Short peptide composition for inhibiting inclusion body formation and promoting gag-like protein nanoparticle self-assembly

Abstract

Disclosed in the present invention is a short peptide composition for inhibiting inclusion body formation and promoting Gag-Like protein nanoparticle self-assembly, comprising a C-terminus short peptide and an N-terminus short peptide. The amino acid sequence of the N-terminus short peptide is as shown in SEQ ID NO: 1, and the amino acid sequence of the C-terminus short peptide is as shown in SEQ ID NO: 2. The short peptide composition can effectively avoid the formation of an inclusion body of a prokaryotically expressed protein and / or promote the solubilization of the prokaryotically expressed protein. Furthermore, by binding the composition to HIV-1 p24 and human endogenous retrovirus K capsid protein CA, nanoparticles are respectively prepared, which can be used as carriers for antigen surface display or drug delivery. Nanoparticles formed from HIV-1 capsid protein p24 and human HERV K CA both have cavities significantly larger than those of ferritin, leading to a greater loading capacity, and also exhibit good stability and wide applicability as antigen display platforms.

Description

A short peptide composition for inhibiting inclusion body formation and promoting self-assembly of Gag-like protein nanoparticles TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, more particularly, to a short peptide composition for inhibiting inclusion body formation and promoting self-assembly of Gag-like protein nanoparticles. BACKGROUND

[0002] A complete viral particle is called "virion" (also known as viral particle, virion), which is composed of a protective "capsid" (also known as shell, protein coat) composed of proteins and nucleic acid wrapped by the capsid. The protein capsid of retrovirus is composed of Gag polyprotein encoded by viral genes, including matrix (MA), capsid structural protein (CA) and nucleocapsid protein (NC). Gag protein aggregates on the inner side of the host cell membrane and self-assembles to form a viral particle, and the capsid wraps and protects the viral RNA genome, ensuring its stability during the maturation of the viral particle. When infecting host cells, the capsid participates in the process of uncoating, releasing viral RNA into the cytoplasm for reverse transcription. In addition, the capsid protein also interacts with host cell proteins, helping the transport and localization of viral RNA.

[0003] Virus-like particles (VLPs) are hollow particles containing one or more structural proteins of a certain virus, without viral nucleic acid, and cannot replicate autonomously, and are identical or similar in morphology to real virus particles, commonly known as pseudo-virus, which is widely used in vaccine production. The capsid protein does not contain viral genome, has high safety, can stimulate strong immune response, and the capsid protein can self-assemble into capsid, therefore, the preparation of VLPs using the capsid protein has obvious advantages.

[0004] Prokaryotic expression system is a commonly used protein expression system, and its advantages in production include fast growth and production cycle, low cost and simple culture conditions, easy gene manipulation and expression optimization, high expression amount, and adaptation to large-scale production demand, which makes it an ideal choice for producing VLPs.

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

[0006] The capsid of retroviridae is composed of Gag protein, and the Gag protein is very similar in structure, and this class of proteins is collectively referred to as Gag-like protein. The Gag-encoded capsid protein CA (p24) of HIV virus (denoted as HIV p24) as part of the capsid protein, which can self-assemble to form a hexamer, and the stable hexamer further assembles to form a tubular structure, and the amino acids at positions 21 and 22 are mutated to cysteine to form a spherical structure. The capsid protein of human endogenous retrovirus K family (HERV-K-CA) has the property of self-assembly, and can self-assemble to form spherical nanoparticles. However, there is a common problem between it and HIV p24, that is, the solubility is low in prokaryotic expression system, the self-assembly ability is poor, and a small amount of nanoparticles are formed, which is a great limitation for large-scale production of stable nanoparticles. SUMMARY

[0007] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide a short peptide composition for inhibiting inclusion body formation.

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

[0009] The first purpose of the present application is to provide a short peptide composition for avoiding the formation of prokaryotic expression protein inclusion bodies and / or promoting the solubility of prokaryotic expression protein.

[0010] The second purpose of the present application is to provide a biological material.

[0011] The third purpose of the present application is to provide the use of the short peptide composition and / or the biological material in inhibiting the formation of prokaryotic expression protein inclusion bodies, promoting the solubility of prokaryotic expression protein, or preparing self-assembled nanoparticles.

[0012] The fourth purpose of the present application is to provide a method for inhibiting the formation of prokaryotic expression protein inclusion bodies and / or promoting the solubility of prokaryotic expression protein.

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

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

[0015] A seventh object of the present application is to provide another biomaterial.

[0016] An eighth object of the present application is to provide 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- assembled nanoparticles.

[0017] A ninth object of the present application is to provide a nanoparticle.

[0018] A tenth object of the present application is to provide a conjugate.

[0019] An eleventh object of the present application is to provide another nanoparticle.

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

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

[0022] A fourteenth object of the present application is to provide 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 a drug or a vaccine.

[0023] A fifteenth object of the present application is to provide another conjugate.

[0024] A sixteenth object of the present application is to provide a further nanoparticle.

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

[0026] An eighteenth object of the present application is to provide 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 or the protein nanoparticle delivery system in the preparation of a drug.

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

[0028] The present application fuses two short peptides with the N-terminal domain and the C-terminal domain of the Gag-CA protein (p24) of human immunodeficiency virus (HIV) respectively, introduces a pair of cysteine mutations, and generates a kind of nanoparticle with strong stability and strong assembly force; and the two short peptides are fused to the N-terminal domain and the C-terminal domain of the Gag-CA protein of human endogenous retrovirus K family (HERV-K) in the same way, and the same better stability and assembly force are achieved.

[0029] The nanoparticle prepared by the above method can be used as an antigen display platform; wherein the HIV-p24 nanoparticle enhances the phagocytosis and presentation of surface antigens in lymph node macrophages, and produces strong humoral immune response against the surface antigens in the mouse body; meanwhile, the nanoparticle can also package heterologous proteins, and can be used as a large-capacity protein delivery nanoparticle carrier. In view of its high conservation and potential T cell immunogen, it can be used as an antigen display platform for vaccine, and can solve the scientific problem of high mutation of retroviruses, and has high application value.

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

[0031] The C-terminal short peptide and N-terminal short peptide can effectively inhibit the formation of inclusion body in the expression process of recombinant protein using prokaryotic expression system, and promote the solubility of recombinant protein.

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

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

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

[0035] (3) recombinant vector, containing nucleotide molecule combination in (1) or expression cassette in (2);

[0036] (4) recombinant microorganism, containing recombinant vector in (3).

[0037] The short peptide combination and / or the biological material are also within the protection scope of the present application.

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

[0039] As a specific solution, the present application claims a recombinant HIV-1 capsid protein p24 (N20-p24(21C / 22C)-C20) with an amino acid sequence as shown in SEQ ID NO: 3.

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

[0041] Further claimed is a biological material, which 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 complement sequence of the nucleic acid molecule in (1);

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

[0045] (4) a recombinant vector containing the nucleic acid molecule in (3);

[0046] (5) a recombinant microorganism containing the recombinant vector in (4).

[0047] The recombinant HIV-1 capsid protein p24, the recombinant human endogenous retrovirus K capsid protein CA, or the biological material for preparing self-assembled nanoparticles are also within the protection scope of the present application.

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

[0049] Further claimed is a nanoparticle self-assembled by the recombinant HIV-1 capsid protein p24 or the recombinant human endogenous retrovirus K capsid protein CA.

[0050] A nanoparticle N20-p24(21C / 22C)-C20 is self-assembled from the recombinant protein N20-p24(21C / 22C)-C20, and a nanoparticle N20-HERV-K-CA-C20 is self-assembled from the recombinant protein N20-HERV-K-CA-C20.

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

[0052] A conjugate comprising a protein part and a coupling part, the coupling part being coupled to the N-terminus of the protein part, the protein part being the recombinant HIV-1 capsid protein p24 or the recombinant human endogenous retrovirus K capsid protein CA.

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

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

[0055] Further, the protein part and the coupling part in the conjugate are coupled by a Linker linker short peptide.

[0056] Further, the protein part and the coupling part in the conjugate are coupled by an SD-GVopti ligation system (doi:10.1038 / s41423-021-00736-2) or a SpyTag / SpyCatcher ligation system.

[0057] A nanoparticle is self-assembled from the conjugate.

[0058] A surface display system comprises the nanoparticle.

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

[0060] Specifically, the surface display antigen is self-assembled from the conjugate, and the conjugate is the N-terminus of the protein of N20-p24(21C / 22C)-C20 with an amino acid sequence as shown in SEQ ID NO: 3 being coupled to a substance with antigen activity.

[0061] Preferably, the coupling is performed by a Linker linker short peptide, an SD-GVopti ligation system (doi:10.1038 / s41423-021-00736-2) or a SpyTag / SpyCatcher ligation system.

[0062] A method for surface display of a protein with antigenic activity, the 5' end of the coding gene of N20-p24(21C / 22C)-C20 with the amino acid sequence shown as SEQ ID NO: 3 is connected with the coding gene of Gv connection element (the amino acid sequence is shown as SEQ ID NO: 16), after cloning into a prokaryotic expression vector, prokaryotic expression is carried out, and purification is carried out to obtain component 1; the 5' end of the coding gene of the protein with antigenic activity is connected with the coding gene of SD connection element (the amino acid sequence is shown as SEQ ID NO: 17), after cloning into a prokaryotic expression vector, prokaryotic expression is carried out, and purification is carried out to obtain component 2; component 1 and component 2 are incubated, and the protein with antigenic activity is surface displayed.

[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 as SEQ ID NO: 18).

[0064] 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 are used for preparing a drug or a vaccine.

[0065] A conjugate comprising a protein part and a coupling part, the coupling part is coupled to the C-terminal 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.

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

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

[0068] Further, the protein part and the coupling part in the conjugate are coupled by a Linker linker short peptide.

[0069] Further, the protein part and the coupling part in the conjugate are coupled by an SD-GVopti connection system (doi:

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

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

[0072] Preferably, the self-assembly is performed by using the conjugate and the 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 containing the nanoparticle, the coupling moiety in the conjugate being the substance to be delivered.

[0074] A method for delivering a protein, the coding gene of N20-p24(21C / 22C)-C20 with the amino acid sequence shown in SEQ ID NO: 3 being connected with the coding gene of Linker linker (GGGGSGGGGS) at the 3' end, and the coding gene of the protein to be delivered being connected at the 3' end of the coding gene of the Linker linker, after being cloned into a prokaryotic expression vector, prokaryotic expression is performed, and after purification, incubation is performed to obtain a nanoparticle for delivering the protein to be delivered.

[0075] A method for delivering a protein,

[0076] The coding gene of N20-p24(21C / 22C)-C20 with the amino acid sequence shown in SEQ ID NO: 3 is connected with the coding gene of Linker linker (GGGGSGGGGS) at the 3' end, and the coding gene of the protein to be delivered is connected at the 3' end of the coding gene of the Linker linker, after being cloned into a prokaryotic expression vector, to obtain vector 1; N20-p24(21C / 22C)-C20 is cloned into a prokaryotic expression vector to obtain vector 2, vector 1 and vector 2 are co-expressed, and after purification, incubation is performed to obtain a nanoparticle for delivering the protein to be delivered.

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

[0078] 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 protein nanoparticle delivery system for use in the preparation of a medicament.

[0079] A method for delivering a substance by using the cavity of the nanoparticle, the C-terminal of the recombinant HIV-1 capsid protein p24 or the recombinant human endogenous retrovirus K capsid protein CA being coupled with the substance to be delivered, and then being mixed with the corresponding recombinant HIV-1 capsid protein p24 or the recombinant human endogenous retrovirus K capsid protein CA which is not coupled with the substance to be delivered to perform self-assembly to obtain a nanoparticle for delivering the substance.

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

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

[0082] Preferably, when the substance to be delivered is a protein, 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 linker, and is cloned into a prokaryotic expression vector; the coding gene of the corresponding recombinant HIV-1 capsid protein p24 or the recombinant human endogenous retrovirus K capsid protein CA is also cloned into a prokaryotic expression vector; and the two obtained expression vectors are co-transformed and expressed to self-assemble.

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

[0084] The present application discloses a kind of short peptide composition to avoid prokaryotic expression protein inclusion body formation and / or promote prokaryotic expression protein dissolution, including C-terminal short peptide and N-terminal short peptide, the amino acid sequence of the N-terminal short peptide is as shown in SEQ ID NO:1, the amino acid sequence of the C-terminal short peptide is as shown in SEQ ID NO:2.The short peptide composition can effectively inhibit prokaryotic expression protein inclusion body formation and / or promote prokaryotic expression protein dissolution, further utilize its combination HIV-1 capsid protein p24 and human endogenous retrovirus K capsid protein CA, respectively prepared to have obtained nanoparticle, it can be used as antigen surface display or drug transport carrier.HIV-1 capsid protein p24 human and endogenous retrovirus K capsid protein CA formation nanoparticle respectively has the large cavity of 40nm and 17~19nm diameter, significantly greater than the 11~13nm cavity of ferritin, with stronger loading capacity;At different pH, different surface proteins (SD-GFP) still can realize stable connection and self-assembly of nanoparticle, and it has good stability and wide applicability as antigen display platform BRIEF DESCRIPTION OF DRAWINGS

[0085] Figure 1 is the preparation and characterization of N20-p24(21C / 22C)-C20 nanoparticle;

[0086] A is the structure diagram of Alphafold prediction, left: WT p24, form hexamer structure (PDB: 3H47);Middle: p24(21C / 22C), after introducing cysteine mutations at positions 21 and 22, a pentamer structure is formed (PDB: 3P05), red represents the formation of disulfide bond;Right: N20-p24(21C / 22C)-C20, N20 (orange) promotes the aggregation of pentamer, C20 (yellow) promotes the aggregation of C-terminal trimer.

[0087] B is N20-p24(21C / 22C)-C20 nanoparticle: left is UV absorption peak chart of molecular sieve of N20-p24(21C / 22C)-C20, dotted line is effective elution volume; right is PAGE electrophoretogram of N20-p24(21C / 22C)-C20 of denaturation and non-denaturation;

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

[0089] D is UV absorption peak chart of molecular sieve, left chart, top is WT p24, middle is p24(21C / 22C), bottom is N20-p24(21C / 22C)-C20; right chart, from top to bottom are p24(21C / 22C) assembled for 24 h, p24(21C / 22C) assembled for 48 h, N20-p24(21C / 22C)-C20 assembled for 24 h and N20-p24(21C / 22C)-C20 assembled for 48 h, dotted line: effective elution volume of nanoparticles, 440 kda and 669 kda are both standards.

[0090] E is PAGE electrophoretogram of soluble supernatant of recombinant protein WT-p24, recombinant protein N20-WT-p24-C20, recombinant protein p24(21C / 22C), recombinant protein N20-p24(21C / 22C)-C20 after induced expression in E. coli;

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

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

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

[0094] Figure 3. Cytotoxicity test of N20-HIV p24(21C / 22C)-C20 nanoparticle; concentration gradient range from 12.5 to 200 pg / ml, n = 8.

[0095] Figure 4. Preparation and characterization of N20-HERV-K-CA-C20 nanoparticle.

[0096] A is the structure diagram 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, which shows that N20 (orange) promotes the pentamer, and C20 (yellow) promotes the C-terminal trimer;

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

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

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

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

[0101] F is the PAGE electrophoresis diagram of the soluble supernatant of the recombinant protein WT-HERC-K-CA and the recombinant protein N20-HERV-K-CA-C20 after 37°C induction expression in E. coli.

[0102] Figure 5. Humoral immune effect of N20-p24(21C / 22C)-C20 nanoparticle; the upper figure is the immunization strategy, and the lower figure is the titer of Anti-p24 in the serum of the mice detected at the sixth week after immunization.

[0103] Figure 6 is the antigen display function of N20-p24(21C / 22C)-C20 nanoparticles; A is PAGE electrophoresis and transmission electron microscopy, the left panel is the result of coupling excess SD-RFP with Gv-N20-p24(21C / 22C)-C20, and the right panel is the result of coupling excess SD-GFP with Gv-N20-HIV p24(21C / 22C)-C20, scale: 500 nm; B: Stability of N20-HIV p24(21C / 22C)-C20 as an antigen display platform; the left panel is the PAGE electrophoresis of coupling excess recombinant protein SD-GFP with recombinant protein Gv-N20-p24(21C / 22C)-C20 or recombinant protein Gv-HPF respectively (p24-NP and HPF-NP); the middle panel is the UV absorption peak chart of the molecular sieve of p24-GFP-NP (p24-NP) and HPF-GFP-NP (HPF-NP); the right panel is the PAGE electrophoresis of p24-NP and HPF-NP after protein elution of the molecular sieve (excess SD-GFP monomers do not form nanoparticles); C is PAGE electrophoresis and transmission electron microscopy; the left panel is the PAGE electrophoresis and transmission electron microscopy of excess SD-GFP and Gv-N20-p24(21C / 22C)-C20 in a system with pH = 6.5; right: PAGE electrophoresis and transmission electron microscopy of excess SD-GFP and Gv-N20-p24(21C / 22C)-C20 in a system with pH = 8.5, scale: 500 nm.

[0104] Figure 7 is the immune effect of N20-p24(21C / 22C)-C20 nanoparticles displaying antibodies on the surface; the upper panel is the immunization strategy, the lower left panel is the PAGE electrophoresis of coupling HPF and N20-p24(21C / 22C)-C20 with JN.1 RBD respectively; the lower right panel is the titer of Anti-JN.1 RBD in the serum of mice after immunization for six weeks.

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

[0106] Figure 9 is the nuclear localization of p24 nanoparticles with GFP packed in the lumen.

[0107] Figure 10 is the construction of a one-component delivery carrier; A is a schematic diagram of the construction of a one-component delivery carrier; B is the UV absorption peak chart of the molecular sieve and its eluate PAGE electrophoresis chart, (dotted line: effective elution volume of nanoparticles); C: Transmission electron microscopy chart (scale: 500 nm). DETAILED DESCRIPTION

[0108] The present application will be further described in conjunction with the drawings and specific examples in the description, which are only used to explain the present application and are not intended to limit the scope of the present application. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

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

[0110] I. Experimental Methods

[0111] 1. Design of short peptides

[0112] In order to avoid the formation of protein inclusion bodies in the production of proteins using prokaryotic expression systems (such as E. coli), two short peptides were designed: short peptide N20 (amino acid sequence as SEQ ID NO: 1) and short peptide C20 (amino acid sequence 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; further to prevent the formation of long tubular structures of HIV p24 protein, a cysteine was introduced at positions 21 and 22. The amino acid sequences of the two short peptides and the HIV p24 with a cysteine introduced at positions 21 and 22 were used to construct the N20-p24(21C / 22C)-C20 fusion protein (amino acid sequence as shown in SEQ ID NO: 3) according to the schematic diagram of A in FIG. 1, and a 6xHis and a translation termination codon were added to the 3' end of the nucleotide sequence of N20-p24(21C / 22C)-C20. It was cloned between the NcoI and XhoI enzyme digestion sites of the prokaryotic expression vector pET-28a, and the recombinant plasmid pET-28a-N20-p24(21C / 22C)-C20-His was constructed.

[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 DH5a competent cells, which were cultured overnight at 37°C, and positive clones were screened and identified by PCR. The plasmid was extracted, transformed into BL21 competent cells, cultured overnight at 37°C, and the next day, the culture was expanded, and the expression was induced by IPTG at 16°C overnight. On the third day, the bacterial cells were harvested by centrifugation, and the supernatant was collected after disruption for purification of the target protein. The supernatant was incubated with Ni-NTA agarose (GE Healthcare), which enriched the His-tagged target protein, and then the protein was eluted with imidazole-containing Tris buffer.

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

[0118] Then, the recombinant protein N20-p24(21C / 22C)-C20 was denatured (100mM DTT, 100°C for 10min), and PAGE electrophoresis was performed with the denatured and non-denatured recombinant protein N20-p24(21C / 22C)-C20, respectively, and Coomassie blue staining was used.

[0119] II. Experimental results

[0120] As shown in Figure 1A, Alphafold prediction of wild-type p24 mainly forms a hexameric structure; after introducing cysteine mutations at positions 21 and 22, a pentameric 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] As shown in Figure 1B (right), after denaturation, PAGE electrophoresis shows that the purified recombinant protein N20-p24(21C / 22C)-C20 has very high purity; without denaturation, PAGE electrophoresis shows that the purified recombinant protein N20-p24(21C / 22C)-C20 presents a gradient assembly, with the largest assembly unit being pentamer. It is shown that the recombinant protein N20-p24(21C / 22C)-C20 is prepared, which can assemble N20-p24(21C / 22C)-C20 nanoparticles, consistent with the prediction results.

[0122] Comparative Example 1: Preparation of Recombinant Protein HIV p24

[0123] I. Experimental methods

[0124] The nucleotide sequence of the editing sequence of HIV p24 (amino acid sequence as SEQ ID NO: 4) was cloned into the Ncol and Xhol enzyme cutting sites of the prokaryotic expression vector pET-28a after adding 6xHis and a translation termination codon to the 3' end of the nucleotide sequence, to construct the expression vector pET-28a-HIV p24-His.

[0125] Expression and purification were performed according to the method of Example 1.

[0126] II. Experimental results

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

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

[0129] I. Experimental methods

[0130] HIV p24 was introduced with cysteine at positions 21 and 22 (amino acid sequence as SEQ ID NO: 5), and then 6xHis and a translation termination codon were added to the 3' end of the nucleotide sequence, which was cloned into the Ncol and Xhol enzyme cutting sites of the prokaryotic expression vector pET-28a, to construct the expression vector pET-28a-p24 (21C / 22C)-His.

[0131] Expression and purification were performed according to the method of Example 1.

[0132] II. Experimental results

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

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

[0135] I. 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 the p24 protein; the N20-WT-p24-C20 fusion protein (amino acid sequence as shown in SEQ ID NO: 6) was constructed, and then 6xHis and a 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 into the Ncol and Xhol enzyme cutting sites of the prokaryotic expression vector pET-28a, to construct the expression vector pET-28a-N20-WT-p24-C20-His.

[0137] Expression and purification were performed according to the method of Example 1.

[0138] II. 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] I. 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 into pH = 7.5 50 mM Tri-HCl 1 M NaCl assembly buffer to prepare N20-p24(21C / 22C)-C20 nanoparticles.

[0143] II. Identification by molecular sieve chromatography

[0144] 1. Experimental method

[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), and 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] As shown in Fig. 1B left, the dashed line represents the effective elution volume of the nanoparticles, and the left side of the dashed line is the nanoparticles, and most of the N20-p24(21C / 22C)-C20 formed nanoparticles.

[0148] III. Identification by transmission electron microscopy

[0149] 1. Experimental method

[0150] The transmission electron microscopy sample preparation method was as follows: 10 μL of the N20-p24(21C / 22C)-C20 nanoparticles shown in Fig. 1B left, at a concentration of 0.04-0.08 μg / μL, was suspended on a copper mesh, and after standing for 1 min, the sample liquid was absorbed along the edge of the copper mesh with filter paper, and 10 μL of phosphotungstic acid staining solution was then applied to the copper mesh, and after standing for 2 min, the liquid was absorbed along the edge of the copper mesh with filter paper, and natural drying was performed, and observation was performed by 100 kv transmission electron microscopy.

[0151] 2. Experimental results

[0152] The results are shown in Fig. 1C. The recombinant protein N20-p24(21C / 22C)-C20 prepared in Example 1 formed an assembly of 40 nm spherical particles (N20-p24(21C / 22C)-C20 nanoparticles).

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

[0154] I. Assembly ability of different recombinant proteins

[0155] 1. Experimental method

[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 each quantified at 500 μg and incubated in pH 7.5 20 mM Tri-HCl 50 mM NaCl for assembly.

[0157] Subsequently, each was subjected to molecular sieve analysis using a Superose 6 Increase 10 / 300 GL column (GE), and the mobile phase buffer for the molecular sieve was pH 7.5 20 mM Tri-HCl 50 mM NaCl.

[0158] 2. Experimental results

[0159] It has been reported that HIV p24, as part of the HIV capsid protein, self-assembles into a hexamer, and the stable hexamer further assembles into a tubular structure as a basic unit. In the present application, the amino acids at positions 21 and 22 are mutated to cysteine (N21C / A22C), so that the stable hexamer assembles into a pentamer, and the stable pentamer forms a certain spatial curvature.

[0160] The results are shown in the left panel of Fig. 1D. WT p24 was shown by molecular sieve analysis to form mostly protein monomers. When cysteine was introduced at positions 21 and 22, the recombinant protein p24(21C / 22C) formed mostly oligomers due to the cross-linking properties of the disulfide bond formed by cysteine. When the short peptide N20 and the short peptide C20 were introduced, the recombinant protein N20-p24(21C / 22C)-C20 further assembled into nanoparticles on the basis of the oligomers, but the proportion of nanoparticles was low.

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

[0162] 1. Experimental method

[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 each diluted to a mass of 500 μg in a buffer (assembly buffer) of pH = 7.5 50 mM Tris-HCl 1 M NaCl and incubated for assembly for 24 and 48 h.

[0164] Subsequently, each was subjected to molecular sieve analysis using a Superose 6 Increase 10 / 300 GL column (GE), and the mobile phase buffer for the molecular sieve was: pH = 7.5, 20 mM Tri-HCl 50 mM NaCl.

[0165] 2. Experimental results

[0166] As shown in the right-hand graph in Fig. 1D, the dotted line represents the effective elution volume of the nanoparticles, and the nanoparticles to the left of the dotted line are assembled nanoparticles. Under high-salt conditions, the proportion of N20-p24(21C / 22C)-C20 nanoparticles increased significantly; after 48 h of assembly, the proportion of N20-p24(21C / 22C)-C20 nanoparticles was significantly higher than that of p24(21C / 22C).

[0167] The results show that the short peptide combination having the amino acid sequences of SEQ ID NOs: 1 and 2 has strong assembly-promoting ability; and incubation in a high-salt ion environment (1 M NaCl) can promote the assembly of nanoparticles.

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

[0169] I. 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 each subjected to expression induction in BL21 competent cells, and the bacterial cells were collected after disruption, and the insoluble protein inclusion bodies were removed, and the soluble supernatant was subjected to PAGE identification.

[0171] II. Experimental results

[0172] As shown in E of FIG. 1, the results show that both WT p24 or p24(21C / 22C) form protein inclusion bodies, and almost no soluble protein is detected in the supernatant. However, after adding the combination of short peptide N20 and short peptide C20, a large amount of soluble protein is detected in the supernatant of the expression of recombinant protein N20-p24(21C / 22C)-C20, indicating that short peptide N20 and short peptide C20 promote the solubilization of recombinant protein N20-p24(21C / 22C)-C20, and increase the expression amount thereof.

[0173] Preparation of H. pylori ferritin HPF nanoparticles

[0174] I. Experimental method

[0175] The 3' end of the nucleotide sequence of HPF (amino acid sequence as shown in SEQ ID NO: 7) was added with 6xHis and a translation termination codon, and was cloned into the Ncol and Xhol enzyme digestion sites of the prokaryotic expression vector pET-28a, to construct the expression vector pET-28a-HPF-His.

[0176] And the expression and purification were performed according to the method of Example 1.

[0177] II. Experimental results

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

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

[0180] I. Experimental method

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

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

[0183] II. Experimental results

[0184] As shown in FIG. 2, the N20-p24(21C / 22C)-C20 nanoparticles (p24) prepared in Example 2 exhibit high stability at 4℃ environment, and do not undergo depolymerization of nanoparticles; but they are not resistant to high temperature, and visible precipitation occurs with time.

[0185] The HPF nanoparticles (HPF) prepared in Comparative Example 4, N20-p24(21C / 22C)-C20 and HPF 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 the p24 nanoparticles also have this condition as a nanoparticle carrier.

[0186] Example 6 Cell safety of N20-p24(21C / 22C)-C20 nanoparticles

[0187] I. Experimental method

[0188] The cytotoxicity of the N20-p24(21C / 22C)-C20 nanoparticles prepared in Example 2 was detected by CCK-8 test. Specifically, Hela cells were plated in a 96-well plate, 100 microliters of culture medium was added to each well, and a total of 5000 cells were added to each well. After the cells were attached 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 detection was performed at 24 h and 72 h, respectively. 10 μL of CCK-8 reagent was added to each well, and after incubation in a 37°C incubator for 1 h, the OD 450 readings were measured.

[0189] II. Experimental results

[0190] The results are shown in Figure 3. The results show that the N20-p24(21C / 22C)-C20 nanoparticles prepared in Example 2 did not show any cytotoxicity after incubation with cells for 72 h at a concentration of 200 μg / ml, 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] I. Experimental method

[0193] 1. Design of short peptides

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

[0195] 2. Preparation of expression vector

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

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

[0198] The same as Example 1.

[0199] II. Experimental results

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

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

[0202] I. Experimental methods

[0203] The 3' end of the HERV-K-CA (the amino acid sequence is shown as SEQ ID NO: 9) nucleotide sequence was added with 6xHis and translation termination codon, and it was cloned into the NcoI and XhoI enzyme cutting sites between the prokaryotic expression vector pET-28a, and the expression vector pET-28a-HERV-K-CA-His was constructed.

[0204] And the expression and purification were carried out according to the method of Example 1.

[0205] II. 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] I. Purification and assembly of N20-HERV-K-CA-C20 nanoparticles

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

[0210] II. Molecular sieve chromatography

[0211] 1. Experimental method

[0212] The same as Example 2.

[0213] 2. Experimental results

[0214] As shown in B of FIG. 4, 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 realizes the assembly of the nanoparticles, and the assembly efficiency is very high.

[0215] III. Transmission electron microscopy identification

[0216] 1. Experimental method

[0217] The same as Example 2.

[0218] 2. Experimental results

[0219] As shown in C of FIG. 4, the transmission electron microscopy observes that N20-HERV-K-CA-C20 is assembled to form 17-19 nm nanoparticles; the dynamic light scattering particle size distribution of N20-HERV-K-CA-C20 nanoparticles is shown in D of FIG. 4, which illustrates that the self-assembly of N20-HERV-K-CA-C20 nanoparticles is successful, and the morphology is complete.

[0220] Example 9 Assembly ability of N20-HERV-K-CA-C20 nanoparticles

[0221] I. Experimental method

[0222] The expression vectors WT HERV-K-CA obtained from Comparative Example 5 and the expression vector of Example 7 were purified and replaced after induced expression under different salt ion concentrations (20 mM Tris-HCl 0 mM, 20 mM Tris-HCl 50 mM or 20 mM Tris-HCl 1 M) protein storage buffer, and were self-assembled, and the obtained N20-HERV-K-CA-C20 was subjected to molecular sieve analysis using a Superose6 Increase 10 / 300 GL chromatography column (GE), and the mobile phase buffer of the molecular sieve chromatography was: pH = 7.5, 20 mM Tri-HCl 50 mM NaCl.

[0223] II. Experimental results

[0224] As shown in Fig. 4, E, the results show that WT HERV-K-CA does not assemble into nanoparticles even at a high salt ion concentration of 20 mM Tris-HCl 1 M, while N20-HERV-K-CA-C20 achieves stable nanoparticle assembly at different salt ion concentrations, and even forms stable nanoparticles at a condition of 0 mM NaCl, proving the assembly stability and non-salt ion concentration dependence of N20-HERV-K-CA-C20.

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

[0226] I. Experimental method

[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 at 37°C in E. coli, and then the bacterial cells were broken, the protein inclusion bodies were removed, and the soluble supernatant was taken for PAGE electrophoresis.

[0228] II. Experimental results

[0229] As shown in Fig. 4, F, the results show that WT HERV-K-CA forms insoluble protein inclusion bodies under the condition of rapid induction expression at 37°C, while N20-HERV-K-CA-C20 detects a large amount of soluble protein in the soluble supernatant, proving that the short peptide N20 and the short peptide C20 promote the solubilization effect of the recombinant protein and increase the protein expression amount.

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

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

[0232] I. Preparation of nanoparticles coupled with fluorescent proteins

[0233] 1. Experimental method

[0234] 1.1 Construction of recombinant plasmid

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

[0236] Gv linker and N20-p24(21C / 22C)-C20 fusion expression, the fusion protein Gv-N20-p24(21C / 22C)-C20 was constructed, the amino acid sequence of which is shown as SEQ ID NO: 10, and 6xHis and a translation termination codon were added to the 3' end of the Gv-N20-p24(21C / 22C)-C20 nucleotide sequence. It was cloned between the NcoI and XhoI enzyme cutting sites of the prokaryotic expression vector pET-28a, and the recombinant plasmid pET-28a-Gv-N20-p24(21C / 22C)-C20-His was constructed.

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

[0238] SD linker and RFP (the amino acid sequence is shown as SEQ ID NO: 20) fusion expression, the fusion protein SD-RFP was constructed, the amino acid sequence of which is shown as SEQ ID NO: 11, and 6xHis 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 enzyme cutting sites of the prokaryotic expression vector pET-28a, and the recombinant plasmid pET-28a-SD-RFP-His was constructed.

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

[0240] SD linker and GFP (the amino acid sequence is shown as SEQ ID NO: 19) fusion expression, the fusion protein SD-GFP was constructed, the amino acid sequence of which is shown as SEQ ID NO: 12, and 6xHis 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 enzyme cutting sites of the prokaryotic expression vector pET-28a, and the recombinant plasmid pET-28a-SD-GFP-His was constructed.

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

[0242] Gv linker and HPF fusion expression, the fusion protein Gv-HPF was constructed, the amino acid sequence of which is shown as SEQ ID NO: 13, and 6xHis and a translation termination codon were added to the 3' end of the Gv-HPF nucleotide sequence. It was cloned between the NcoI and XhoI enzyme cutting sites of the prokaryotic expression vector pET-28a, and the recombinant plasmid pET-28a-Gv-HPF-His was constructed.

[0243] 1.2 Expression and purification of recombinant proteins

[0244] The 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 respectively to couple in 600 μL pH=7.5 50mM Tris-HCl 1M NaCl, the molar ratio was 3nmol:1nmol.

[0247] 2, Experimental results

[0248] The results are shown in Figure 6A, N20-p24(21C / 22C)-C20 was completely consumed, which means N20-p24(21C / 22C)-C20 was completely coupled with SD-GFP or SD-RFP protein.

[0249] II. Transmission electron microscopy analysis of nanoparticles

[0250] 1, Experimental method

[0251] The same as Example 2.

[0252] 2, Experimental results

[0253] The results are shown in Figure 5A, the results show that when N20-p24(21C / 22C)-C20 is completely consumed, i.e. completely coupled with RFP or GFP, N20-p24(21C / 22C)-C20 can form nanoparticles, which indicates that the nanoparticles have good stability.

[0254] III. Molecular sieve and PAGE electrophoresis analysis

[0255] 1, Experimental method

[0256] HPF was used as a positive control for verification, the prepared N20-p24(21C / 22C)-C20 protein nanoparticle coupled with GFP and the HPF protein nanoparticle coupled with GFP were subjected to molecular sieve chromatography using a Superose6 Increase 10 / 300 GL chromatography column (GE), and 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 shown in Figure 6, part B. The results show that elution of nanoparticles can be observed for both GFP-conjugated HPF and p24. The nanoparticle peaks were collected and identified by PAGE, and it was found that both were composed of GFP-conjugated protein components, proving that the nanoparticle peaks were composed of GFP-conjugated nanoparticles rather than the remaining HPF or p24 that had not been conjugated. This proves that the RFP- or GFP-conjugated N20-p24(21C / 22C)-C20 nanoparticles and HPF nanoparticles can still form stable nanoparticles and have good stability.

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

[0260] 1. Experimental method

[0261] The Gv-N20-p24(21C / 22C)-C20 nanoparticles were respectively conjugated with excess SD-GFP or SD-RFP in a 50 mM Tris-HCl 1 M NaCl pH = 6.5 and 50 mM Tris-HCl 1 M NaCl pH = 8.5 system, and were observed by transmission electron microscopy.

[0262] 2. Experimental results

[0263] The results are shown in Figure 6, part C. After the N20-p24(21C / 22C)-C20 nanoparticles were conjugated with different surface proteins (SD-GFP or SD-RFP) at different pH, stable nanoparticle morphologies were still achieved. This proves the stability and broad applicability of the nanoparticles as an antigen display platform.

[0264] Example 12. Effect of N20-p24(21C / 22C)-C20 nanoparticles on humoral immunity

[0265] I. Experimental method

[0266] The N20-p24(21C / 22C)-C20 nanoparticles prepared in Example 2 and the recombinant protein WT-p24 prepared in Comparative Example 1 were respectively used to immunize BALB / c mice (immunization strategy shown in Figure 5). The BALB / c mice were injected intramuscularly with 5 μg of the N20-p24(21C / 22C)-C20 nanoparticles prepared in Example 2 and the recombinant protein WT-p24 prepared in Comparative Example 1 at day 0 and day 28.

[0267] II. Experimental results

[0268] The results are shown in Figure 5. The N20-p24(21C / 22C)-C20 nanoparticles can significantly improve the humoral immune level of mice.

[0269] Example 13 Antigen delivery using N20-p24(21C / 22C)-C20 nanoparticle as an antigen display platform

[0270] Because the immunogens of HIV, especially the envelope protein, have dense glycan shielding and low immunogenicity, the receptor binding domain (RBD) protein of the novel coronavirus JN.1 strain was coupled with the N20-p24(21C / 22C)-C20 nanoparticle, and the C57 mice were immunized to investigate the antigen delivery effect.

[0271] I. 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 and expressed with the receptor binding domain (RBD) of the novel coronavirus JN.1 strain to construct a fusion protein SD-JN.1, and the amino acid sequence is shown as SEQ ID NO: 14. The 3' end of the SD-JN.1 nucleotide sequence was added with 6xHis and a translation termination codon. It was cloned into the EcoRI and XbaI enzyme digestion 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] The same as Example 11.

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

[0279] The same as Example 11.

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

[0281] The 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 at a ratio of 1 nmol:1 nmol in 600 μL pH=7.5 50 mM Tris-Hcl 1 M NaCl, respectively. Nanoparticles HPF-JN.1 and p24-JN.1 were prepared, respectively.

[0284] 2. Experimental results

[0285] The results are shown in Figure 6, which show that both p24 and HPF were effectively coupled with JN.1 to obtain nanoparticles HPF-JN.1 and p24-JN.1.

[0286] II. Nanoparticle immunization of C57 mice

[0287] 1. Experimental method

[0288] The prepared nanoparticles HPF-JN.1 and p24-JN.1 were used to immunize C57 mice, and 5 μg dose of nanoparticles HPF-JN.1, nanoparticles p24-JN.1 and uncoupled nanoparticle control group JN.1 monomer protein (JN.1 monomer) were injected intramuscularly on day 0 and day 28, and orbital blood was taken on day 42.

[0289] The mouse serum was allowed to stand for 1 h, and was obtained by centrifugation at 4°C, 2800 rpm for 10 min, and was used for Anti-RBD IgG ELISA detection experiment.

[0290] 2. Experimental results

[0291] The results are shown in Figure 7, which show that the mice immunized with the N20-p24(21C / 22C)-C20 nanoparticles and HPF nanoparticles of the application after coupling with the receptor binding domain (RBD) protein of the novel coronavirus JN.1 strain have similar titers of JN.1 RBD, and have antibody titers superior to the JN.1 monomer control group JN.1 monomer protein. This shows that the N20-p24(21C / 22C)-C20 nanoparticles as an antigen presentation platform have the advantage of enhancing humoral immunity.

[0292] Example 13 N20-p24(21C / 22C)-C20 nanoparticles as a protein delivery platform

[0293] As shown in A of FIG. 8, 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 E. coli to induce expression, two-component assembly was performed, so that the nanoparticles cavity carried GFP.

[0294] I. Preparation of Nanoparticles

[0295] 1. Experimental Method

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

[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 E. coli in a mass ratio of 1:1 to induce expression.

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

[0299] 2. Experimental Results

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

[0301] II. Analysis of Nanoparticles by Molecular Sieve

[0302] 1. Experimental Method

[0303] Molecular sieve analysis of N20-p24(21C / 22C)-C20 nanoparticles conjugated with GFP was performed using a Superose 6 Increase 10 / 300 GL column (GE) with a mobile phase buffer of pH = 7.5, 20 mM Tri-HCl 50 mM NaCl.

[0304] 2. Experimental results

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

[0306] III. Transmission electron microscopy detection

[0307] 1. Experimental method

[0308] The same as Example 2.

[0309] 2. Experimental results

[0310] The results are shown in Figure 8C, which show that a two-component delivery vector was successfully constructed using recombinant proteins N20-p24(21C / 22C)-C20 and N20-p24(21C / 22C)-C20-Linker-GFP, forming a stable nanoparticle (GFP-encapsulated p24 nanoparticle), which can achieve packaging of GFP protein into the cavity of N20-p24(21C / 22C)-C20 nanoparticles.

[0311] IV. Intracellular localization of nanoparticles

[0312] 1. Experimental method

[0313] GFP-encapsulated p24 nanoparticles were introduced into 293T and Hela cells treated with digitonin to break the membrane, and DAPI staining was performed to observe whether GFP was co-localized with the nucleus.

[0314] 2. Experimental results

[0315] The results are shown in Figure 9. The nuclei after DAPI staining and GFP fluorescence achieved co-localization, indicating that the lumen packaged with GFP p24 nanoparticles were located in the nucleus rather than the cytoplasm in the cell, indicating that the two-component delivery vector constructed using the recombinant protein N20-p24(21C / 22C)-C20 N20-p24(21C / 22C)-C20 and N20-p24(21C / 22C)-C20-Linker-P(target protein) has the ability to deliver cargo P to the nucleus, and is a large-capacity nuclear delivery vector.

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

[0317] As shown in Figure 10A, only the recombinant plasmid pET-28a-N20-p24(21C / 22C)-C20-Linker-GFP-His was transformed into BL21 E. coli for expression induction, and then self-assembly was performed.

[0318] I. Preparation of Nanoparticles

[0319] 1. Experimental Method

[0320] The recombinant plasmid pET-28a-N20-p24(21C / 22C)-C20-Linker-GFP-His was expressed and purified according to the method of Example 1, and then concentrated and replaced with buffer using a ultrafiltration tube, and the buffer was replaced with a 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] II. Molecular Sieve Analysis of Nanoparticles

[0324] 1. Experimental Method

[0325] Superose6 Increase 10 / 300 GL chromatography column (GE) was used to perform molecular sieve analysis of N20-p24(21C / 22C)-C20 nanoparticles coupled with GFP, and the mobile phase buffer for molecular sieve chromatography was: pH = 7.5, 20mM Tri-HCl 50M NaCl.

[0326] 2. Experimental Results

[0327] Results are shown in Figure 10, panel B, with the dotted line representing the effective elution volume of the nanoparticles, and the left side of the dotted line representing the nanoparticles. The results show that the assembly strategy of the single component N20-p24(21C / 22C)-C20-Linker-GFP forms protein oligomers and fails to form nanoparticles.

[0328] III. Transmission Electron Microscopy

[0329] 1. Experimental Methods

[0330] Example 2.

[0331] 2. Experimental Results

[0332] Results are shown in Figure 10, panel C, showing that the presence of GFP at the C-terminus sterically hinders the assembly of the single component nanoparticles. The recombinant protein N20-p24(21C / 22C)-C20 N20-p24(21C / 22C)-C20-Linker-GFP single component fails to form stable nanoparticles and instead forms protein oligomers.

Claims

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

2.

2. Biomaterial, characterized in that, It is any one 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 combination of claim 1, or reverse complementary nucleic acid molecules encoding the C-terminal short peptide and the N-terminal short peptide in the short peptide combination; (2) an expression cassette, the 5' end of which 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 which 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 in (1) or the expression cassette in (2); (4) a recombinant microorganism comprising the recombinant vector in (3).

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

4. A method of inhibiting inclusion body formation and / or promoting solubilization of a prokaryotically expressed protein, characterized by, constructing a recombinant expression vector for prokaryotic expression of a protein to be expressed, wherein the 5' end of the coding gene of 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 coding gene of 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, The amino acid sequence of the N-terminal short peptide is shown as SEQ ID NO:

1.

6. A recombinant human endogenous retrovirus K capsid protein CA, characterized in that, The amino acid sequence of the C-terminal short peptide is shown as SEQ ID NO:

2.

7. A biomaterial, characterized in that, It is any one 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 in (1); (3) an expression cassette comprising the nucleic acid molecule in (1) or (2); (4) a recombinant vector comprising the nucleotide molecule in (3); (5) a recombinant microorganism comprising the recombinant vector in (4).

8. Use of the recombinant HIV-1 capsid protein p24 of claim 5, the recombinant human endogenous retrovirus K capsid protein CA of claim 6, or the biological material of claim 6 in preparing self-assembled nanoparticles.

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

10. A conjugate, characterized in that, The conjugate comprises a protein part and a coupling part, 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 of claim 5 or the recombinant human endogenous retrovirus K capsid protein CA of claim 6.

11. A nanoparticle, characterized in that, obtained by self-assembly of the conjugate of claim 10.

12. A surface display system, characterized by comprising the nanoparticles of claim 11.

13. A surface display antigen system, characterized in that, comprising the nanoparticles of claim 11, and the coupling part in the conjugate is an antigen.

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

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

16. A nanoparticle, characterized in that, Self-assembly using the conjugate of claim 15.

17. The nanoparticle of claim 16, wherein, Self-assembly using the conjugate of claim 15 and a corresponding one of the recombinant HIV-1 capsid protein p24 of claim 5 or the recombinant human endogenous retrovirus K capsid protein CA of claim 6.

18. A protein nanoparticle delivery system, comprising: The nanoparticle of claim 16 or 17, the coupling part in the conjugate being the substance to be delivered.

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

20. A method of delivering a substance using a nanoparticle cavity delivery, comprising: 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 is mixed with a corresponding recombinant HIV-1 capsid protein p24 of claim 5 or the recombinant human endogenous retrovirus K capsid protein CA of claim 6 which is not coupled with the substance to be delivered, to self-assemble into a nanoparticle of the substance to be delivered.

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