Short peptide composition for inhibiting inclusion body formation and promoting self-assembly of Gag-Like protein nanoparticles

Abstract

The invention discloses an oligopeptide composition for inhibiting inclusion body formation and promoting Gag-Like protein nanoparticle self-assembly, the oligopeptide composition comprises a C-terminal oligopeptide and an N-terminal oligopeptide, the amino acid sequence of the N-terminal oligopeptide is as shown in SEQ ID NO: 1, and the amino acid sequence of the C-terminal oligopeptide is as shown in SEQ ID NO: 2. The oligopeptide composition can effectively avoid formation of a prokaryotic expression protein inclusion body and / or promote dissolution of prokaryotic expression protein, the oligopeptide composition is further utilized to be combined with HIV-1p24 and human endogenous retrovirus K capsid protein CA, nanoparticles are prepared respectively, and the oligopeptide composition can be used as a carrier for antigen surface display or drug transportation. The nanoparticles formed by the HIV-1 capsid protein p24 and the human HERV K CA have cavities which are obviously larger than those of ferritin, so that the nanoparticles have higher loading capacity; as an antigen display platform, the antibody also has good stability and wide applicability.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and more specifically to a short peptide composition capable of inhibiting the formation of inclusion bodies and promoting the self-assembly of Gag-Like protein nanoparticles. Background Art

[0002] A complete virus particle is called a "virion" (also known as a virus particle or virus particle), which is composed of a protective "capsid" (also known as a shell or protein shell) composed of proteins and the nucleic acid enclosed by the capsid. The protein capsid of a retrovirus is composed of the Gag polyprotein encoded by the viral gene, which includes the matrix (MA), capsid structural protein (CA) and nucleocapsid protein (NC). Gag proteins aggregate and self-assemble on the inner side of the host cell membrane to form virus particles. The capsid encapsulates and protects the viral RNA genome, ensuring its stability during the maturation of the virus particles. When infecting host cells, the capsid participates in the uncoating process, releasing the viral RNA into the cytoplasm for reverse transcription. In addition, the capsid protein also interacts with host cell proteins to help transport and locate the viral RNA.

[0003] Virus-like particles (VLPs) are hollow particles containing one or more structural proteins of a virus. They do not have viral nucleic acid and cannot replicate autonomously. They are identical or similar in morphology to real virus particles. They are commonly known as pseudoviruses and are widely used in vaccine production. Capsid protein does not contain viral genome, has high safety, can stimulate strong immune response, and can self-assemble into capsid. Therefore, using it to prepare VLPs has obvious advantages.

[0004] The prokaryotic expression system is a commonly used protein expression system. Its advantages in production include rapid growth and production cycle, low cost and simple culture conditions, easy gene manipulation and expression optimization, high expression yield and adaptability to large-scale production needs. These characteristics make it an ideal choice for the production of VLPs.

[0005] However, most retroviral capsid proteins often form protein inclusion bodies during production in prokaryotic expression systems (such as E. coli). The inclusion bodies need to be dissolved with urea and purified under denaturation conditions, and then renatured to obtain active proteins. They usually form monomers or oligomers, which limits their further assembly into stable nanoparticles. This seriously restricts the preparation of VLPs using retroviral capsid proteins.

[0006] The capsids of the retroviridae are all composed of Gag proteins, and Gag proteins are very similar in structure. This type of protein is collectively called Gag-like proteins. The capsid protein CA (p24) encoded by HIV virus Gag (denoted as HIV p24) is part of the capsid protein, which will self-assemble into a hexamer. The stable hexamer as a basic unit will further assemble to form a tubular structure. After the amino acids at positions 21 and 22 are mutated to cysteine, a spherical structure can be formed. The capsid protein of the human endogenous retrovirus K family (HERV-K-CA) has the property of self-assembly and can self-assemble into spherical nanoparticles. However, it has the same problem as HIVp24, that is, it has low solubility in the prokaryotic expression system, poor self-assembly ability, and forms a small number of nanoparticles, which is a huge limitation for the large-scale production of stable nanoparticles. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a short peptide composition for inhibiting the formation of inclusion bodies.

[0008] The present invention is aimed at the scientific and technological difficulties that Gag-like protein (such as HIV p24 and HERV-K-CA) nanoparticles form protein inclusion bodies in prokaryotic expression systems as a delivery system, have low solubility, are unstable, and have poor nanoparticle assembly ability. The present invention achieves the ability of nanoparticles to promote dissolution and assembly while introducing as few exogenous sequences as possible by introducing designed short peptides. Among them, the optimized HIV p24 nanoparticles can be used as a delivery carrier to package goods in its cavity, and as an antigen display platform to enhance the humoral immune response of surface antigens. It can also be used as an immunogen for HIV vaccines, providing a new idea and insight for the design of HIV vaccines. The optimized human endogenous retrovirus K capsid protein also has the same effect of promoting dissolution and assembly, and can be used as an endogenous delivery carrier to play its role.

[0009] The first object of the present invention is to provide a short peptide composition that can avoid the formation of inclusion bodies of prokaryotic expressed proteins and / or promote the solubilization of prokaryotic expressed proteins.

[0010] The second object of the present invention is to provide a biomaterial.

[0011] The third object of the present invention is to provide the use of the short peptide combination and / or the biomaterial in inhibiting the formation of prokaryotic expression protein inclusion bodies, promoting the dissolution of prokaryotic expression proteins or preparing self-assembled nanoparticles.

[0012] The fourth object of the present invention is to provide a method for inhibiting the formation of inclusion bodies of prokaryotic expressed proteins and / or promoting the dissolution of prokaryotic expressed proteins.

[0013] The fifth object of the present invention is to provide a recombinant HIV-1 capsid protein p24.

[0014] The sixth object of the present invention is to provide a recombinant human endogenous retrovirus K capsid protein CA.

[0015] The seventh object of the present invention is to provide another biomaterial.

[0016] The eighth objective of the present invention is to provide the use of the recombinant HIV-1 capsid protein p24, the recombinant human endogenous retrovirus K capsid protein CA or the biomaterial in the preparation of self-packaging nanoparticles.

[0017] The ninth object of the present invention is to provide a nanoparticle.

[0018] The tenth object of the present invention is to provide a conjugate.

[0019] The eleventh object of the present invention is to provide another nanoparticle.

[0020] A twelfth object of the present invention is to provide a surface display system.

[0021] The thirteenth object of the present invention is to provide a surface display antigen system.

[0022] The fourteenth object of the present invention is to provide the use of the recombinant HIV-1 capsid protein p24, the recombinant human endogenous retrovirus K capsid protein CA, the biomaterial, the nanoparticle, the conjugate, the nanoparticle, the surface display system or the surface display antigen system in the preparation of drugs or vaccines.

[0023] The fifteenth object of the present invention is to provide another conjugate.

[0024] The sixteenth object of the present invention is to provide another nanoparticle.

[0025] The seventeenth object of the present invention is to provide a protein nanoparticle delivery system.

[0026] The eighteenth objective of the present invention is to provide the use of the recombinant HIV-1 capsid protein p24, the recombinant human endogenous retrovirus K capsid protein CA, the biomaterial, the nanoparticles, the conjugate, the nanoparticles, and the protein nanoparticle delivery system in the preparation of drugs.

[0027] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0028] The present invention fuses two short peptides with the N-terminal domain and the C-terminal domain of the Gag-CA protein (p24) of the human immunodeficiency virus (HIV) for expression, and introduces a pair of cysteine ​​mutations to generate a nanoparticle with strong stability and strong assembly force; and fuses the two short peptides at both ends of the N-terminal domain and the C-terminal domain of the Gag-CA protein of the human endogenous retrovirus K family (HERV-K) in the same way, thus achieving better stability and assembly force.

[0029] The nanoparticles prepared by the above method can be used as an antigen display platform; HIV-p24 nanoparticles enhance the phagocytosis and presentation of surface antigens by macrophages in lymph nodes, and produce a strong humoral immune response against surface antigens in mice; at the same time, the nanoparticles can also package heterologous proteins and can be used as a large-capacity protein delivery nanoparticle carrier. Given that it is highly conserved and a potential T cell immunogen, it can be used as an antigen display platform for vaccines to address the scientific problem of high mutation of retroviruses, and has high application value.

[0030] The present invention claims protection for a short peptide composition for inhibiting inclusion body formation, comprising a C-terminal short peptide and an N-terminal short peptide, wherein the amino acid sequence of the N-terminal short peptide is shown in SEQ ID NO: 1, and the amino acid sequence of the C-terminal short peptide is shown in SEQ ID NO: 2.

[0031] The C-terminal short peptide and the N-terminal short peptide can effectively inhibit the formation of inclusion bodies during the expression of recombinant proteins using a prokaryotic expression system, and promote the solubility of the recombinant proteins.

[0032] Biological material, which is any of the following:

[0033] (1) a nucleotide molecule combination, comprising nucleic acid molecules encoding the C-terminal short peptide and the N-terminal short peptide in the short peptide composition, or reverse complementary nucleotide molecules of nucleic acid molecules encoding the C-terminal short peptide and the N-terminal short peptide in the short peptide composition;

[0034] (2) An expression cassette, wherein the 5' end is provided with a nucleotide molecule encoding the N-terminal short peptide in the short peptide combination, and the 3' end is provided with a nucleotide molecule encoding the C-terminal short peptide in the short peptide combination.

[0035] (3) A recombinant vector comprising the nucleotide molecule combination described in (1) or the expression cassette described in (2);

[0036] (4) A recombinant microorganism comprising the recombinant vector described in (3).

[0037] The use of the short peptide combination and / or the biomaterial in inhibiting the formation of prokaryotic expression protein inclusion bodies, promoting the dissolution of prokaryotic expression proteins or preparing self-assembled nanoparticles also falls within the protection scope of the present invention.

[0038] Also claimed is a method for inhibiting the formation of inclusion bodies of prokaryotic expressed proteins and / or promoting the dissolution of prokaryotic expressed proteins, constructing a recombinant expression vector for prokaryotic expression of the protein to be expressed, wherein the 5' end of the gene encoding the protein to be expressed is provided with a nucleotide molecule encoding the N-terminal short peptide in the short peptide combination, and the 3' end of the gene encoding the protein to be expressed is provided with a nucleotide molecule encoding the C-terminal short peptide in the short peptide combination.

[0039] As a specific embodiment, the present invention claims protection for a recombinant HIV-1 capsid protein p24 (N20-p24 (21C / 22C)-C20), whose amino acid sequence is shown in SEQ ID NO: 3.

[0040] And as a specific embodiment, a recombinant human endogenous retrovirus K capsid protein CA (N20-HERV-K-CA-C20), whose amino acid sequence is shown in SEQ ID NO: 8.

[0041] It is further claimed that the biological material is any one of the following:

[0042] (1) a nucleotide molecule encoding the recombinant HIV-1 capsid protein p24 or the recombinant human endogenous retrovirus K capsid protein CA;

[0043] (2) a nucleotide molecule which is the reverse complementary sequence of the nucleic acid molecule described in (1);

[0044] (3) an expression cassette comprising the nucleic acid molecule described in (1) or (2);

[0045] (4) a recombinant vector comprising the nucleotide molecule described in (3);

[0046] (5) A recombinant microorganism comprising the recombinant vector described in (4).

[0047] The use of the recombinant HIV-1 capsid protein p24, the recombinant human endogenous retrovirus K capsid protein CA or the biomaterial in preparing self-assembled nanoparticles also falls within the protection scope of the present invention.

[0048] Preferably, the nanoparticles are nanoparticles of antigen display carriers or protein transport carriers.

[0049] The invention further claims protection for a nanoparticle obtained by self-assembly of the recombinant HIV-1 capsid protein p24 or the recombinant human endogenous retrovirus K capsid protein CA.

[0050] The recombinant protein N20-p24(21C / 22C)-C20 and the recombinant protein N20-HERV-K-CA-C20 were used to self-assemble the nanoparticles N20-p24(21C / 22C)-C20 and nanoparticles

[0051] N20-HERV-K-CA-C20.

[0052] And a conjugate, which comprises a protein part and a coupling part, wherein the coupling part is coupled to the N-terminus of the protein part, and the protein part is the recombinant HIV-1 capsid protein p24 or the recombinant human endogenous retrovirus K capsid protein CA.

[0053] As a specific embodiment, the protein part and the coupling part in the conjugate are coupled via a covalent bond.

[0054] Furthermore, the covalent bond is a peptide bond or an isopeptide bond.

[0055] Furthermore, the protein part and the coupling part in the conjugate are coupled using a linker short peptide.

[0056] Furthermore, the protein part and the coupling part in the conjugate utilize the SD-GVopti connection system (doi:10.1038 / s41423-021-00736-2) or the SpyTag / SpyCatcher connection system.

[0057] A nanoparticle is obtained by self-assembly using the conjugate.

[0058] A surface display system contains the nanoparticles.

[0059] A surface display antigen system comprises the nanoparticles, wherein the coupling part in the conjugate is an antigen.

[0060] Specifically, the surface displayed antigen is obtained by self-assembly of a conjugate, wherein the conjugate is a protein having an amino acid sequence of N20-p24(21C / 22C)-C20 as shown in SEQ ID NO: 3, and a substance having antigenic activity is coupled to the N-terminus.

[0061] Preferably, coupling is performed using a linker peptide, SD-GVopti connection system (doi: 10.1038 / s41423-021-00736-2) or SpyTag / SpyCatcher connection system.

[0062] A method for surface displaying a protein with antigenic activity, wherein the 5' end of the gene encoding N20-p24(21C / 22C)-C20 with an amino acid sequence as shown in SEQ ID NO: 3 is connected to a gene encoding a Gv linker (amino acid sequence as shown in SEQ ID NO: 16), cloned into a prokaryotic expression vector, and then prokaryotic expression and purification are performed to obtain component 1; the 5' end of the gene encoding a protein with antigenic activity is connected to a gene encoding a SD linker (amino acid sequence as shown in SEQ ID NO: 17), cloned into a prokaryotic expression vector, and then prokaryotic expression and purification are performed to obtain component 2; component 1 and component 2 are incubated to display the protein with antigenic activity on the surface.

[0063] As a specific scheme, the protein with antigenic activity is the RBD protein of JN.1 of the new coronavirus (the amino acid sequence is shown in SEQ ID NO: 18).

[0064] Application of the recombinant HIV-1 capsid protein p24, the recombinant human endogenous retrovirus K capsid protein CA, the biomaterial, the nanoparticle, the conjugate, the nanoparticle, the surface display system or the surface display antigen system in the preparation of drugs or vaccines.

[0065] A conjugate comprises a protein portion and a coupling portion, wherein the coupling portion is coupled to the C-terminus of the protein portion, and the protein portion is the recombinant HIV-1 capsid protein p24 or the recombinant human endogenous retrovirus K capsid protein CA.

[0066] As a specific embodiment, the protein part and the coupling part in the conjugate are coupled via a covalent bond.

[0067] Furthermore, the covalent bond is a peptide bond or an isopeptide bond.

[0068] Furthermore, the protein part and the coupling part in the conjugate are coupled using a linker short peptide.

[0069] Furthermore, the protein part and the coupling part in the conjugate were connected using the SD-GVopti connection system (doi:

[0070] 10.1038 / s41423-021-00736-2) or SpyTag / SpyCatcher connection system.

[0071] A nanoparticle is obtained by self-assembly using the conjugate.

[0072] Preferably, the conjugate is self-assembled using the conjugate and a corresponding one of the recombinant HIV-1 capsid protein p24 or the recombinant human endogenous retrovirus K capsid protein CA.

[0073] A protein nanoparticle delivery system contains the nanoparticles, and the coupling part in the conjugate is a substance to be delivered.

[0074] A method for protein delivery, wherein the 3' end of the gene encoding N20-p24(21C / 22C)-C20 of the amino acid sequence as shown in SEQ ID NO: 3 is connected to a gene encoding a linker connector (GGGGSGGGGS), and then a gene encoding a protein to be delivered is connected to the 3' end of the gene encoding the linker connector, cloned into a prokaryotic expression vector, and then prokaryotically expressed, purified, and incubated to obtain nanoparticles for delivering the protein to be delivered.

[0075] and, a method for protein delivery,

[0076] The 3' end of the gene encoding N20-p24(21C / 22C)-C20 with the amino acid sequence as shown in SEQ ID NO: 3 is connected to the gene encoding the linker connector (GGGGSGGGGS), and then the 3' end of the gene encoding the linker connector is connected to the gene encoding the protein to be delivered. After cloning into a prokaryotic expression vector, vector 1 is obtained; after N20-p24(21C / 22C)-C20 is cloned into the prokaryotic expression vector, vector 2 is obtained, and vector 1 and vector 2 are jointly subjected to prokaryotic expression, purification, and incubation to obtain nanoparticles that deliver the protein to be delivered.

[0077] As a specific embodiment, the protein to be delivered is GFP (amino acid sequence is shown in SEQ ID NO: 19).

[0078] Application of the recombinant HIV-1 capsid protein p24, the recombinant human endogenous retrovirus K capsid protein CA, the biomaterial, the nanoparticle, the conjugate, the nanoparticle, and the protein nanoparticle delivery system in preparing drugs.

[0079] A method for delivering substances using the cavity of nanoparticles, wherein the C-terminus of the recombinant HIV-1 capsid protein p24 or the recombinant human endogenous retrovirus K capsid protein CA is coupled with a substance to be delivered, and then mixed and self-assembled with the corresponding recombinant HIV-1 capsid protein p24 or the recombinant human endogenous retrovirus K capsid protein CA that is not coupled with the substance to be delivered, to obtain nanoparticles for delivering the substance.

[0080] Preferably, coupling is performed using a linker.

[0081] More preferably, the amino acid sequence of the Linker is GGGGSGGGGS.

[0082] Preferably, the substance to be delivered is a protein, and the 3' end of the coding gene of the recombinant HIV-1 capsid protein p24 or the recombinant human endogenous retrovirus K capsid protein CA is connected to the coding gene of the protein through the coding gene of the Linker connector and cloned into a prokaryotic expression vector; the corresponding coding gene of the recombinant HIV-1 capsid protein p24 or the recombinant human endogenous retrovirus K capsid protein CA is also cloned into a prokaryotic expression vector; the obtained two expression vectors are co-transformed and expressed and then self-assembled to obtain.

[0083] Compared with the prior art, the present invention has the following beneficial effects:

[0084] The present invention discloses a short peptide composition for preventing the formation of inclusion bodies of prokaryotic expression proteins and / or promoting the dissolution of prokaryotic expression proteins, comprising a C-terminal short peptide and an N-terminal short peptide, wherein the amino acid sequence of the N-terminal short peptide is shown in SEQ ID NO: 1, and the amino acid sequence of the C-terminal short peptide is shown in SEQ ID NO: 2. The short peptide composition can effectively inhibit the formation of inclusion bodies of prokaryotic expression proteins and / or promote the dissolution of prokaryotic expression proteins, and further utilizes the short peptide composition to combine with HIV-1 capsid protein p24 and human endogenous retrovirus K capsid protein CA to prepare nanoparticles, which can be used as carriers for antigen surface display or drug transport. The nanoparticles formed by HIV-1 capsid protein p24 and human endogenous retrovirus K capsid protein CA have large cavities with diameters of 40nm and 17-19nm, respectively, which are significantly larger than the 11-13nm cavity of ferritin and have stronger loading capacity; at different pH, different surface proteins (SD-GFP) can still achieve stable connection and self-assembly of nanoparticles, and it has good stability and wide applicability as an antigen display platform. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 Preparation and characterization of N20-p24(21C / 22C)-C20 nanoparticles;

[0086] A is a schematic diagram of the structure predicted by Alphafold. On the left: WT p24, forming a hexameric structure (PDB: 3H47); in the middle: p24 (21C / 22C), after introducing cysteine ​​mutations at positions 21 and 22, a pentamer structure is formed (PDB: 3P05), and red indicates the formation of disulfide bonds; on the right: N20-p24 (21C / 22C)-C20, N20 (orange) promotes the aggregation of pentamers, and C20 (yellow) promotes the aggregation of C-terminal trimers.

[0087] B is N20-p24(21C / 22C)-C20 nanoparticles: left is the UV absorption peak diagram of the molecular sieve of N20-p24(21C / 22C)-C20, and the dotted line is the effective elution volume; right is the PAGE electrophoresis diagram of denatured and non-denatured N20-p24(21C / 22C)-C20;

[0088] C is the transmission electron microscopy result, showing that N20-p24(21C / 22C)-C20 assembled to form 40nm nanoparticles, scale bar: 500nm;

[0089] D is the ultraviolet absorption peak diagram of the molecular sieve. In the left figure, the top is: WT p24, the middle is: p24 (21C / 22C), and the bottom is: N20-p24 (21C / 22C)-C20; in the right figure, from top to bottom are: p24 (21C / 22C) at assembly 24h, p24 (21C / 22C) at assembly 48h, N20-p24 (21C / 22C)-C20 at assembly 24h and N20-p24 (21C / 22C)-C20 at assembly 48h. The dotted line: the effective elution volume of the nanoparticles, 440kda and 669kda are both standard products.

[0090] E is the PAGE electrophoresis of the soluble supernatant of the recombinant protein WT-p24, recombinant protein N20-WT-p24-C20, recombinant protein p24 (21C / 22C), and recombinant protein N20-p24 (21C / 22C)-C20 after induction expression in Escherichia coli;

[0091] F is a cryo-EM image of N20-HIV p24(21C / 22C)-C20 nanoparticles;

[0092] G is: dynamic light scattering particle size distribution of N20-HIV p24 (21C / 22C)-C20 nanoparticles.

[0093] Figure 2 Figure 2 is the stability of N20-HIV p24 (21C / 22C)-C20 nanoparticles; A is the PAGE electrophoresis diagram of N20-HIV p24 (21C / 22C)-C20 nanoparticles or Helicobacter pylori ferritin (H. pylori ferritin, HFP) nanoparticles treated at 4°C or 37°C under shaking or static conditions (0d, 2d, 4d, 6d, 8d and 10d); B is the ultraviolet absorption peak diagram of the molecular sieve of N20-HIV p24 (21C / 22C)-C20 nanoparticles treated at 4°C or 37°C under shaking conditions (2d, 4d, 6d, 8d and 10d).

[0094] Figure 3 This is a cytotoxicity test of N20-HIV p24 (21C / 22C)-C20 nanoparticles; the concentration gradient range is 12.5-200 μg / ml, n=8.

[0095] Figure 4 Preparation and characterization of N20-HERV-K-CA-C20 nanoparticles;

[0096] A is a schematic diagram of the structure predicted by Alphafold. The left figure is the wild-type HERV-K-CA, which mainly forms a pentamer structure; the right figure is N20-HERV-K-CA-C20, showing that N20 (orange) promotes the aggregation of pentamers, and C20 (yellow) promotes the aggregation of C-terminal trimers;

[0097] B is the ultraviolet absorption peak diagram of the molecular sieve N20-HERV-K-CA-C20, and the dotted line is the effective elution volume of the nanoparticles;

[0098] C is the transmission electron microscopy result, N20-HERV-K-CA-C20 assembled to form nanoparticles with a diameter of 17 to 19 nm, scale bar: 500 nm;

[0099] D is the dynamic light scattering particle size distribution of N20-HERV-K-CA-C20 nanoparticles;

[0100] E is the ultraviolet absorption peak diagram of molecular sieve; the mobile phase buffer of molecular sieve is pH=7.5 20mM Tri-HCl50mM NaCl, from top to bottom: WT HERV-K-CA, protein storage buffer is pH=7.5 20mM Tri-HCl 1MNaCl, N20-HERV-K-CA-C20, protein storage buffer is pH=7.5 20mM Tri-HCl 1M NaCl, N20-HERV-K-CA-C20, protein storage buffer is pH=7.5 20mM Tri-HCl 50mM NaCl and N20-HERV-K-CA-C20, protein storage buffer is pH=7.5 20mM Tri-HCl 0mM NaCl;

[0101] F is the PAGE electrophoresis of the soluble supernatant after the recombinant protein WT-HERC-K-CA and the recombinant protein N20-HERV-K-CA-C20 were induced to express in Escherichia coli at 37°C.

[0102] Figure 5 The figure above shows the humoral immune effect of N20-p24 (21C / 22C)-C20 nanoparticles. The figure below shows the titer of Anti-p24 in mouse serum detected in the sixth week after immunization.

[0103] Figure 6 The antigen display function of N20-p24(21C / 22C)-C20 nanoparticles; A is PAGE electrophoresis and transmission electron microscopy, the left picture is the result of coupling of excess SD-RFP with Gv-N20-p24(21C / 22C)-C20, the right picture is the result of coupling of excess SD-GFP with Gv-N20-HIV p24(21C / 22C)-C20, scale: 500nm; B: N20-HIV The stability of p24(21C / 22C)-C20 as an antigen display platform; the left figure is the PAGE electrophoresis of the excess recombinant protein SD-GFP and the recombinant protein Gv-N20-p24(21C / 22C)-C20 or the recombinant protein Gv-HPF after incubation and coupling (p24-NP and HPF-NP); the middle figure is the UV absorption peak diagram of the molecular sieve of p24-GFP-NP (p24-NP) and HPF-GFP-NP (HPF-NP); the right figure is the PAGE electrophoresis of the excess recombinant protein SD-GFP and the recombinant protein Gv-N20-p24(21C / 22C)-C20 or the recombinant protein Gv-HPF after incubation and coupling (p24-NP and HPF-NP); the middle figure is the UV absorption peak diagram of the molecular sieve of p24-GFP-NP (p24-NP) and HPF-GFP-NP (HPF-NP); P is the PAGE electrophoresis of the molecular sieve protein eluate (the excess SD-GFP monomer did not form nanoparticles); C is the PAGE electrophoresis and transmission electron microscopy; the left picture is the PAGE electrophoresis and transmission electron microscopy of the excess SD-GFP and Gv-N20-p24 (21C / 22C)-C20 in the system of pH = 6.5; right: PAGE electrophoresis and transmission electron microscopy of the excess SD-GFP and Gv-N20-p24 (21C / 22C)-C20 in the system of pH = 8.5, scale: 500nm.

[0104] Figure 7 The immune effect of antibodies displayed on the surface of N20-p24(21C / 22C)-C20 nanoparticles; the upper figure shows the immunization strategy, the lower left figure shows the PAGE electrophoresis of HPF and N20-p24(21C / 22C)-C20 coupled with JN.1RBD respectively; the lower right figure shows the titer of Anti-JN.1RBD in mouse serum in the sixth week after immunization.

[0105] Figure 8 A is the construction of a two-component delivery vector; A is a schematic diagram of the construction of a two-component delivery vector; B is the ultraviolet absorption peak diagram of the molecular sieve and the PAGE electrophoresis diagram of its eluate (dashed line: effective elution volume of nanoparticles); C is a transmission electron microscopy image (scale: 500nm).

[0106] Fig. 9 Nuclear localization of p24 nanoparticles packaged with GFP in the lumen.

[0107] Fig.10A is the construction of a single-component delivery vector; A is a schematic diagram of the construction of a single-component delivery vector; B is the ultraviolet absorption peak diagram of the molecular sieve and the PAGE electrophoresis diagram of its eluate (dashed line: effective elution volume of nanoparticles); C: transmission electron microscopy image (scale: 500nm). DETAILED DESCRIPTION

[0108] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0109] Example 1 Preparation of N20-p24 (21C / 22C)-C20 Nanoparticles

[0110] 1. Experimental Methods

[0111] 1. Design of short peptides

[0112] In order to avoid the formation of protein inclusion bodies during the production of proteins in prokaryotic expression systems (such as Escherichia coli), two short peptides were designed: short peptide N20 (amino acid sequence such as SEQ ID NO: 1) and short peptide C20 (amino acid sequence such as SEQ ID NO: 2).

[0113] 2. Construction of expression vector

[0114] N20 was fused to the N-terminal domain of HIV p24 protein, and C20 was fused to the C-terminal domain of p24 protein; cysteine ​​was introduced at positions 21 and 22 to prevent HIV p24 protein from forming a long tubular structure. Figure 1 The schematic diagram of A in the figure is to construct the N20-p24(21C / 22C)-C20 fusion protein (amino acid sequence is shown in SEQ ID NO: 3), and 6×His and translation termination codon are added to the 3' end of the N20-p24(21C / 22C)-C20 nucleotide sequence. It is cloned between the NcoI and XhoI restriction sites of the prokaryotic expression vector pET-28a to construct the recombinant plasmid pET-28a-N20-p24(21C / 22C)-C20-His.

[0115] 3. Expression and purification of N20-p24(21C / 22C)-C20 protein

[0116] The prepared recombinant plasmid pET-28a-N20-p24(21C / 22C)-C20-His was transformed into DH5α competent cells, cultured overnight at 37°C, and positive clones were screened and identified by PCR. The plasmid was extracted and transformed into BL21 competent cells, cultured overnight at 37°C, expanded the culture on the second day, induced expression at low temperature of 16°C overnight with IPTG, and harvested the cells by centrifugation on the third day. After crushing, the supernatant was collected for purification of the target protein. The supernatant was incubated with Ni-NTA agarose (GE Healthcare) to enrich the His-tagged target protein, and then the protein was eluted with Tris buffer containing imidazole.

[0117] The purified recombinant protein N20-p24(21C / 22C)-C20 was obtained by concentrating the solution using a 10 kDa ultrafiltration tube and replacing it with a pH = 7.5 20 mM Tri-HCl 50 mM NaCl buffer. The protein concentration was determined by the BCA method.

[0118] Afterwards, the recombinant protein N20-p24(21C / 22C)-C20 was denatured (under the condition of 100 mM DTT, 100°C for 10 min) and subjected to PAGE electrophoresis separately from the undenatured recombinant protein N20-p24(21C / 22C)-C20 and stained with Coomassie blue.

[0119] 2. Experimental Results

[0120] like Figure 1 A in the figure shows that Alphafold predicts that the wild-type p24 mainly forms a hexameric structure; after introducing cysteine ​​mutations at positions 21 and 22, a pentamer structure is mainly formed, and the red color shows the formation of disulfide bonds; N20 (orange) promotes the aggregation of pentamers, and C20 (yellow) promotes the aggregation of C-terminal trimers.

[0121] like Figure 1 As shown in B (right) in the figure, after denaturation treatment, PAGE electrophoresis showed that the purified recombinant protein N20-p24(21C / 22C)-C20 had a high purity; while without denaturation treatment, PAGE electrophoresis showed that the purified recombinant protein N20-p24(21C / 22C)-C20 presented a gradient assembly, and the largest assembly unit was pentamer. This indicates that the recombinant protein N20-p24(21C / 22C)-C20 was prepared, and it can be assembled into N20-p24(21C / 22C)-C20 nanoparticles, which is consistent with the predicted results.

[0122] Comparative Example 1 Preparation of recombinant protein HIV p24

[0123] 1. Experimental Methods

[0124] The nucleotide sequence of HIV p24 (amino acid sequence such as SEQ ID NO: 4) editing sequence was added with 6×His and translation termination codon at the 3′ end and cloned into the prokaryotic expression vector pET-28a between the NcoI and XhoI restriction sites to construct the expression vector pET-28a-HIV p24-His.

[0125] The expression and purification were performed according to the method in Example 1.

[0126] 2. Experimental Results

[0127] PAGE electrophoresis results showed that the recombinant protein HIV p24 (hereinafter referred to as WT p24) was successfully prepared.

[0128] Comparative Example 2 Preparation of recombinant protein p24 (21C / 22C)

[0129] 1. Experimental Methods

[0130] HIV p24 introduced cysteine ​​at positions 21 and 22 (amino acid sequence such as SEQ ID NO: 5), and then added 6×His and a translation termination codon at the 3' end of its nucleotide sequence, and cloned it between the NcoI and XhoI restriction sites of the prokaryotic expression vector pET-28a to construct the expression vector pET-28a-p24(21C / 22C)-His.

[0131] The expression and purification were performed according to the method in Example 1.

[0132] 2. Experimental Results

[0133] PAGE electrophoresis results showed that the recombinant protein p24 (21C / 22C) was successfully prepared.

[0134] Comparative Example 3 Preparation of recombinant protein N20-WT-p24-C20

[0135] 1. Experimental Methods

[0136] N20 was fused to the N-terminal domain of HIV p24 protein, and C20 was fused to the C-terminal domain of p24 protein; N20-WT-p24-C20 fusion protein (amino acid sequence is shown in SEQ ID NO: 6) was constructed, and then 6×His and translation termination codon were added to the 3′ end of the nucleotide sequence of the coding sequence of N20-WT-p24-C20. It was cloned between the NcoI and XhoI restriction sites of the prokaryotic expression vector pET-28a to construct the expression vector pET-28a-N20-WT-p24-C20-His.

[0137] The expression and purification were performed according to the method in Example 1.

[0138] 2. Experimental Results

[0139] PAGE electrophoresis showed that the recombinant protein N20-WT-p24-C20 was successfully prepared.

[0140] Example 2 Assembly and identification of N20-p24(21C / 22C)-C20 nanoparticles

[0141] 1. Assembly of N20-p24(21C / 22C)-C20 Nanoparticles

[0142] The recombinant protein N20-p24(21C / 22C)-C20 purified in Example 1 was diluted or replaced with pH=7.550mM Tri-HCl 1M NaCl assembly buffer to prepare N20-p24(21C / 22C)-C20 nanoparticles.

[0143] 2. Molecular sieve chromatography identification

[0144] 1. Experimental methods

[0145] The prepared N20-p24 (21C / 22C)-C20 protein nanoparticles were subjected to molecular sieve chromatography using a Superose 6 Increase 10 / 300 GL chromatography column (GE). The mobile phase buffer for the molecular sieve chromatography was: pH = 7.5, 20 mM Tri-HCl 50 mM NaCl.

[0146] 2. Experimental results

[0147] The results are as follows Figure 1 As shown in B on the left, the dotted line represents the effective elution volume of the nanoparticles, the nanoparticles are on the left side of the dotted line, and most of N20-p24(21C / 22C)-C20 forms nanoparticles.

[0148] 3. Transmission electron microscopy identification

[0149] 1. Experimental methods

[0150] The sample preparation method for transmission electron microscopy is to absorb Figure 1 The N20-p24(21C / 22C)-C20 nanoparticles on the left side of the dotted line shown in B have a concentration of 0.04-0.08μg / μL. 10μL is dropped on the copper mesh and allowed to stand for 1 min. The sample liquid is then absorbed along the edge of the copper mesh with filter paper. Then 10μL of phosphotungstic acid dye is absorbed and covered on the copper mesh. The mesh is allowed to stand for 2 min. The liquid is absorbed along the edge of the copper mesh with filter paper. The mesh is dried naturally and observed using a 100kV transmission electron microscope.

[0151] 2. Experimental results

[0152] The results are as follows Figure 1 As shown in C, the recombinant protein N20-p24(21C / 22C)-C20 prepared in Example 1 assembled to form spherical particles of 40 nm (N20-p24(21C / 22C)-C20 nanoparticles).

[0153] Example 3 Assembly ability of N20-p24 (21C / 22C)-C20 nanoparticles

[0154] 1. Assembly capacity of different recombinant proteins

[0155] 1. Experimental methods

[0156] The purified recombinant protein N20-p24(21C / 22C)-C20 prepared in Example 1, the recombinant protein WT p24 prepared in Comparative Example 1, and the recombinant protein p24(21C / 22C) prepared in Comparative Example 2 were quantified at 500 μg and incubated in 20 mM Tri-HCl 50 mM NaCl at pH = 7.5 for assembly.

[0157] Then, a Superose6 Increase10 / 300GL chromatography column (GE) was used for molecular sieve analysis. The mobile phase buffer of the molecular sieve was: pH = 7.5, 20mM Tri-HCl 50mM NaCl.

[0158] 2. Experimental results

[0159] Research reports show that HIV p24, as a part of HIV capsid protein, will self-assemble into hexamers, and the stable hexamers as basic units will further assemble into tubular structures. The present invention mutates the amino acids at positions 21 and 22 into cysteine ​​(N21C / A22C) so that the stable hexamers are assembled towards the pentamer direction, and the stable pentamers can form a certain spatial curvature.

[0160] result Figure 1 As shown in the left figure in D, molecular sieve analysis of WT p24 showed that most of the p24 protein formed protein monomers; after the introduction of cysteine ​​at positions 21 and 22, most of the recombinant protein p24 (21C / 22C) formed oligomers due to the cross-linking characteristics of cysteine ​​to form disulfide bonds; and after the introduction of short peptides N20 and C20, the recombinant protein N20-p24 (21C / 22C)-C20 further assembled into nanoparticles based on the oligomers, but the proportion of nanoparticles formed was low.

[0161] 3. Effect of salt ion concentration on assembly

[0162] 1. Experimental methods

[0163] The recombinant protein N20-p24(21C / 22C)-C20 prepared in Example 1 and the recombinant protein p24(21C / 22C) prepared in Comparative Example 2 were diluted at a mass of 500 μg into a buffer solution of pH=7.5 50 mM Tris-HCl 1 M NaCl (assembly buffer) and incubated for assembly for 24 and 48 h.

[0164] Then, a Superose6 Increase10 / 300GL chromatography column (GE) was used for molecular sieve analysis. The mobile phase buffer of the molecular sieve was: pH = 7.5, 20mM Tri-HCl 50mM NaCl.

[0165] 2. Experimental results

[0166] result Figure 1 As shown in the right figure in D, the dotted line represents the effective elution volume of the nanoparticles, and the assembled nanoparticles are on the left side of the dotted line. Under high salt conditions, the production ratio of N20-p24(21C / 22C)-C20 nanoparticles is significantly increased; after 48 hours of assembly, the proportion of N20-p24(21C / 22C)-C20 nanoparticles is significantly higher than that of p24(21C / 22C).

[0167] The results showed that the combination of short peptides with amino acid sequences such as SEQ ID NO: 1 and 2 has a strong ability to promote assembly; and incubation in a high salt ion environment (1M NaCl) can promote the assembly of nanoparticles.

[0168] Example 4 Solubility of N20-p24(21C / 22C)-C20 Nanoparticles

[0169] 1. Experimental Methods

[0170] The recombinant protein WT p24 prepared in Comparative Example 1, the recombinant protein p24 (21C / 22C) prepared in Comparative Example 2, the recombinant protein N20-WT-p24-C20 prepared in Comparative Example 3, and the recombinant protein N20-p24 (21C / 22C) -C20 prepared in Example 1 were induced for expression in the BL21 competent cell, and the cells were collected and broken to remove the insoluble protein inclusion bodies, and the soluble supernatant was identified by PAGE.

[0171] 2. Experimental Results

[0172] result Figure 1As shown in Figure E, the results show that WT p24 or p24(21C / 22C) formed protein inclusion bodies, and almost no soluble protein was detected in the supernatant. After adding the short peptide N20 and short peptide C20 combination, a large amount of soluble protein was detected in the supernatant of the recombinant protein N20-p24(21C / 22C)-C20 expression, indicating that the short peptide N20 and short peptide C20 promoted the solubility of the recombinant protein N20-p24(21C / 22C)-C20 and increased its expression level.

[0173] Comparative Example 4 Preparation of Helicobacter pylori Ferritin HPF Nanoparticles

[0174] 1. Experimental Methods

[0175] The 6×His and translation termination codon were added to the 3′ end of the HPF (amino acid sequence such as SEQ ID NO: 7) nucleotide sequence and cloned into the prokaryotic expression vector pET-28a between the NcoI and XhoI restriction sites to construct the expression vector pET-28a-HPF-His.

[0176] The expression and purification were performed according to the method in Example 1.

[0177] 2. Experimental Results

[0178] PAGE electrophoresis results showed that Helicobacter pylori ferritin HPF nanoparticles were successfully prepared.

[0179] Example 5 Stability of N20-p24(21C / 22C)-C20 Nanoparticles

[0180] 1. Experimental Methods

[0181] The HIV p24 nanoparticles prepared in Example 2 and the HPF nanoparticles prepared in Comparative Example 4 were placed at 37° C. or 4° C. under shaking or static conditions, respectively, and samples were taken every 2 days for PAGE identification and molecular sieve chromatography.

[0182] The mobile phase buffer of the molecular sieve is: pH=7.5, 20mM Tri-HCl 50mM NaCl.

[0183] 2. Experimental Results

[0184] The results are as follows Figure 2 As shown, the N20-p24(21C / 22C)-C20 nanoparticles (p24) prepared in Example 2 exhibited high stability at 4°C, and no depolymerization of the nanoparticles occurred; however, they were not tolerant to high temperatures, and visible precipitation occurred over time.

[0185] The HPF nanoparticles (HPF), N20-p24 (21C / 22C)-C20 and HPF prepared in Comparative Example 4 all achieved strong stability of the protein at 4°C. HPF nanoparticles (HPF) are known to have strong stability and are widely used as nanoparticle carriers for drug delivery, which also proves that p24 nanoparticles, as nanoparticle carriers, also have this condition.

[0186] Example 6 Cellular Safety of N20-p24(21C / 22C)-C20 Nanoparticles

[0187] 1. Experimental Methods

[0188] The cytotoxicity of the N20-p24 (21C / 22C)-C20 nanoparticles prepared in Example 2 was detected by CCK-8 test. The specific method was as follows: Hela cells were plated in a 96-well plate, and 100 μl of culture medium was added to each well, with a total of 5000 cells per well. After the cells adhered overnight, N20-p24 (21C / 22C)-C20 nanoparticles with a final concentration gradient of 12.5 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml and 200 μg / ml were added, and the detection was performed at 24h and 72h, respectively. 10 μL of CCK-8 reagent was added to each well, and after incubation at 37°C for 1h, the OD was measured. 450 Read the value.

[0189] 2. Experimental Results

[0190] The results are as follows Figure 3 As shown, the results showed that the N20-p24(21C / 22C)-C20 nanoparticles prepared in Example 2 at a concentration of 200 μg / ml did not show any cytotoxicity when incubated with cells for 72 hours, proving the safety of the N20-p24(21C / 22C)-C20 nanoparticles prepared in Example 2.

[0191] Example 7 Preparation of recombinant protein N20-HERV-K-CA-C20

[0192] 1. Experimental Methods

[0193] 1. Design of short peptides

[0194] Similar to Example 1, in order to avoid the formation of protein inclusion bodies during the production of proteins in a prokaryotic expression system (such as Escherichia coli), two short peptides were designed: short peptide N20 (amino acid sequence such as SEQ ID NO: 1) and short peptide C20 (amino acid sequence such as SEQ ID NO: 2).

[0195] 2. Preparation of expression vector

[0196] N20 is fused to the N-terminal domain of the CA protein of HERV-K, and C20 is fused to the C-terminal domain of the HERV-K protein; Figure 4 As shown in A in the figure, the coding sequence of N20-HERV-K-CA-C20 fusion protein (amino acid sequence as shown in SEQ ID NO: 8) was constructed, and then the coding sequence of 6×His and translation termination codon was added to the 3' end of the N20-HERV-K-CA-C20 nucleotide sequence. It was cloned between the NcoI and XhoI restriction sites of the prokaryotic expression vector pET-28a to construct the expression vector pET-28a-N20-HERV-K-CA-C20-His.

[0197] 3. Expression and purification of N20-HERV-K-CA-C20 protein

[0198] Same as Example 1.

[0199] 2. Experimental Results

[0200] PAGE electrophoresis showed that the recombinant protein N20-HERV-K-CA-C20 was successfully prepared.

[0201] Comparative Example 5 Preparation of recombinant protein HERV-K-CA

[0202] 1. Experimental Methods

[0203] 6×His and a translation termination codon were added to the 3′ end of the HERV-K-CA (amino acid sequence such as SEQ ID NO: 9) nucleotide sequence and cloned into the prokaryotic expression vector pET-28a between the NcoI and XhoI restriction sites to construct the expression vector pET-28a-HERV-K-CA-His.

[0204] The expression and purification were performed according to the method in Example 1.

[0205] 2. Experimental Results

[0206] PAGE electrophoresis showed that the recombinant protein HERV-K-CA (hereinafter referred to as WT HERV-K-CA) was successfully prepared.

[0207] Example 8 N20-HERV-K-CA-C20 Nanoparticles

[0208] 1. Purification and assembly of N20-HERV-K-CA-C20 nanoparticles

[0209] Example 7 The purified recombinant protein N20-HERV-K-CA-C20 was concentrated using a 10 kDa ultrafiltration tube and the buffer was replaced with a pH = 7.5 50 mM Tri-HCl 1 M NaCl buffer.

[0210] 2. Molecular Sieve Chromatography

[0211] 1. Experimental methods

[0212] Same as Example 2.

[0213] 2. Experimental results

[0214] The results are as follows Figure 4 As shown in B, the dotted line indicates the effective elution volume of the nanoparticles, and the nanoparticles are on the left side of the dotted line. The results show that N20-HERV-K-CA-C20 successfully achieved the assembly of nanoparticles with a very high assembly efficiency.

[0215] 3. Transmission electron microscopy identification

[0216] 1. Experimental methods

[0217] Same as Example 2.

[0218] 2. Experimental results

[0219] like Figure 4 As shown in C, transmission electron microscopy observed that N20-HERV-K-CA-C20 assembled into nanoparticles with a size of 17 to 19 nm; the dynamic light scattering particle size distribution of N20-HERV-K-CA-C20 nanoparticles is shown in Figure 4 As shown in D, it shows that the self-assembly of N20-HERV-K-CA-C20 nanoparticles is successful and their morphology is complete.

[0220] Example 9 Assembly Capability of N20-HERV-K-CA-C20 Nanoparticles

[0221] 1. Experimental Methods

[0222] The expression vector WT HERV-K-CA obtained in Comparative Example 5 and the expression vector of Example 7 were induced to express and purified with protein storage buffer (20mM Tris-HCl 0mM, 20mM Tris-HCl 50mM or 20mMTris-HCl 1M) at different salt ion concentrations, and self-assembled. The obtained N20-HERV-K-CA-C20 was subjected to molecular sieve analysis using a Superose6Increase10 / 300GL chromatography column (GE). The mobile phase buffer for molecular sieve chromatography was: pH = 7.5, 20mM Tri-HCl 50mM NaCl.

[0223] 2. Experimental Results

[0224] result Figure 4 As shown in Figure E, the results show that WT HERV-K-CA basically does not assemble into nanoparticles even under the high salt ion conditions of 20mM Tris-HCl 1M, while N20-HERV-K-CA-C20 achieves stable assembly of nanoparticles under different salt ion concentrations, and even under 0mM NaCl conditions, it still forms stable nanoparticles, proving the assembly stability and salt ion concentration independence of N20-HERV-K-CA-C20.

[0225] Example 10 Solubility of HERV-K-CA Nanoparticles

[0226] 1. Experimental Methods

[0227] The recombinant protein WT HERV-K-CA of Comparative Example 5 and the recombinant protein N20-HERV-K-CA-C20 of Example 7 were induced to express in Escherichia coli at 37°C, and the cells were broken to remove protein inclusion bodies, and the soluble supernatant was subjected to PAGE electrophoresis.

[0228] 2. Experimental Results

[0229] result Figure 4 As shown in F, the results showed that WT HERV-K-CA formed insoluble protein inclusion bodies under the rapid induction expression conditions at 37°C, while a large amount of soluble protein was detected in the soluble supernatant of N20-HERV-K-CA-C20, proving that the short peptides N20 and C20 promoted the solubility of recombinant proteins and increased the protein expression level.

[0230] Example 11RFP or GFP displayed on the surface of N20-p24(21C / 22C)-C20 nanoparticles

[0231] The Gv linker and SD linker in the Gv / SD isopeptide bond connection system were used to fuse with N20-p24(21C / 22C)-C20 and RFP or GFP, respectively, to construct nanoparticles with HIV p24 as the core and carrying RFP protein or GFP protein on the surface.

[0232] 1. Preparation of Nanoparticles Conjugated with Fluorescent Proteins

[0233] 1. Experimental methods

[0234] 1.1 Construction of recombinant plasmid

[0235] (1) Construction of recombinant plasmid pET-28a-Gv-N20-p24(21C / 22C)-C20-His

[0236] The Gv linker was fused with N20-p24(21C / 22C)-C20 to construct a fusion protein Gv-N20-p24(21C / 22C)-C20, whose amino acid sequence is shown in SEQ ID NO: 10, and 6×His and a translation termination codon were added to the 3′ end of the Gv-N20-p24(21C / 22C)-C20 nucleotide sequence. The fusion protein was cloned between the NcoI and XhoI restriction sites of the prokaryotic expression vector pET-28a to construct a recombinant plasmid pET-28a-Gv-N20-p24(21C / 22C)-C20-His.

[0237] (2) Construction of recombinant plasmid pET-28a-SD-RFP-His

[0238] The SD linker was fused with RFP (amino acid sequence as shown in SEQ ID NO: 20) to construct the fusion protein SD-RFP, whose amino acid sequence is shown in SEQ ID NO: 11, and 6×His and a translation termination codon were added to the 3' end of the SD-RFP nucleotide sequence. It was cloned between the NcoI and XhoI restriction sites of the prokaryotic expression vector pET-28a to construct the recombinant plasmid pET-28a-SD-RFP-His.

[0239] (3) Construction of recombinant plasmid pET-28a-SD-GFP-His

[0240] The SD linker was fused with GFP (amino acid sequence as shown in SEQ ID NO: 19) to construct the fusion protein SD-GFP, whose amino acid sequence is shown in SEQ ID NO: 12, and 6×His and a translation termination codon were added to the 3' end of the SD-GFP nucleotide sequence. It was cloned between the NcoI and XhoI restriction sites of the prokaryotic expression vector pET-28a to construct the recombinant plasmid pET-28a-SD-GFP-His.

[0241] (4) Construction of recombinant plasmid pET-28a-Gv-HPF-His

[0242] The Gv linker was fused with HPF to construct the fusion protein Gv-HPF, whose amino acid sequence is shown in SEQ ID NO: 13, and 6×His and a translation termination codon were added to the 3' end of the Gv-HPF nucleotide sequence. The fusion protein was cloned between the NcoI and XhoI restriction sites of the prokaryotic expression vector pET-28a to construct the recombinant plasmid pET-28a-Gv-HPF-His.

[0243] 1.2 Expression and purification of recombinant proteins

[0244] Same as Example 1.

[0245] 1.3. Nanoparticles coupled with fluorescent proteins

[0246] Excess SD-RFP or SD-GFP was incubated with Gv-N20-p24(21C / 22C)-C20 or Gv-HPF-His in 600 μL pH=7.5 50 mM Tris-HCl 1 M NaCl for coupling, with the added molar ratio being 3 nmol:1 nmol.

[0247] 2. Experimental results

[0248] The results are as follows Figure 6 Panel A shows that N20-p24(21C / 22C)-C20 was completely consumed, indicating that N20-p24(21C / 22C)-C20 was completely coupled to GFP or RFP protein with SD.

[0249] 2. Transmission Electron Microscopy Analysis of Nanoparticles

[0250] 1. Experimental methods

[0251] Same as Example 2.

[0252] 2. Experimental results

[0253] The results are as follows Figure 6 A in the figure shows that the results show that when N20-p24(21C / 22C)-C20 is completely consumed, that is, all coupled with RFP or GFP, N20-p24(21C / 22C)-C20 can form nanoparticles, indicating that the nanoparticles have good stability.

[0254] 3. Molecular sieve and PAGE electrophoresis analysis

[0255] 1. Experimental methods

[0256] HPF was used as a positive control for verification. The prepared N20-p24(21C / 22C)-C20 protein nanoparticles coupled with GFP and HPF protein nanoparticles coupled with GFP were subjected to molecular sieve chromatography using a Superose6 Increase10 / 300GL chromatography column (GE). The mobile phase buffer for the molecular sieve chromatography was: pH = 7.5, 20mM Tri-HCl 50mM NaCl.

[0257] 2. Experimental results

[0258] The results are as follows Figure 6 B in the figure shows that the results show that the elution of nanoparticles can be observed regardless of whether it is HPF or p24 coupled with GFP. The nanoparticle peaks were collected for PAGE identification, and it was found that both were protein components coupled with GFP, proving that the nanoparticle peak is composed of nanoparticles coupled with GFP, rather than the remaining HPF or p24 that is not completely coupled. This proves that N20-p24(21C / 22C)-C20 nanoparticles and HPF nanoparticles coupled with RFP or GFP can still form stable nanoparticles with good stability.

[0259] IV. Effect of pH on the stability of N20-p24(21C / 22C)-C20 nanoparticles

[0260] 1. Experimental methods

[0261] Gv-N20-p24(21C / 22C)-C20 nanoparticles were coupled with excess SD-GFP or SD-RFP in Example 11 in 50mM Tris-HCl 1M NaCl pH=6.5 and 50mM Tris-Hcl 1M Nacl pH=8.5 systems, respectively, and the samples were prepared and observed by transmission electron microscopy.

[0262] 2. Experimental results

[0263] The results are as follows Figure 6 Figure C shows that at different pH, N20-p24(21C / 22C)-C20 nanoparticles can still achieve stable nanoparticle morphology after being coupled with different surface proteins (SD-GFP or SD-RFP), indicating its stability and wide applicability as an antigen display platform.

[0264] Example 12 Humoral immune effect of N20-p24 (21C / 22C)-C20 nanoparticles

[0265] 1. Experimental Methods

[0266] BALB / c mice were immunized with the N20-p24(21C / 22C)-C20 nanoparticles prepared in Example 2 and the recombinant protein WT-p24 prepared in Comparative Example 1 (see Immunization strategy for details). Figure 5 ), BALB / c mice were intramuscularly injected with 5 μg of N20-p24(21C / 22C)-C20 nanoparticles prepared in Example 2 and the recombinant protein WT-p24 prepared in Comparative Example 1 on day 0 and day 28.

[0267] 2. Experimental Results

[0268] The results are as follows Figure 5As shown, N20-p24(21C / 22C)-C20 nanoparticles can significantly enhance the humoral immunity level of mice.

[0269] Example 13 N20-p24(21C / 22C)-C20 nanoparticles as antigen display platform for antigen delivery

[0270] Since HIV immunogens, especially envelope proteins, are densely glycan-shielded and have low immunogenicity, the receptor binding domain (RBD) protein of the new coronavirus JN.1 strain was coupled to N20-p24 (21C / 22C)-C20 nanoparticles, which were used to immunize C57 mice to examine their antigen delivery effect.

[0271] 1. Construction of Nanoparticles

[0272] 1. Experimental methods

[0273] (1) Construction of recombinant plasmid

[0274] 1.1 Construction of recombinant plasmid pCDNA3.1-SD-JN.1-His

[0275] The SD linker was fused with the receptor binding domain (RBD) of the novel coronavirus JN.1 strain to construct the fusion protein SD-JN.1, whose amino acid sequence is shown in SEQ ID NO: 14. 6×His and a translation termination codon were added to the 3' end of the SD-JN.1 nucleotide sequence. It was cloned between the EcoRI and XbaI restriction sites of the eukaryotic expression vector pCNDA3.1 to construct the recombinant plasmid pCDNA3.1-SD-JN.1-His.

[0276] 1.2 Construction of recombinant plasmid pET-28a-Gv-N20-p24(21C / 22C)-C20-His

[0277] Same as Example 11.

[0278] 1.3 Construction of recombinant plasmid pET-28a-Gv-HPF-His

[0279] Same as Example 11.

[0280] (2) Expression and purification of recombinant proteins

[0281] Same as Example 1.

[0282] (3) Preparation of nanoparticles

[0283] SD-JN.1 was incubated with Gv-N20-p24(21C / 22C)-C20 or Gv-HPF-His in 600 μL pH=7.5 50 mM Tris-Hcl 1 M NaCl at a ratio of 1 nmol:1 nmol to prepare nanoparticles HPF-JN.1 and p24-JN.1, respectively.

[0284] 2. Experimental results

[0285] Results Figure 6 The results showed that both p24 and HPF were effectively coupled with JN.1 to obtain nanoparticles HPF-JN.1 and p24-JN.1.

[0286] 2. Nanoparticle Immunization of C57 Mice

[0287] 1. Experimental methods

[0288] The prepared nanoparticles HPF-JN.1 and p24-JN.1 were used to immunize C57 mice, respectively. 5 μg of nanoparticles HPF-JN.1, nanoparticles p24-JN.1 and a control group of JN.1 monomer protein (JN.1monomer) not coupled with nanoparticles were injected intramuscularly on days 0 and 28, and blood was collected from the eye sockets on day 42.

[0289] Mouse serum was allowed to stand for 1 h and then centrifuged at 4°C, 2800 rpm for 10 min for use in the Anti-RBD IgG ELISA assay.

[0290] 2. Experimental results

[0291] Results Figure 7 , using the N20-p24(21C / 22C)-C20 nanoparticles and HPF nanoparticles of the present invention, after coupling the receptor binding domain (RBD) protein of the novel coronavirus JN.1 strain, the immunized mice had a similar titer of JN.1RBD, and had an antibody titer superior to that of the JN.1 monomer control group JN.1 monomer protein. This shows that N20-p24(21C / 22C)-C20 nanoparticles have the advantage of enhancing humoral immunity as an antigen presentation platform.

[0292] Example 13 N20-p24(21C / 22C)-C20 Nanoparticles as Protein Delivery Platform

[0293] like Figure 8As shown in A, the recombinant proteins N20-p24(21C / 22C)-C20N20-p24(21C / 22C)-C20 and the recombinant protein N20-p24(21C / 22C)-C20-Linker-GFP were co-transformed into BL21 Escherichia coli to induce expression, and two-component assembly was performed so that GFP was carried in the cavity of the nanoparticle.

[0294] 1. Preparation of Nanoparticles

[0295] 1. Experimental methods

[0296] The C-terminus of N20-p24(21C / 22C)-C20 was covalently linked to a 10-amino acid linker (GGGGSGGGGS), and then the C-terminus of the linker was covalently linked to a GFP protein to construct a recombinant protein N20-p24(21C / 22C)-C20-Linker-GFP, the amino acid sequence of which is shown in SEQ ID NO: 15, and then 6×His and a translation stop codon were added to the 3' end. It was cloned between the NcoI and XhoI restriction sites of the prokaryotic expression vector pET-28a to construct a recombinant plasmid pET-28a-N20-p24(21C / 22C)-C20-Linker-GFP-His.

[0297] The recombinant plasmid pET-28a-N20-p24(21C / 22C)-C20-Linker-GFP-His and the recombinant plasmid pET-28a-N20-p24(21C / 22C)-C20-His obtained in Example 1 were co-transformed into BL21 Escherichia coli at a mass ratio of 1:1 to induce expression.

[0298] The subsequent expression and purification of the recombinant protein was performed according to the method of Example 1, and then the solution was concentrated by ultrafiltration tube and the buffer was replaced by pH=7.5 50 mM Tri-HCl 1 M NaCl buffer.

[0299] 2. Experimental results

[0300] PAGE electrophoresis showed that both recombinant proteins were successfully expressed.

[0301] 2. Molecular Sieve Analysis of Nanoparticles

[0302] 1. Experimental methods

[0303] Superose6 Increase10 / 300GL chromatography column (GE) was used to perform molecular sieve analysis on N20-p24(21C / 22C)-C20 nanoparticles coupled with GFP. The mobile phase buffer for molecular sieve chromatography was: pH = 7.5, 20mM Tri-HCl 50mM NaCl.

[0304] 2. Experimental results

[0305] The results are as follows Figure 8 As shown in B, the results showed that two sizes of protein components were detected in the elution volume of the nanoparticles, indicating that N20-p24(21C / 22C)-C20-Linker-GFP and N20-p24(21C / 22C)-C20 co-assembled to form nanoparticles.

[0306] 3. Transmission electron microscopy

[0307] 1. Experimental methods

[0308] Same as Example 2.

[0309] 2. Experimental results

[0310] The results are as follows Figure 8 As shown in C, a two-component delivery vector was successfully constructed using the recombinant proteins N20-p24(21C / 22C)-C20 and N20-p24(21C / 22C)-C20-Linker-GFP to form stable nanoparticles (p24 nanoparticles with GFP packaged in the inner cavity), which can package the GFP protein into the cavity of the N20-p24(21C / 22C)-C20 nanoparticles.

[0311] 4. Intracellular Localization of Nanoparticles

[0312] 1. Experimental methods

[0313] p24 nanoparticles with GFP packaged in the inner cavity were injected into 293T and Hela cells after digitonin permeabilization, and DAPI staining was performed to observe whether GFP co-localized with the cell nucleus.

[0314] 2. Experimental results

[0315] Results Fig. 9The cell nucleus and GFP fluorescence after DAPI staining achieved co-localization, indicating that the p24 nanoparticles packaged with GFP in the inner cavity were localized in the cell nucleus rather than the cytoplasm. This shows that the two-component delivery vector constructed using the recombinant proteins N20-p24(21C / 22C)-C20N20-p24(21C / 22C)-C20 and N20-p24(21C / 22C)-C20-Linker-P (target protein) has the ability to deliver cargo P to the cell nucleus, and is a large-capacity cell nucleus delivery vector.

[0316] Comparative Example 6N20-p24(21C / 22C)-C20 Nanoparticles as a Protein Delivery Platform

[0317] like Fig.10 As shown in A, only the recombinant plasmid pET-28a-N20-p24(21C / 22C)-C20-Linker-GFP-His was transformed into BL21 Escherichia coli to induce expression, followed by self-assembly.

[0318] 1. Preparation of Nanoparticles

[0319] 1. Experimental methods

[0320] The recombinant plasmid pET-28a-N20-p24(21C / 22C)-C20-Linker-GFP-His was used for subsequent recombinant protein expression and purification according to the method of Example 1, and then concentrated by ultrafiltration tube and replaced with buffer, and the buffer was replaced with pH=7.5 50mM Tri-HCl 1M NaCl buffer.

[0321] 2. Experimental results

[0322] PAGE electrophoresis showed that the N20-p24(21C / 22C)-C20-Linker-GFP-His recombinant protein was successfully expressed and purified.

[0323] 2. Molecular Sieve Analysis of Nanoparticles

[0324] 1. Experimental methods

[0325] Superose6 Increase10 / 300GL chromatography column (GE) was used to perform molecular sieve analysis on N20-p24(21C / 22C)-C20 nanoparticles coupled with GFP. The mobile phase buffer for molecular sieve chromatography was: pH = 7.5, 20mM Tri-HCl 50M NaCl.

[0326] 2. Experimental results

[0327] The results are as follows Fig.10As shown in B, the dotted line represents the effective elution volume of the nanoparticles, and the nanoparticles are on the left side of the dotted line. The results show that the assembly strategy of the single-component N20-p24(21C / 22C)-C20-Linker-GFP formed protein oligomers and failed to form nanoparticles.

[0328] 3. Transmission electron microscopy

[0329] 1. Experimental methods

[0330] Same as Example 2.

[0331] 2. Experimental results

[0332] The results are as follows Fig.10 As shown in Figure C, the presence of GFP at the C-terminus spatially affects the assembly of single-component nanoparticles. The recombinant protein N20-p24(21C / 22C)-C20N20-p24(21C / 22C)-C20-Linker-GFP single component cannot form stable nanoparticles, but instead forms protein oligomers.

Claims

1. A short peptide composition for inhibiting inclusion body formation, characterized in that: It comprises a C-terminal short peptide and an N-terminal short peptide, wherein the amino acid sequence of the N-terminal short peptide is shown in SEQ ID NO: 1, and the amino acid sequence of the C-terminal short peptide is shown in SEQ ID NO:

2.

2. Biomaterial, characterized in that It is any of the following: (1) A nucleotide molecule combination, comprising nucleic acid molecules encoding the C-terminal short peptide and the N-terminal short peptide in the short peptide composition of claim 1, or reverse complementary nucleotide molecules of nucleic acid molecules encoding the C-terminal short peptide and the N-terminal short peptide in the short peptide composition; (2) An expression cassette, wherein the 5' end is provided with a nucleotide molecule encoding the N-terminal short peptide in the short peptide combination of claim 1, and the 3' end is provided with a nucleotide molecule encoding the C-terminal short peptide in the short peptide combination of claim 1. (3) a recombinant vector comprising the nucleotide molecule combination described in (1) or the expression cassette described in (2); (4) A recombinant microorganism comprising the recombinant vector described in (3).

3. Use of the short peptide combination according to claim 1 and / or the biomaterial according to claim 2 in inhibiting the formation of inclusion bodies of prokaryotic expressed proteins, promoting the dissolution of prokaryotic expressed proteins or preparing self-assembled nanoparticles.

4. A method for inhibiting the formation of inclusion bodies of prokaryotic expression proteins and / or promoting the dissolution of prokaryotic expression proteins, characterized in that: A recombinant expression vector for prokaryotic expression of the protein to be expressed is constructed, wherein the 5' end of the gene encoding the protein to be expressed is provided with a nucleotide molecule encoding the N-terminal short peptide in the short peptide combination of claim 1, and the 3' end of the gene encoding the protein to be expressed is provided with a nucleotide molecule encoding the C-terminal short peptide in the short peptide combination of claim 1.

5. A recombinant HIV-1 capsid protein p24, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

3.

6. A recombinant human endogenous retrovirus K capsid protein CA, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

8.

7. Biomaterial, characterized in that It is any of the following: (1) a nucleotide molecule encoding the recombinant HIV-1 capsid protein p24 of claim 5 or the recombinant human endogenous retrovirus K capsid protein CA of claim 6; (2) a nucleotide molecule which is the reverse complementary sequence of the nucleic acid molecule described in (1); (3) an expression cassette comprising the nucleic acid molecule described in (1) or (2); (4) a recombinant vector comprising the nucleotide molecule described in (3); (5) A recombinant microorganism comprising the recombinant vector described in (4).

8. Use of the recombinant HIV-1 capsid protein p24 according to claim 5, the recombinant human endogenous retrovirus K capsid protein CA according to claim 6, or the biomaterial according to claim 6 in the preparation of self-assembling nanoparticles.

9. A nanoparticle, characterized in that: It is obtained by self-assembly using the recombinant HIV-1 capsid protein p24 described in claim 5 or the recombinant human endogenous retrovirus K capsid protein CA described in claim 6.

10. A conjugate, characterized in that It comprises a protein part and a coupling part, wherein the coupling part is coupled to the N-terminus of the protein part, and the protein part is the recombinant HIV-1 capsid protein p24 according to claim 5 or the recombinant human endogenous retrovirus K capsid protein CA according to claim 6.

11. A nanoparticle, characterized in that: The conjugate according to claim 10 is obtained by self-assembly.

12. A surface display system, characterized in that: Containing the nanoparticles according to claim 11.

13. A surface display antigen system, characterized in that: The nanoparticles according to claim 11, wherein the coupling part in the conjugate is an antigen.

14. Use of the recombinant HIV-1 capsid protein p24 according to claim 5, the recombinant human endogenous retrovirus K capsid protein CA according to claim 6, the biomaterial according to claim 7, the nanoparticle according to claim 9, the conjugate according to claim 10, the nanoparticle according to claim 11, the surface display system according to claim 12 or the surface display antigen system according to claim 13 in the preparation of drugs or vaccines.

15. A conjugate, characterized in that It comprises a protein part and a coupling part, wherein the coupling part is coupled to the C-terminus of the protein part, and the protein part is the recombinant HIV-1 capsid protein p24 according to claim 5 or the recombinant human endogenous retrovirus K capsid protein CA according to claim 6.

16. A nanoparticle, characterized in that: Obtained by self-assembly of the conjugate according to claim 15.

17. The nanoparticle according to claim 16, characterized in that It is obtained by self-assembly using the conjugate described in claim 15 and a corresponding one of the recombinant HIV-1 capsid protein p24 described in claim 5 or the recombinant human endogenous retrovirus K capsid protein CA described in claim 6.

18. A protein nanoparticle delivery system, characterized in that: The nanoparticles according to claim 16 or 17, wherein the coupling part in the conjugate is a substance to be delivered.

19. Use of the recombinant HIV-1 capsid protein p24 according to claim 5, the recombinant human endogenous retrovirus K capsid protein CA according to claim 6, the biomaterial according to claim 7, the nanoparticles according to claim 9, the conjugate according to claim 15, the nanoparticles according to claim 16 or 17, and the protein nanoparticle delivery system according to claim 18 in the preparation of drugs.

20. A method for delivering substances using nanoparticle cavities, characterized in that: The C-terminus of the recombinant HIV-1 capsid protein p24 of claim 5 or the recombinant human endogenous retrovirus K capsid protein CA of claim 6 is coupled with a substance to be delivered, and then mixed and self-assembled with the corresponding recombinant HIV-1 capsid protein p24 of claim 5 or the recombinant human endogenous retrovirus K capsid protein CA of claim 6 that is not coupled with a substance to be delivered, to obtain nanoparticles for delivering the substance.

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