (its 5' end introduces a restriction endonuclease Nde I site (CATATG), ATG is the start codon in the E. coli expression system ); 460R (SEQ ID NO: 64): 5'-GGA AGA TCT AGA GGA TCC GCG CGA TGG GGC TGG-3' (for amplification of MIP1) or 451R (SEQ ID NO: 65): 5'-GGA AGA TCT ATG GGA TCC CGG TCG GTCCTG CTC-3' (used to amplify MIP2) is the reverse primer (in which the restriction endonuclease BamHI site is introduced), and the PCR thermal cycler (Biometra T3) is carried out according to the following conditions PCR reaction:

The amplified product is a DNA fragment of about 300bp. The PCR product was connected with a commercially available pMD18-T vector (produced by TAKARA Company), and identified by NdeI/BamHI digestion to obtain positive clone plasmids pMD18-T-MIP1 and pMD18-T-MIP2 inserted into MIP1 and MIP2 genes. In Shanghai Boya Bioengineering Company, the pMD18-T-MIP1 and pMD 18-T-MIP2 plasmids were sequenced using M13(+) primers, and the results showed that the nucleotide sequences of MIP1 and MIP2 are SEQ ID NO: 1 and SEQ ID NO: 3, the encoded amino acid sequences are SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

The above-mentioned pMD 18-T-MIP1 and pMD 18-T-MIP2 plasmids were digested with NdeI/BamHI, and pTO-T7 prokaryotic expression vector (Luo Wenxin et al., Bioengineering Journal, 2000, 16: 53-57), and then identified by NdeI/BamHI digestion to obtain positive expression plasmids pTO-T7-MIP1 and pTO-T7-MIP2 inserted with MIP1 and MIP2 gene fragments. Take 1 μL of the plasmid (0.15 mg/ml) to transform 40 μL of competent Escherichia coli ER2566 (purchased from Invitrogen) prepared by the calcium chloride method, and spread it on a medium containing kanamycin (final concentration 100 mg/ml, the same below) solid LB medium (ingredients: 10g/L peptone, 5g/L yeast powder, 10g/L sodium chloride, the same below), and cultured at 37°C for 10-12 hours until a single colony is clearly identifiable. Pick a single colony to a test tube containing 4 mL of liquid LB medium (containing kanamycin), shake it at 180 rpm at 37°C for 10 hours, and take 1 mL of the bacterial solution and store it at -70°C.

Example 2: Construction of the expression vector of the H5 subtype avian influenza virus HA1 protein fused to MIP1/MIP2

The present invention relates to MIP1/MIP2 capable of hydrophobic interaction aggregation, which can induce the target protein fused thereto or covalently coupled to assume aggregated or granulated form. This example shows that the target protein can be fused to the C-terminal or N-terminal or both of the N-terminal and C-terminal of MIP1/MIP2, and the resulting fusion protein can be assembled into particles, and MIP1/MIP2 can also be appropriately mutated Or missing while still maintaining the property of inducing aggregation or granulation.

As shown in Figure 1, design and construct a construct containing MIP1/MIP2 and HA1 protein, wherein the target protein can be fused to the N-terminal or C-terminal of MIP1/MIP2. In addition, mutated MIP1 was also designed by truncating the C-terminus of MIP1 or replacing or deleting the 12A10 epitope of MIP1 (He et al. JGV, 2008). Structurally, MIP1 has 9 more amino acid residues at the C-terminus than MIP2 (see Example 1). In order to allow the target protein to fold freely, a flexible linking peptide (whose amino acid sequence is GGGGSGGGGSGGGGS, other flexible linking peptides can also be used, as exemplified above) is also used to link HA1 and MIP1. In addition, the truncated fragment of HA1 was also connected to the N-terminal and C-terminal of MIP1 at the same time, and the N-terminal was connected using a flexible linker peptide (the amino acid sequence of which was GGGGSGGGGSGGGGS).

The gene cloning of the fusion protein was carried out using three PCR reactions, and the initial templates used included pTO-T7-p239 plasmid (abbreviated as 239 in Table 1) (Li, Vaccine, 2005), pTO-T7-p239Δ12A10 plasmid (in Table 1 Abbreviated as 239-12A10Δ), pTO-T7-p239his12A10 plasmid (abbreviated as 239-12A10 in Table 1) (He, JGV, 2008) and HA gene (GenBank accession number: AAT39077, abbreviated as HA1-Yu777 in Table 1) ( Chen, JID, 2009). The templates, primers and annealing conditions of each PCR reaction are shown in Table 1. The amplification conditions of the PCR reaction are set as follows: denaturation at 94°C for 10 minutes, 25 cycles (denaturation at 94°C for 50 seconds, annealing at a specified temperature for a certain period of time, extension at 72°C for 50 seconds ), and a final 72°C extension for 10 minutes. The sequences of the PCR primers used are listed in Table 2.

Table 2: The primer sequence (SEQ ID NO:66-99) that is used to construct the HA protein of fusion MIP1/MIP2

The final product of the amplified DNA fragment was connected to a commercially available pMD 18-T vector (manufactured by TAKARA Company), and the positive clone containing the target fragment was identified by EcoRI/Not I digestion. Then, the obtained restriction fragment was connected with the pTO-T7 prokaryotic expression vector digested with EcoRI/NotI, and the positive expression clone containing the target protein gene fragment was obtained through NdeI/NotI digestion. In Shanghai Boya Bioengineering Co., Ltd., using T7 primers, the nucleotide sequences of the target fragments inserted in the pTO-T7 plasmid were measured as SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, the amino acid sequence encoded by it is SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 (respectively with 24 fusion proteins in Table 1 one-to-one correspondence). Wherein, SEQ ID NO:51 encodes the HA1 protein that does not fuse MIP, HA1-N63C51, which is used as a control to study the beneficial effect of MIP on the expression of HA1 protein.

Take 1 μL of each of the above-mentioned expression plasmids (0.15 mg/ml) and transform 40 μL of competent Escherichia coli ER2566 prepared by the calcium chloride method, and spread the bacteria on a medium containing kanamycin (final concentration 100 mg/ml, the same below). Solid LB medium, static culture at 37°C for 10-12 hours until a single colony is clearly identifiable. Pick a single colony to a test tube containing 4 mL of liquid LB medium (containing kanamycin), shake it at 250 rpm at 37°C for 10 hours, and take 1 mL of the bacterial liquid from it and store it at -70°C.

Example 3: Expression and purification of H5 avian influenza virus HA1 protein fused with MIP1/MIP2

Expression of HA1 protein of H5 subtype avian influenza virus fused with MIP1/MIP2

Take out 5 μL of bacterial liquid from the ultra-low temperature refrigerator at -70°C, inoculate it into 5 mL of liquid LB medium containing kanamycin, culture it with shaking at 37°C and 250 rpm until the OD600 reaches about 0.5, and then transfer it to 500 mL of liquid medium containing kanamycin In the LB medium, shake culture at 37°C and 250rpm for 4-5 hours. When the OD600 reached about 1.5, IPTG was added to a final concentration of 0.4 mM, and induced by shaking at 250 rpm at 37°C for 4 hours.

Purification of H5 subtype avian influenza virus HA1 protein fused with MIP1/MIP2

After induction, collect the thalline by centrifugation at 8000rpm for 5min, then resuspend the thalline with the lysate at the ratio of 1g of the thallus to 10mL lysate (20mM Tris buffer pH 7.2, 300mM NaCl), ice bath, and use an ultrasonic breaker ( Sonics VCX750 Ultrasonic Breaker) to process the bacteria (processing conditions: working time 15min, pulse 2s, pause 4s, output power 55%). Cell lysate at 12000rpm, centrifuge at 4°C for 5min (the same below), discard the supernatant to retain the precipitate (i.e. the inclusion body); then wash the precipitate with an equal volume of 2% Triton-100, shake for 30min, discard the supernatant by centrifugation; then use Buffer I (20mM Tris-HCl pH8.0, 100mM NaCl, 5mMEDTA) to resuspend the pellet, shake for 30min, and centrifuge to discard the supernatant; then resuspend the pellet with BufferI containing 2M urea, shake at 37°C for 30min, and centrifuge to obtain the supernatant and pellet; Then resuspend the pellet with an equal volume of Buffer I containing 4M urea, shake at 37°C for 30 minutes, and centrifuge at 12,000 rpm at 4°C for 15 minutes to obtain the supernatant and pellet; keep the supernatant and continue to resuspend with an equal volume of Buffer I containing 8M urea. Suspend the pellet, shake at 37°C for 30 minutes, centrifuge and keep the supernatant. The SDS-PAGE analysis of the various fractions obtained (visualized by Coomassie brilliant blue staining, the same below, the method refers to the second edition of "Molecular Cloning") is shown in Figure 2. The results showed that the fusion proteins all existed in the form of inclusion bodies, and after washing the inclusion bodies, most of the fusion proteins could be dissolved in Buffer I containing 4M urea.

Refolding and particle assembly of H5 subtype avian influenza virus HA1 protein fused with MIP1/MIP2

Protein refolding and particle assembly are carried out by dialysis (small amount) or using CENTRASETTE 5 tangential flow device (PALL company product) (large amount). 7.45 phosphate buffer, 0.15M NaCl) and 4M urea dissolved supernatant for solution exchange, the operating pressure is 0.5psi, the circulation flow rate is 500mL/min, and the tangential flow rate is 200mL/min. After sufficient exchange, the samples were sterilized and filtered with a 0.22 μm filter and stored at 4°C. As shown in Figure 2, all proteins dissolved in 4M urea can be refolded into PBS solution.

Example 4: Particle analysis of H5 subtype avian influenza virus HA1 protein fused with MIP1/MIP2

Molecular sieve chromatography of fusion proteins

The 1120Compact LC high-performance liquid chromatography system of Agilent Corporation of the United States was used for chromatography, and the analytical molecular sieve chromatography column used was TSK GelPW5000xl (specification: 7.8x300mm) of TOSOH Corporation of Japan. Pre-equilibrate the chromatographic column with 2 times the column volume of PBS until the absorbance at 280 nm does not change significantly, and reset the absorbance of the detector to zero. Samples were injected by an autosampler and the results analyzed. As shown in Figure 3, except for MIP1(C 35)n-HA1-N63C51 and MIP1(C45)n-HA1-N63C51, the retention time of the main peak of other refolding proteins on the molecular sieve chromatography column is comparable to that of 239 particles (Li et al , Vaccine, 2005), which indicated that these proteins could form granules after removing 4M urea and refolding into PBS solution; however, when the C-terminus of MIP was truncated to HEV ORF2aa425 or aa415, it could not induce HA1 protein granulation , suggesting that the C-terminus of MIP can be located at least at aa435.

TEM Observation of Fusion Protein

The instrument used is a 120kV transmission electron microscope produced by Japan Electronics Corporation, with a magnification of 100,000 times. The assembled fusion protein particles were negatively stained with 2% phosphotungstic acid pH 7.0, fixed on a carbon-sprayed copper grid, and observed. The results are shown in Figure 4, all fusion proteins present granular components in the visual field, among them, MIP1-HA1-N63, MIP1-HA1-N63C51, MIP1Δ-HA1-N63C51, MIP2-HA1-N57C67, MIP1nL-HA1-N63C51 and MIP1nLc-HA1-N63C51 can form a better particle shape with a diameter of about 20nm; while MIP1h-HA1-N63C51, MIP2-HA1-N57C51 and MIP1n-HA1-N63C51 can form a large number of spherical particles with a diameter of 20-30nm, The shape is relatively uniform and the outline is clear. This shows that no matter what kind of fusion is performed, even a small amount of amino acid replacement, MIP1/MIP2 can induce the aggregation of fragments of different sizes of HA1 to form particles. Of course, aggregation may be affected by local conformation, and the morphology of the formed particles may also be different. Linker peptides may also act to separate domains and facilitate protein folding.

Example 5: Immunoreactivity of H5 subtype avian influenza virus HA1 protein particles fused with MIP1/MIP2

Western blotting of fusion protein and anti-H5 specific monoclonal antibody immunoreactivity

After refolding and particle assembly, the fusion protein sample was separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane, blocked with 5% skimmed milk at room temperature for 2 hours; added diluted monoclonal antibody (1:1000), placed in React at room temperature for 1 h (both 3G4 and 13D4 are H5N1-specific antibodies prepared in our laboratory, and 13D4 is an H5 neutralizing antibody that can be used to treat avian influenza, see Chen et al., JID, 199(1): 49-58, 2009, which is incorporated herein by reference); wash the membrane 3 times with TNT solution (8.765g NaCl, 1.21g Tris Base and 0.5ml Tween-20, add deionized water to 1L, adjust the pH to 8.0), each 10min; Then goat anti-mouse alkaline phosphatase (1:5000) (KPL product) was added and reacted at room temperature for 1 h; the membrane was washed 3 times with TNT, 10 min each time; 4-chloro-3-indolyl phosphate) substrate for color development. As shown in Figure 5, the results of Western blot analysis showed that MIP1-HA1-N63C51, MIP1-HA1-N63C46, MIP1-HA1-N57C51, MIP1-HA1-N52C51, MIP2-HA1-N57C51 and MIP1nLc-HA1-N63C51 were all It can react with specific H5 monoclonal antibodies 3G4 and 13D4, but HA1-N63C51 (lane 7) without MIP fusion has no obvious reaction. This shows that the fusion of MIP can not only induce the aggregation of HA1 protein, but also facilitate its formation of natural H5 epitope conformation.

Dot blot immunoreactivity of fusion protein in particulate form with H5 broadly neutralizing monoclonal antibody

Compared to western blotting, dot blots maintain the granular form of proteins and preserve conformational epitopes. A P91E hybrid spotter from BioRad, USA was used. Cut off a piece of film that can cover all the voids on the spotter according to the ruler, and cut off a corner as a mark. The membrane was pre-soaked with sterilized water, and then MIP1-HA1-N63C51 protein particles (1mg/ml) were spotted on the membrane according to the original dilution of 1/10, 1/50, 1/250, 1/1250, 1/6250 , the loading volume of each band was 200 μl. The HA1 protein (which is a monomeric protein) expressed by yeast cells was used as a control. Suction filtration was performed with vacuum to adsorb the particulate protein to the membrane. Leave the membrane at room temperature for 2 hours, block with 5% skimmed milk at room temperature for 2 hours; then add H5-specific monoclonal antibody diluted with 5% skimmed milk, and leave it at room temperature for 1 hour; wash the membrane with TNT solution for 3 times, each time for 10 minutes; then add alkali The goat anti-mouse secondary antibody (KPL product) labeled with sex phosphatase (AP) was placed at room temperature for 30 minutes; then the membrane was washed 3 times with TNT solution, 10 minutes each time; and then developed with NBT and BCIP. The results are shown in Figure 6, where MIP1-HA1-N63C51 particles are combined with H5 broad-spectrum neutralizing mAbs 13D4, 8G9 and 10F7 (see Chen et al., JID, 199(1): 49-58, 2009, which is incorporated by reference The reactivity of this article) is not lower than that of the HA1 monomeric protein, which indicates that the HA1 part of the particle formed by the MIP1 polypeptide induces the HA1 protein still maintains a good conformation.

ELISA immunoreactivity of fusion protein in particulate form with H5 mAb disc

Each protein was diluted with carbonate buffer (20 mM Na ₂ CO ₃ , pH 9.6) to a final concentration of 1 ng/μl, then coated on a 96-well plate (coating volume 100 μl), and incubated at 37° C. for 2 h. After blocking at 37°C for 2 hours, drained, and added the purified H5 monoclonal antibody prepared by our laboratory (obtained by immunizing mice with avian influenza virus and then screening monoclonal antibody by hemagglutination inhibition method) diluted to a final concentration of 100ng/ml These monoclonal antibodies, these monoclonal antibodies can refer to Chinese patent ZL200510007308.X, Chinese patent application 200710006098.1, Chinese patent application 200810110869.6, which are incorporated herein by reference), react at 37°C for 30min; wash the plate, and then add HRP-labeled anti-mouse IgG Secondary antibody (KPL product), react at 37°C for 30min; wash the plate, develop color at 37°C for 15min, and then read the absorbance value at 450nm (the reference wavelength is 620nm). The result is shown in Figure 7. Figure 7A performs clustering analysis based on the reactivity of each protein antigen and antibody, among which MIP1-HA1-N63C51, MIP1-HA1-N57C51, MIP2-HA1-N57C51 and MIP1-HA1-N52C51 maintained a good anti-H5 monoclonal antibody panel The reaction spectrum of the HA1 protein from yeast is comparable to the monoclonal antibody reaction spectrum of the yeast-derived HA1 protein, while the control protein HA1-N63C51 that is not fused with MIP hardly reacts with the monoclonal antibody disk; Figure 7B performs cluster analysis based on the reactivity of each antibody and antigen , in which the reactivity of various antibodies with MIP fusion protein was comparable, all higher than the reactivity of HA1-N63C51 which was not fused with MIP with the control protein. This suggests that MIP facilitates the correct folding of proteins while inducing granulation.

Example 6: Immunogenicity of H5 subtype avian influenza virus HA1 protein particles fused with MIP1/MIP2

Immunogenicity experiments were performed on fusion particles such as MIP1-HA1-N63C51, MIP1-HA1-N57C51, and MIP2-HA1-N57C51. The experimental animals were female, 6-week-old BALB/c mice. The way of immunization is intramuscular injection into the deltoid muscle of the limbs. Fused particles were mixed with aluminum adjuvant at a final concentration of 5 μg/100 μl. HA1 protein not fused with MIP was mixed with Freund's adjuvant at the same dose as a control. It is generally believed that Freund's adjuvant has a better effect of promoting immune response than aluminum adjuvant. The same dose was used to boost at 2 weeks and 4 weeks, the isolated serum was collected every week, and the serum antibody titer was detected by ELISA with HA1. Serum was diluted as follows: 0 week blood, 1:100 dilution; 1-5 week blood, 1:100, 1:300, 1:900, 1:2700 serial dilution; 6 week blood, 1:100, :400, 1:1600, 1:6400 serial dilutions. The ELISA positive cut-off value was set at 0.3. The growth curve of the detected antibody titers is shown in FIG. 8 . The results showed that the antibody titers elicited by fusion particles at 6 weeks were comparable to those of Freund's adjuvant/HA1, which indicated that the granulated HA1 fusion protein had comparable immunogenicity to Freund's adjuvant/HA1, thus indicating that MIP1/MI P2-induced granulation preserves and enhances the immunogenicity of the target protein.

Embodiment 7: the construction of the expression vector of the HIV-1 env protein fragment of fusion MIP1/MIP2

HIV-1-env-gp41-26 and HIV-1-env-gp120-222 fused with MIP1/MIP2 build

Use the pNL 4-3 plasmid (preserved in our laboratory, gene accession number: AF 324493) as the template for the PCR reaction, and HIV-1-env-gp41-26F (SEQ ID NO: 100): 5′ -GGA TCC GGA AGC ACT ATG GGC GC-3' or HIV-1-env-gp120-222F (SEQ ID NO: 102): 5'- GGA TCC GAA TCT GTA GAG-3' is the forward primer (its 5' end is introduced into a restriction Dicer BamH I site (underlined)); HIV-1-env-gp41-26R (SEQ ID NO: 101): 5'- GAA TTC ACT TGC CCA TTT ATC T-3' or HIV-1- env-gp120-222R (SEQ ID NO: 103): 5'- GAA TTC CTA TCT TTTTTC TCT CTC-3' is a reverse primer (the 5' end introduces a restriction endonuclease EcoR I site (underlined) ), the PCR reaction was carried out in a PCR thermal cycler (Biometra T3) according to the following conditions.

The amplified products were size-specific DNA fragments of about 700bp and 400bp respectively. The PCR product was connected to a commercially available pMD 18-T carrier (produced by TAKARA Company), and after identification by BamHI/EcoR I digestion, positive clones pMD 18-T-HIV-1-env-gp41-26 and pMD 18 were obtained -T-HIV-1-env-gp120-222.

In Shanghai Boya Bioengineering Co., Ltd., using M13(+)/(-) primers, the nucleotide sequence of the target fragment inserted in the pMD 18-T-HIV-1-env-gp41-26 plasmid was measured as sequence SEQID NO : 53, the amino acid sequence encoded by it is SEQ ID NO: 54; the nucleotide sequence of the target fragment inserted in pMD 18-T-HIV-1-env-gp120-222 plasmid is sequence SEQ ID NO: 55, which encode The amino acid sequence is the sequence SEQ ID NO:56.

The above pMD 18-T-HIV-1-env-gp41-26, gp120-222 plasmids were digested with BamHI/EcoRI to obtain HIV-1-env-gp41-26, gp120-222 gene fragments. Then the obtained fragments were connected with the pTO-T7-MIP1 and pTO-T7-MIP2 expression vectors digested by BamH I/EcoR I, and the positive expression clone pTO-T7- MIP1-gp41-26, pTO-T7-MIP2-gp41-26, pTO-T7-MIP1-gp120-222 and pTO-T7-MIP2-gp120-222. Take 1 μL of the plasmid (0.15 mg/ml) to transform 40 μL of competent Escherichia coli ER2566 (purchased from Invitrogen) prepared by the calcium chloride method, and spread it on a medium containing kanamycin (final concentration 100 mg/ml, the same below) solid LB medium, and cultured at 37°C for 10-12 hours until a single colony is clearly identifiable. Pick a single colony to a test tube containing 4 mL of liquid LB medium (containing kanamycin), shake it at 180 rpm at 37°C for 10 hours, and take 1 mL of the bacterial liquid from it and store it at -70°C.

Example 8: Expression and purification of HIV-1 env protein fragments fused to NIP1/MIP2

Expression of HIV-1-env-gp41-26 and gp120-222 proteins fused with MIP1/MIP2

Remove large intestine carrying recombinant plasmids pTO-T7-MIP1-gp41-26, pTO-T7-MIP2-gp41-26, pTO-T7-MIP1-gp120-222 or pTO-T7-MIP2-gp120-222 from -70°C Bacteria liquid, inoculated in 50ml of LB liquid medium containing kanamycin, cultivated at 180rpm, 37°C for about 12 hours; then transferred into 10 bottles of 500ml LB medium containing kanamycin (each bottle received Add 5ml bacterial solution), culture at 180rpm at 37°C until the OD value at 600nm is about 1.2; add 500uL IPTG (concentration 0.5mM/L), and shake at 37°C for 4 hours.

Purification of HIV-1-env-gp41-26, gp120-222 proteins fused to MIP1/MIP2 and particle assembly

After being induced at 37°C for 4 hours, centrifuge the culture at 8000rpm and 4°C for 5min, collect the bacteria, and then resuspend with the lysate at the ratio of 1g of the bacteria to 10mL of the lysate (20mM Tris buffer pH 7.2, 300mM NaCl) Bacteria, and ultrasonically disrupted. The cell lysate was centrifuged at 9500rpm at 4°C for 5min, and the precipitate (i.e. the inclusion body) was retained; the inclusion body was washed with 1% Triton-100 and Buffer I, then resuspended with 4M urea, stirred for 30min, and centrifuged at 9500rpm at 4°C for 5min ; The precipitate obtained was resuspended with 8M urea, stirred for 30min, and centrifuged at 12000rpm at 4°C for 15min; harvested supernatant (that is, 8M urea dissolved supernatant), and dialyzed it to PBS so that the recombinant protein was assembled into particles; further Centrifuge at 12000 rpm at 4° C. for 15 min, and harvest the supernatant (ie, renatured supernatant). The results of SDS-PAGE analysis of the expression, purification and renaturation of the fusion proteins MIP1-gp41-26 and MIP2-gp41-26 are shown in lanes 2, 4, 6 and lanes 1, 3, and 5 of Figure 9A, respectively, obtained The molecular weight of the purified protein is about 30kD, and the purity is greater than 70%. The results of SDS-PAGE analysis of the expression, purification and renaturation of the fusion proteins MIP1-gp120-222 and MIP2-gp120-222 are shown in lanes 2, 4, 6 and lanes 1, 3, and 5 of Figure 9B, respectively. The molecular weight of the purified protein is about 45kD, and the purity is greater than 80%.

Example 9: Particle analysis of HIV-1 env protein fused to MIP1/MIP2

Molecular sieve chromatography of fusion proteins

The 1120 Compact LC high-performance liquid chromatography system of Agilent Corporation of the United States was used for chromatography, and the analytical column used was TSK Gel PW5000xl 7.8x300mm. Pre-equilibrate the chromatographic column with 2 times the column volume of PBS until the absorbance at 280 nm does not change significantly, and reset the absorbance of the detector to zero. Samples were injected by an autosampler and the results analyzed. The results are shown in Figure 10, the main peak retention time of MIP1-gp41-26, MIP2-gp41-26, MIP1-gp120-222 and MIP2-gp120-222 protein in molecular sieve chromatography column is all less than 239 particles (Li et al, Vaccine , 2005), which shows that these four fusion proteins can form particles (the smaller the retention time in the molecular sieve chromatogram, the larger the molecular weight or diameter of the sample).

Dynamic Light Scattering Analysis of Granularity of Fusion Proteins

The instrument used is DynaPro MS/X dynamic light scattering instrument (including temperature controller) produced by American Protein Solutions Company, and the algorithm used is Regulation algorithm. The samples were particle assembled MIP1-gp41-26, MIP2-gp41-26, MIP1-gp120-222 and MIP2-gp120-222 proteins. The samples were measured after being filtered through a 0.22 μm filter membrane. The measurement results are shown in Figure 11. The results showed that after env protein fragments gp41-26 and gp120-222 were fused with MIP1 or MIP2, they could form particle components with a radius of 20-30 nm in solution.

Particle TEM Observation of Fusion Protein

The instrument used is a 120kV transmission electron microscope (model: JEM2100) produced by Japan Electronics Corporation, with a magnification of 100,000 times. The assembled fusion proteins MIP1-gp41-26, MIP2-gp41-26, MIP1-gp120-222 and MIP2-gp120-222 were negatively stained with 2% phosphotungstic acid pH 7.0, and fixed on the carbon-sprayed copper grid. Make observations. The results of the electron microscope are shown in Figure 12, where it can be seen that the fusion protein forms a large number of particles with a radius of about 20 nm.

Example 10: Immunogenicity analysis of HIV-1 env protein fused to MIP1/MIP2

Immunogenicity Analysis of MIP1-gp41-26 and MIP2-gp41-26 Particles

Experimental animals: BALB/c mice, female, 6 weeks old, 3 mice in each group. Immunization method: Intramuscular injection into the deltoid muscle of the limbs. Immunization volume: 100 μl. Immunization schedule: primary immunization at 0 weeks, booster at 2 weeks and 4 weeks. MIP1-gp41-26 and MIP2-gp41-26 particles were respectively set at 0.5 μg/no adjuvant group (Ad-) and 5 μg/aluminum adjuvant group (Al+). 0.5 μg/no adjuvant (Ad-) and 0.5 μg/Freund’s adjuvant (F+) of gp41-26 protein not fused with MIP were used as controls. In the Freund's adjuvant experimental group, complete Freund's adjuvant (1:1 dilution) was used for initial immunization, and incomplete Freund's adjuvant (1:1 dilution) was used for booster immunization. The content of aluminum adjuvant was 1.6mg/ml.

Blood was drawn every week to separate serum, and 100 ng of gp41-26 protein was used to perform antibody enzyme-linked immunosorbent assay (ELISA) to determine the titer of the antibody. The logarithmic mean of the antibody titers from 3 mice per group is plotted against time. The results are shown in Figure 13, where MIP1-gp41-26 and MIP2-gp41-26 particles can produce antibodies in the second week under the condition of low dose of 0.5 μg/no adjuvant, and after two boosters, the The antibody titer of 5 weeks can reach 10 ^3.7 ; while the 0.5μg/no adjuvant control group can produce antibodies in the 3rd week, and the antibody titer of the 5th week is only 10 ^2.3 , the 0.5μg/Freund's adjuvant control group Also can only produce the antibody titer of 10 ^2.4 in the 5th week. It can be seen that the granulation induced by MIP can increase the antibody titer induced by gp41-26 protein by more than 1 order of magnitude, which is higher than the immunogenicity of the control protein/Freund's adjuvant, which indicates that MIP can significantly increase the antibody titer of the target protein immunogenicity. The results also showed that by using the aluminum adjuvant that can be used in humans to assist the MIP fusion protein and increasing the immunization dose to 5 μg, the antibody titer can be increased by another order of magnitude to 10 ^5.5 .

Immunogenicity Analysis of MIP1-gp120-222 and MIP2-gp120-222 Particles

Experimental animals: BALB/c mice, female, 6 weeks old, 3 mice in each group. Immunization method: Intramuscular injection into the deltoid muscle of the limbs. Immunization volume: 100 μl. Immunization schedule: primary immunization at 0 weeks, booster at 2 weeks and 4 weeks. MIP1-gp120-222 and MIP2-gp120-222 particles were respectively set at 0.5 μg/no adjuvant group (Ad-) and 5 μg/aluminum adjuvant group (Al+). 0.5 μg/no adjuvant (Ad-) and 0.5 μg/Freund’s adjuvant (F+) of gp120-222 protein not fused with MIP were used as controls. In the Freund's adjuvant experimental group, complete Freund's adjuvant (1:1 dilution) was used for initial immunization, and incomplete Freund's adjuvant (1:1 dilution) was used for booster immunization. The content of aluminum adjuvant was 1.6mg/ml.

Blood was drawn every week to separate serum, and 100 ng of gp120-222 protein was used to perform antibody enzyme-linked immunosorbent assay (ELISA) to determine the titer of the antibody. The logarithmic mean of the antibody titers of 3 mice per group was plotted against the time of immunization. The results are shown in Figure 14, where MIP1-gp120-222 and MIP2-gp120-222 particles were boosted twice at a low dose of 0.5 μg/no adjuvant, and the antibody titers at week 5 could reach 10 ^3.5 ; while the 0.5 μg/no adjuvant control group had an antibody titer of only 102 ^.1 at week 5, and the 0.5 μg/Freund's adjuvant control group could only produce an antibody titer of 10 ^3.5 . It can be seen that the granulation induced by MIP can increase the antibody titer induced by gp120-222 protein by more than 1 order of magnitude, and the immunogenicity is comparable to that of the control protein/Freund's adjuvant, which indicates that MIP can significantly improve the immunity of the target protein Originality. The results also showed that by using the aluminum adjuvant that can be used in humans to assist the MIP fusion protein and increasing the immune dose to 5 μg, the antibody titer can be increased by another order of magnitude to about 10 ⁵ .

Example 11: Expression and purification of HIV-1B subtype virus strain (named HIV-1-22) P24 protein fused with MIP1/MIP2

Construction of expression vector of HIV-1-22-P24 fused with MIP1/MIP2

The P24 gene of HIV-1-22 strain (the capsid protein of its coding HIV, also known as CA protein) is cloned from HIV-1 positive serum, and its full length is 693bp, and its nucleotide sequence is as SEQ ID NO: 57 As shown, the encoded amino acid sequence is shown in SEQ ID NO: 58. The above-mentioned gene fragment was used as the template of the PCR reaction, and P24F (SEQ ID NO: 104): 5′-GGATCC AAT TTC CCT ATA GTG CAG AAC ATC C-3′ was used as the forward primer (the 5′ end of which introduced a restriction endonuclease enzyme BamH I site); P24R (SEQ ID NO: 105): 5'-GAATTC TTA TGC CTC AGC CAA TAC TCT TGC-3' is a reverse primer (its 5' end introduces a restriction endonuclease EcoR I site ), carry out the PCR reaction according to the following conditions in a PCR thermal cycler (Biometra T3).

The amplified product is a size-specific DNA fragment of about 700bp. Using a method similar to that described in Example 7, insert the PCR product into pTO-T7-MIP1 and pTO-T7-MIP2 prokaryotic expression vectors to obtain a positive expression clone pTO-T7-MIP1-P24 inserted with the P24 protein gene fragment and pTO-T7-MIP2-P24.

Take 1 μL of the plasmid (0.15 mg/ml) to transform 40 μL of competent Escherichia coli ER2566 (purchased from Invitrogen) prepared by the calcium chloride method, and spread it on a medium containing kanamycin (final concentration 100 mg/ml, the same below) solid LB medium, and cultured at 37°C for 10-12 hours until a single colony is clearly identifiable. Pick a single colony to a test tube containing 4 mL of liquid LB medium (containing kanamycin), shake it at 180 rpm at 37°C for 10 hours, and take 1 mL of the bacterial liquid from it and store it at -70°C.

Expression of MIP1-P24 and MIP2-P24 fusion proteins

Take out the Escherichia coli liquid carrying the recombinant plasmid pTO-T7-MIP1-P24 or pTO-T7-MIP2-P24 from -70°C, inoculate 50ml of LB liquid medium containing kanamycin, at 180rpm, 37°C Cultivate for about 12 hours; then transfer to 10 bottles of 500ml kanamycin-containing LB medium (each bottle is inserted with 5ml bacterial solution), cultivate at 180rpm and 37°C until the OD value at 600nm is about 1.2; add 500uL IPTG (concentration 0.5mM/L), shaking culture at 37°C for 4 hours.

Purification of MIP1-P24 and MIP2-P24 fusion proteins

After being induced at 37°C for 4 hours, centrifuge the culture at 8000rpm and 4°C for 5min, collect the cells, and then weigh 1g cells with 10mL lysate (20mM Tris buffer pH 7.2, 300mM NaCl) with the lysate. Suspended bacteria were subjected to ultrasonic disruption. The cell lysate was centrifuged at 9500rpm at 4°C for 5min, and the precipitate (i.e. the inclusion body) was retained; the inclusion body was washed with 1% Triton-100 and Buffer I, then resuspended with 2M urea, stirred for 30min, and centrifuged at 9500rpm at 4°C for 5min; The obtained pellet was resuspended with 4M urea, stirred for 30min, and centrifuged at 12000rpm at 4°C for 15min; the supernatant (that is, 4M urea dissolving supernatant) was harvested and dialyzed into PBS to allow the recombinant protein to be assembled into particles; Centrifuge at 12000 rpm at 4°C for 15 min, and harvest the supernatant (ie, refolding supernatant).

The results of the SDS-PAGE analysis of the expression, purification and renaturation of the fusion proteins MIP1-P24 and MIP2-P24 are shown in Figure 15, wherein lanes 1-6 show characteristic protein bands around 35kD, and lanes 3 and 6 show the obtained The purity of the purified protein is about 80%.

Example 12: Particle analysis of HIV-1 P24 protein fused to MIP1/MIP2

Molecular sieve chromatography analysis of fusion protein granularity

The 1120Compact LC high-performance liquid chromatography system of Agilent Corporation of the United States was used for chromatography, and the analytical column used was TSK Gel PW5000xl 7.8x300mm. Pre-equilibrate the chromatographic column with 2 times the column volume of PBS until the absorbance at 280 nm does not change significantly, and reset the absorbance of the detector to zero. Samples were injected by an autosampler and the results analyzed. The results are shown in Figure 16, the first protein peaks of MIP1-P24 and MIP2-P24 fusion proteins have a similar retention time to the protein peaks of 239 particles (Li et al, Vaccine, 2005), which indicates that fusion proteins can form In granular form comparable in size to 239, the component content is approximately 40%.

Dynamic Light Scattering Analysis of Granularity of Fusion Proteins

The instrument used is DynaPro MS/X dynamic light scattering instrument (including temperature controller) produced by American Protein Solutions Company, and the algorithm used is Regulation algorithm. The samples are MIP1-P24 and MIP2-P24 proteins assembled by particles. The samples were measured after being filtered through a 0.22 μm filter membrane. The measurement results are shown in Figure 17. The results showed that after fusion with MIP1 or MIP2, P24 could form particle components with a radius of about 18nm or 24nm in solution.

Particle TEM Observation of Fusion Protein

The instrument used is a 120kV transmission electron microscope (model: JEM2100) produced by Japan Electronics Corporation, with a magnification of 100,000 times. The fusion protein MIP1-P24 and MIP2-P24 particles were negatively stained with 2% phosphotungstic acid pH7.0, fixed on the carbon-sprayed copper grid, and observed. The results of the electron microscope are shown in Figure 18, where it can be seen that the fusion protein forms a large number of particles with a radius of about 20 nm.

Example 13: Immunogenicity analysis of HIV-1 P24 protein fused to MIP1/MIP2

Experimental animals: BALB/c mice, female, 6 weeks old, 3 mice in each group. Immunization method: Intramuscular injection into the deltoid muscle of the limbs. Immunization volume: 100 μl. Immunization schedule: primary immunization at 0 weeks, booster at 2 weeks and 4 weeks. MIP1-P24 and MIP2-P24 particles were set at 0.5 μg/no adjuvant group (Ad-) and 5 μg/aluminum adjuvant group (Al+), respectively. 0.5 μg/no adjuvant (Ad-) and 0.5 μg/Freund’s adjuvant (F+) of P24 protein not fused with MIP were used as controls. In the Freund's adjuvant experimental group, complete Freund's adjuvant (1:1 dilution) was used for initial immunization, and incomplete Freund's adjuvant (1:1 dilution) was used for booster immunization. The content of aluminum adjuvant was 1.6mg/ml.

Blood was drawn every week to separate serum, and 100 ng of P24 protein was used to perform antibody enzyme-linked immunosorbent assay (ELISA) to determine the titer of the antibody. The logarithmic mean of the antibody titers from 3 mice per group is plotted against time. The results are shown in Figure 19, where MIP1-P24 and MIP2-P24 particles can produce antibodies in the second week or the first week under the condition of a low dose of 0.5 μg/no adjuvant, and after two boosters, the The antibody titers at 5 weeks can reach 10 ^3.9 and 10 ^4.6 ; while the 0.5μg/no adjuvant control group can only produce antibodies at the 3rd week, and the antibody at the 5th week is only 10 ^3.3 , 0.5μg/Freund's adjuvant control Group also can only produce the antibody titre of 10 ^2.9 in the 5th week. It can be seen that the granulation induced by MIP can increase the antibody titer induced by P24 protein by more than 1 order of magnitude, which is higher than the immunogenicity of the control protein/Freund's adjuvant, which indicates that MIP can significantly improve the immunity of the target protein. Originality. The results also showed that by using the aluminum adjuvant that can be used in humans to assist the MIP fusion protein and increasing the immunization dose to 5 μg, the antibody titer can be increased by another order of magnitude to 10 ^5.4 .

Example 14: Expression and purification of HBV-HBsAg antigen fused to MIP2

Construction of expression vector of HBV-HBsAg antigen fused with MIP2

A plasmid containing the coding sequence of the HBV-HBs Ag antigen aa 100-165 peptide (named 1021) (see Gao Yi et al., Acta Virology, 2003, 19 (1): 31-35, which is incorporated herein by reference) As a template for the PCR reaction, HBVSAg-1021F (SEQ ID NO: 106): 5'-TTT GGA TCC TAT CAA GGT ATG TTG CCC GTT-3' was used as the forward primer (the 5' end of which was introduced with the restriction enzyme BamH I site); with HBVSAg-1021R (SEQID NO: 107): 5'-TTT GAA TTC TTA CAC TCC CAT AGG AAT CTT GC-3' as reverse primer (its 5' end introduces restriction endonuclease EcoR I Site), PCR reaction was carried out in a PCR thermal cycler (Biometra T3) according to the following conditions.

The amplified product is a size-specific DNA fragment of about 200 bp. Using a method similar to that described in Example 7, the PCR product was inserted into the pTO-T7-MIP2 prokaryotic expression vector to obtain a positive expression clone pTO-T7-MIP2-HBsAg-1021 inserted with the HBsAg-1021 gene fragment. The nucleotide sequence of the inserted target fragment is shown in SEQ ID NO:59, and the encoded amino acid sequence is shown in SEQ ID NO:60.

The HBsAg-1021 protein fused with MIP2 was expressed in Escherichia coli and purified using a method similar to that described in Example 11, except that the purification steps were partially changed. Specifically, in the purification step, the inclusion body pellet was first washed with 20mM Tris buffer containing 2% Triton100, then resuspended with 2M urea, stirred for 30min, and centrifuged at 9500rpm at 4°C for 5min; the obtained pellet was then washed with 4M urea , 20mM Tris buffer, resuspended in 1‰β-mercaptoethanol, stirred for 30min, centrifuged at 12000rpm, 4°C for 15min; harvested the supernatant, and dialyzed it into 2M urea, 20mM carbonate buffer, 1‰β-mercapto Ethanol, then dialyzed into 1M urea, 20mM Tris buffer, 1‰β-mercaptoethanol, and finally dialyzed into 10mM phosphate buffer, pH7.5, 0.5M NaCl; further centrifuged at 12000rpm, 4°C for 15min, The supernatant (ie, renaturation supernatant) was harvested. The SDS-PAGE analysis results of the renatured MIP2-HBsAg-1021 fusion protein are shown in Figure 20, wherein the purity of the target protein (about 17kD) is about 60%.

Example 15: Particle analysis of HBsAg-1021 protein fused to MIP2

Dynamic Light Scattering Analysis of Granularity of Fusion Proteins

The instrument used is DynaPro MS/X dynamic light scattering instrument (including temperature controller) produced by American Protein Solutions Company, and the algorithm used is Regulation algorithm. The sample is particle-assembled MIP2-HBsAg-1021 protein. The samples were measured after being filtered through a 0.22 μm filter membrane. The measurement results are shown in Figure 21. The results showed that after HBsAg-1021 polypeptide was fused with MIP2, it could form particle components with a diameter of about 15nm in solution.

Particle TEM Observation of Fusion Protein

The instrument used is a 120kV transmission electron microscope (model: JEM2100) produced by Japan Electronics Corporation, with a magnification of 100,000 times. The particles of the fusion protein MIP2-HBsAg-1021 were negatively stained with 2% phosphotungstic acid pH 7.0, fixed on the carbon-sprayed copper grid, and observed. The electron microscope results are shown in Figure 22, where it can be seen that the fusion protein forms a large number of particles with a diameter of about 15-20 nm.

Example 16: Expression and purification of HPV16L2 protein fragments fused to MIP1

Construction of Expression Vector of HPV16L2 Protein Fragment Fused with MIP1

The full-length gene of HPV16L2 protein (gene accession number: AF125673_7) was synthesized by Shanghai Yingjun Company. Using the synthetic gene as a template, L2FP (SEQ ID NO: 108): 5'- GGA TCC ATG CGA CAC AAA CG-3' was used as a forward primer (the 5' end of which was introduced into the restriction endonuclease BamH I site ), with L2RP (SEQ ID NO: 109): 5'- GTC GAC CTATTG GTC AGT TAT ATT AAT G-3' as reverse primer (its 5' end introduces restriction endonuclease Sal I site), in PCR The PCR reaction was carried out in a thermal cycler (Biometra T3) according to the following conditions.

The amplified product is a size-specific DNA fragment of about 1500bp. Using a method similar to that described in Example 7, the PCR product was inserted into the pTO-T7-MIP1 prokaryotic expression vector to obtain a positive expression clone pTO-T7-MIP1-L2C50 inserted with the target gene fragment. The nucleotide sequence of the inserted target fragment is shown in SEQ ID NO: 61, and the encoded amino acid sequence is shown in SEQ ID NO: 62. This amino acid sequence lacks 50 C-terminal amino acids compared with the full-length L2 protein, and the corresponding The protein was named L2C50.

The method similar to that described in Example 11 was used to express and purify the L2C50 protein fused with MIP1 in Escherichia coli except that the purification steps were partially changed. Particularly, in the purification step, the 4M urea dissolving supernatant is dialyzed into 20mM phosphate buffer saline, pH7.5, in 4M urea solution; Then use Q-FF anion exchange chromatography (the medium is Q Fast Flow produced by GE Sepharose, the instrument is the Purifier 100 liquid chromatograph of GE Company) purifies, with 20mM phosphate buffer saline, pH7.5, 4M urea, 80mM NaCl elutes impurity protein, with 20mM phosphate buffer saline, pH7.5, 4M Elute the target protein with urea and 150mM NaCl; then dialyze the target protein fraction into PBS for renaturation; further centrifuge at 12000rpm, 4°C for 15min, and harvest the supernatant (ie, renaturation supernatant). The SDS-PAGE analysis results of the fractions in each purification step are shown in FIG. 23 . The finally obtained renatured fusion protein MIP1-L2C50 has a purity of about 70% ( FIG. 23 , lane 8).

Example 17: Particle analysis of HPV16 L2 protein fragments fused to MIP1

Molecular sieve chromatography analysis of fusion protein granularity

The 1120Compact LC high-performance liquid chromatography system of Agilent Corporation of the United States was used for chromatography, and the analytical column used was TSK Gel PW5000xl 7.8x300mm. Pre-equilibrate the chromatographic column with 2 times the column volume of PBS until the absorbance at 280 nm does not change significantly, and reset the absorbance of the detector to zero. Samples were injected by an autosampler and the results analyzed. The results are shown in Figure 24, the first protein peak of the MIP1-L2C50 fusion protein is earlier than the retention time of the protein peak of 239 particles (Lietal, Vaccine, 2005), which shows that the fusion protein can form particles, and the component content About 50%.

Dynamic Light Scattering Analysis of Granularity of Fusion Proteins

The instrument used is DynaPro MS/X dynamic light scattering instrument (including temperature controller) produced by American Protein Solutions Company, and the algorithm used is Regulation algorithm. The sample is particle-assembled MIP1-L2C50 protein. The samples were measured after being filtered through a 0.22 μm filter membrane. The measurement results are shown in Figure 25. The results showed that after HPV16 L2C50 protein was fused with MIP1, it could form particle components with a diameter of about 18nm in solution.

Particle TEM Observation of Fusion Protein

The instrument used is a 120kV transmission electron microscope (model: JEM2100) produced by Japan Electronics Corporation, with a magnification of 100,000 times. The fusion protein MIP1-L2C50 particles were negatively stained with 2% phosphotungstic acid pH 7.0, fixed on the carbon-sprayed copper grid, and observed. The electron microscope results are shown in Figure 26, where it can be seen that the fusion protein forms a large number of particles with a diameter of about 20-30nm.

Although the specific implementation of the present invention has been described in detail, those skilled in the art will understand that: according to all the teachings that have been disclosed, various modifications and changes can be made to the details, and these changes are all within the protection scope of the present invention . The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (13)

1. one kind can make target protein take place to assemble or granular peptide, and it comprises

(1) aminoacid sequence, said aminoacid sequence are the fragments of HEV ORF2 encoded polypeptides, and comprise the 370-435 amino acids residue of HEV ORF2 encoded polypeptides at least;

For example; The N end of said aminoacid sequence can be positioned between 345-370 position (the for example 345-368 position) amino-acid residue of HEV ORF2 encoded polypeptides, and/or the C of said aminoacid sequence end can be positioned between 435-460 position (the for example 451-460 position) amino-acid residue of HEV ORF2 encoded polypeptides;

For example, said aminoacid sequence can comprise the 368-451 position or the 368-460 amino acids residue of HEV ORF2 encoded polypeptides, perhaps is made up of the 368-451 position or the 368-460 amino acids residue of HEV ORF2 encoded polypeptides;

(2) defined aminoacid sequence different are disappearance, substitute and/or have added 1-20 (like 1-15,1-10,1-5,1,2,3, or 4) amino-acid residue in the variant of defined aminoacid sequence in (1), said variant and (1);

For example, the different 12A10 of the being epi-positions of defined aminoacid sequence are deleted or are suddenlyd change in said variant and (1);

For example, said variant is to comprise the peptide of being deleted or being suddenlyd change by 368-460 amino acids residue and its 12A10 epi-position of HEV ORF2 encoded polypeptides; Or

(3) defined aminoacid sequence have at least 80% identity of (as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) in the variant of defined aminoacid sequence in (1), said variant and (1);

For example, in (2) or (3) in defined variant and (1) defined aminoacid sequence different be one or more conservative amino acid replacements, for example 1-20 (like 1-15,1-10,1-5,1,2,3, or 4) conservative amino acid replacement.

2. fusion rotein, it comprises the peptide and the target polypeptides of claim 1, and said peptide is connected through joint with target polypeptides is optional,

For example, said target polypeptides is connected to the N end and/or the C end of said peptide,

For example, said joint can be a flexible peptide linker.

3. conjugate, it comprises the peptide and the target polypeptides of claim 1, and wherein, said peptide is connected through covalent manner or non-covalent mode with target polypeptides, and said peptide is connected through joint with target polypeptides is optional.

4. the polynucleotide of the peptide of the claim 1 of encoding or the fusion rotein of claim 2.

5. the construct that comprises the polynucleotide of claim 4.

6. comprise the carrier of construct of polynucleotide or the claim 5 of claim 4, for example said carrier is an expression vector.

7. the host cell or the organism of carrier of construct or claim 6 that comprises polynucleotide or the claim 5 of claim 4.

8. aggregate or particle; Wherein said aggregate or particle comprise the fusion rotein of at least 2 claims 2 or the conjugate of claim 3; Perhaps, said aggregate or particle are formed by the fusion rotein of 2 claims 2 or the conjugate of claim 3 at least.

The peptide of claim 1 be used to improve polypeptide activity, or improve polypeptide immunogenicity, or produce polypeptide aggregate or particle, or make the polypeptide prilling or form the purposes of aggregate.

10. improve polypeptide activity, or improve polypeptide immunogenicity, or produce polypeptide aggregate or particle, or make the polypeptide prilling or form the method for aggregate; It comprises that the peptide with said polypeptide and claim 1 links together; For example put together or merge (randomly using joint, for example flexible peptide linker).

11. prepare the method for particulate antigen or vaccine, it comprises that the peptide that makes as antigenic polypeptide and claim 1 links together, and for example puts together or merges (randomly using joint, for example flexible peptide linker), forms particulate antigen or vaccine afterwards.

12. prepare the method for nano particle, it comprises links together the peptide of polypeptide and claim 1, for example puts together or merges (randomly using joint, for example flexible peptide linker), forms nano particle afterwards.

13. the protein grain that obtains through each method of claim 10-12.

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