Virus-like particle presenting IL-10, nano vaccine of virus-like particle and application of virus-like particle in anti-tumor and / or immunotherapy drugs

Abstract

The invention provides a virus-like particle presenting IL-10, a nano vaccine of the virus-like particle and application of the virus-like particle in anti-tumor and / or immunotherapy drugs, and belongs to the technical field of biological drugs or biological products. The invention provides a virus-like particle for presenting IL-10. The virus-like particle is a virus-like particle of which the surface is modified with IL-10 or a virus-like particle of which the surface is modified with IL-10 and E7 antigen peptide. According to the embodiment of the invention, the virus-like particles are adopted to present the IL-10 or simultaneously present the E7 antigen peptide, so that compared with free IL-10, the virus-like particles have a better effect of inhibiting tumor growth, and the purpose of tumor immunotherapy is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and particularly relates to virus-like particles presenting IL-10 and nano vaccines thereof, and applications thereof in anti-tumor and / or immunotherapy drugs. Background Art

[0002] IL-10 is a cytokine with immunomodulatory function, and its role in tumor immunity is dual. On the one hand, IL-10 inhibits the production of proinflammatory cytokines and the function of antigen-presenting cells, leading to an immunosuppressive state and promoting tumor immune escape and growth. On the other hand, IL-10 also shows an immunostimulatory effect, which can enhance CD8 + The activity of T cells and natural killer cells promotes anti-tumor immune responses. However, the administration of IL-10 cytokines alone results in a short half-life in the body and often fails to achieve the desired tumor-killing effect. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a virus-like particle that presents IL-10, which targets tumors and lymph nodes by presenting IL-10 through the virus-like particle, thereby greatly improving the tumor-killing effect of IL-10.

[0004] The present invention provides a virus-like particle presenting IL-10, including a virus-like particle with IL-10 modified on the surface.

[0005] Preferably, the virus-like particles include virus-like particles of norovirus S protein.

[0006] Preferably, the surface of the IL-10 presenting virus-like particle is further modified with an E7 antigen peptide.

[0007] Preferably, the modification between the IL-10 or E7 antigen peptide and the virus-like particle is linked via a Spycatcher-SpyTag system or a biotin-streptavidin system.

[0008] The present invention provides a method for preparing the IL-10 presenting virus-like particles, comprising the following steps:

[0009] Recombinantly expressing the first handshake molecule-modified IL-10 by genetic engineering means, or recombinantly expressing IL-10 by genetic engineering means and then modifying the first handshake molecule to obtain the first handshake molecule-modified IL-10;

[0010] Recombinant expression of a virus protein modified with a second handshake molecule by genetic engineering means, and self-assembly to obtain virus-like particles modified with the second handshake molecule; or recombinant expression of a virus protein by genetic engineering means, followed by self-assembly, and then modification of the virus-like particles with the second handshake molecule to obtain virus-like particles modified with the second handshake molecule.

[0011] Mix the IL-10 modified with the first handshake molecule and the virus-like particles modified with the second handshake molecule to allow the first handshake molecule and the second handshake molecule to undergo a ligation reaction, resulting in virus-like particles with IL-10 on the surface.

[0012] Preferably, during the recombinant expression of the IL-10 modified with the first handshake molecule, the gene fragment encoding the IL-10 modified with the first handshake molecule includes a TF tag.

[0013] Preferably, during the self-assembly of the virus-like particles modified with the second handshake molecule, the assembly solution used includes 45 - 55 mM Tris and 180 - 220 mM NaCl; the pH value of the assembly solution is 6 - 8.

[0014] Preferably, when the virus-like particles presenting IL-10 are further modified with an E7 antigen peptide on the surface, recombinant expression of the E7 antigen peptide modified with the first handshake molecule by genetic engineering means, or preparation of the E7 antigen peptide modified with the first handshake molecule by chemical synthesis; ligate the E7 antigen peptide modified with the first handshake molecule and the IL-10 modified with the first handshake molecule together with the second handshake molecule to obtain virus-like particles with IL-10 and the E7 antigen peptide on the surface.

[0015] The present invention provides the use of the virus-like particles presenting IL-10 or the virus-like particles presenting IL-10 prepared by the preparation method in the preparation of an anti-tumor vaccine or drug;

[0016] The tumors include at least one of the following: HPV-related cancers, colon cancer, melanoma, breast cancer, non-small cell lung cancer, and laryngeal epidermoid carcinoma.

[0017] The present invention provides an anti-tumor vaccine or drug, the active ingredient of which includes the virus-like particles presenting IL-10 or the virus-like particles presenting IL-10 prepared by the preparation method.

[0018] The present invention provides a virus-like particle presenting IL-10, which comprises a virus-like particle with IL-10 surface-modified thereon. The present invention innovatively uses the virus-like particle as a delivery platform for the cytokine IL-10, enabling IL-10 to enter the body in the nanostructure of the virus-like particle, and it is relatively easy to reach the tumor and lymph node sites through the in vivo circulation to achieve targeted action, realizing the efficient, stable delivery and precise expression of IL-10. The virus-like particle presenting IL-10 provided by the present invention gives full play to the high safety of the virus-like particle, effectively overcomes the limitations of the traditional cytokine delivery system in terms of stability and targeting; meanwhile, effectively regulates the tumor microenvironment and enhances the anti-tumor immune response. The technical solution of the present invention provides a new idea for the combination of cytokine therapy and nano-vaccine technology, demonstrating the application potential of IL-10 in tumor immunotherapy and its important value in precision medicine. Description of the Drawings

[0019] Figure 1 Schematic diagram of cloning the IL-10 gene fragment with Spytag on the pThioHisA vector;

[0020] Figure 2 For the construction method and results of the recombinant vector, where A is the spectrum of the recombinant vector without adding an extra tag; B is the spectrum of the recombinant vector containing the Trx-tagged Spytag-IL-10 gene fragment; C is the protein detection result of the recombinant expression of the recombinant vector;

[0021] Figure 3 For the protein detection result of the TF-tagged Spytag-IL-10 gene fragment obtained by recombinant expression, where A is the protein detection result of TF-spytag-IL-10 obtained by recombinant expression, and B is the schematic diagram of the result of purifying TF-spytag-IL-10 with a nickel column;

[0022] Figure 4 For the electron microscopy observation result of the virus-like particle NC purified in the optimal assembly solution;

[0023] Figure 5 For the electron microscopy observation result of the virus-like particle NC purified in the control assembly solution;

[0024] Figure 6 For the electron microscopy observation result of the virus-like particle NC purified in the control assembly solution 2;

[0025] Figure 7 For the result of the change in the secretion of IFN-γ in the tumors of mice after immunization by different methods;

[0026] Figure 8 For the result of the change in the proportion of NK cells in the spleens of mice after immunization by different methods;

[0027] Figure 9 Results of the proportion change of NK cells in the spleens of mice after immunization by different methods; Figure 10 Results of the proportion change of MDSC cells in the spleens of mice after immunization by different methods. Detailed implementation manners

[0028] The present invention provides a virus-like particle presenting IL-10, including a virus-like particle surface-modified with IL-10.

[0029] In the present invention, the virus-like particle preferably includes a virus-like particle of norovirus S protein. As a delivery vector, the virus-like particle relies on the advantages of nanostructure to deliver the surface-modified IL-10 into the body. Through the action of in vivo circulation, it is more likely to reach the tumor and lymph node sites, achieve targeted tumor sites, and improve the tumor killing ability of IL-10; at the same time, the delivery of the virus-like particle quickly reaches the targeted site, solving the problem that the tumor killing ability of IL-10 is weak due to its short half-life when used alone in the body.

[0030] In the present invention, the surface of the virus-like particle presenting IL-10 preferably further includes an E7 antigen peptide surface-modified. The E7 antigen peptide combined with IL-10 exerts the dual effects of cytokine therapy and tumor immunotherapy, can further improve the killing effect on tumors, and shows more excellent anti-tumor drug efficacy.

[0031] In the present invention, the modification between the IL-10 or E7 antigen peptide and the virus-like particle is preferably connected through the Spycatcher-SpyTag system or the biotin-streptavidin system. In the examples of the present invention, in the Spycatcher-SpyTag system, a covalent isopeptide bond is formed between SpyCatcher and SpyTag through a side-chain amino group, thereby realizing the stable connection between molecules. The present invention uses the Spycatcher-SpyTag system to display proteins or polypeptide antigens on the surface of virus-like particles, which not only improves the stability of vaccines or drugs, but also enhances the immune response effect of vaccines, and further improves the tumor immunotherapy effect.

[0032] The present invention provides a preparation method of the virus-like particle presenting IL-10, including the following steps:

[0033] Recombinantly express IL-10 modified with a first handshake molecule by genetic engineering means or perform first handshake molecule modification after recombinantly expressing IL-10 by genetic engineering means to obtain IL-10 modified with a first handshake molecule;

[0034] Recombinant expression of a virus protein modified with a second handshake molecule by genetic engineering means, and self-assembly to obtain virus-like particles modified with the second handshake molecule; or recombinant expression of a virus protein by genetic engineering means, followed by self-assembly, and then modification of the virus-like particles with the second handshake molecule to obtain virus-like particles modified with the second handshake molecule.

[0035] Mix the IL-10 modified with the first handshake molecule and the virus-like particles modified with the second handshake molecule to allow the first handshake molecule and the second handshake molecule to undergo a ligation reaction, thereby obtaining virus-like particles with IL-10 on the surface.

[0036] The present invention recombinantly expresses IL-10 modified with the first handshake molecule by genetic engineering means or performs modification with the first handshake molecule after recombinantly expressing IL-10 by genetic engineering means to obtain IL-10 modified with the first handshake molecule.

[0037] In the present invention, the method for recombinantly expressing IL-10 modified with the first handshake molecule by genetic engineering means preferably involves cloning the nucleotide sequence encoding IL-10 modified with the first handshake molecule into a vector, transforming the resulting recombinant vector into a prokaryotic expression system for recombinant expression, screening and culturing, inducing culturing, separating and purifying the recombinant protein to obtain IL-10 modified with the first handshake molecule. The first handshake molecule is preferably one of SpyCatcher and SpyTag. The first handshake molecule is linked to IL-10 through a linker peptide. The linker peptide is preferably GGGGSGS. The present invention places no special restrictions on the methods of transformation, screening culture, and induction culture, and well-known methods in the art can be used.

[0038] In the present invention, during the recombinant expression of IL-10 modified with the first handshake molecule, the gene fragment encoding IL-10 modified with the first handshake molecule preferably includes a TF tag. The TF tag is co-expressed with the gene fragment encoding IL-10 modified with the first handshake molecule, which is beneficial to improving the expression level of IL-10 modified with the first handshake molecule. In the examples of the present invention, in order to further improve the expression level of the target protein, the effects of different tags on the target protein were explored. The results showed that the TF tag can better improve the expression level of IL-10 modified with the first handshake molecule than the Trx tag, which is beneficial to obtaining a large number of virus-like particles presenting IL-10.

[0039] Recombinant expression of a virus protein modified with a second handshake molecule by genetic engineering means, and self-assembly to obtain virus-like particles modified with the second handshake molecule; or recombinant expression of a virus protein by genetic engineering means, followed by self-assembly, and then modification of the virus-like particles with the second handshake molecule to obtain virus-like particles modified with the second handshake molecule.

[0040] In the present invention, the method for recombinantly expressing a virus protein modified with a second handshake molecule by genetic engineering means preferably involves cloning the coding gene of the virus protein modified with the second handshake molecule into a plasmid, transforming the obtained recombinant plasmid into a prokaryotic expression vector for expression, and separating and purifying the recombinant protein through screening culture and induction culture. The self-assembly method further purifies the above-separated and purified recombinant protein in the molecular sieve of the assembly solution to obtain purified virus-like particles. During the self-assembly process of the virus-like particles modified with the second handshake molecule, the assembly solution used preferably includes 45 - 55 mM Tris and 180 - 220 mM NaCl; the pH value of the assembly solution is 6 - 8, or it can be 50 mM Tris and 200 mM NaCl; the pH value of the assembly solution is 7.6. Compared with other assembly solutions, the assembly solution is more conducive to improving the assembly efficiency of virus-like particles, obtaining a large number of virus-like particles, and ensuring the preparation materials for the subsequent virus-like particles presenting IL-10. The purification method of the molecular sieve is preferably gel filtration chromatography. The packing material for the gel filtration chromatography is preferably Sepharose 6 Fast Flow packing material.

[0041] After obtaining the IL-10 modified with the first handshake molecule and the virus-like particles modified with the second handshake molecule, the present invention mixes the IL-10 modified with the first handshake molecule and the virus-like particles modified with the second handshake molecule to enable the first handshake molecule and the second handshake molecule to undergo a ligation reaction, resulting in virus-like particles with IL-10 modified on their surfaces.

[0042] In the present invention, the ligation reaction is preferably incubated at 4 - 8 °C for 10 - 14 h, or it can be incubated at 4 °C for 12 h.

[0043] In the present invention, when the surface of the virus-like particles presenting IL-10 is further modified with an E7 antigen peptide, preferably, the E7 antigen peptide modified with the first handshake molecule is recombinantly expressed by genetic engineering means or prepared by an artificial synthesis method; the E7 antigen peptide modified with the first handshake molecule and the IL-10 modified with the first handshake molecule are ligated with the second handshake molecule together to obtain virus-like particles with IL-10 and the E7 antigen peptide modified on their surfaces.

[0044] The present invention provides the use of the IL-10-presenting virus-like particles or the IL-10-presenting virus-like particles prepared by the preparation method in the preparation of anti-tumor vaccines or drugs; the tumors include at least one of the following: HPV-related cancers, colon cancer, melanoma, breast cancer, non-small cell lung cancer, and laryngeal epidermoid carcinoma. The HPV-related cancers preferably include cervical cancer, anal cancer, oropharyngeal cancer, vaginal cancer, vulvar cancer, and penile cancer. The HPV-related cancers are tumor-bearing animal models developed with TC-1 cells (mouse lung epithelial cells) transformed with the E6 and E7 genes of human papillomavirus (HPV) type 16 and the ras gene. TC-1 cells not only retain the characteristics of lung epithelial tissue but also exhibit tumor characteristics through genetic engineering means, enabling them to have good growth characteristics in vitro.

[0045] In the embodiments of the present invention, using the tumor-bearing mouse tumor model constructed by inoculating mice with TC-1 cells as the experimental object, the immune response and anti-tumor activity of the vaccine prepared from the IL-10-presenting virus-like particles were carried out. The results showed that the vaccine prepared from the IL-10-presenting virus-like particles could effectively regulate the tumor microenvironment and enhance the anti-tumor immune response, improve the anti-tumor activity of IL-10, and greatly improve the application potential in tumor immunotherapy and the important value in precision medicine.

[0046] The present invention provides an anti-tumor vaccine or drug, the active ingredient of which includes the IL-10-presenting virus-like particles or the IL-10-presenting virus-like particles prepared by the preparation method.

[0047] In the present invention, in the vaccine or drug, the concentration of the IL-10-presenting virus-like particles is preferably 0.1-5 mg / mL, and it can be 1 mg / mL. The present invention has no special restrictions on the preparation method of the vaccine or drug, and the well-known preparation methods of vaccines or drugs in the art can be used.

[0048] The following combines examples to elaborate in detail on an IL-10-presenting virus-like particle and its nano-vaccine provided by the present invention and its application in anti-tumor and / or immunotherapy drugs, but they cannot be understood as limiting the protection scope of the present invention.

[0049] Plasmid source description:

[0050] The pThioHisA plasmid was purchased from Invitrogen;

[0051] pCold TM The TF plasmid was purchased from Takara;

[0052] pThioHisA-4S-Spycatcher plasmid: Previously constructed in our laboratory, it was obtained by inserting the S protein of norovirus fused with SpyCatcher into the pThioHisA expression vector through double digestion with Nde I and EcoR I. The specific reference document is the patent with the publication number CN116063578A.

[0053] Example 1

[0054] In vitro expression method of Spytag-IL-10 recombinant protein

[0055] 1. Target gene sequence and codon optimization

[0056] 1.1 The amino acid sequence of IL-10 protein is:

[0057] SRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSL

[0058] MQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRM

[0059] RLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS(SEQ ID NO:1);

[0060] The nucleotide sequence obtained by codon optimization of the IL-10 protein coding sequence is as follows: TCTCGTGGTCAATATTCTCGTGAAGATAACAACTGCACGCACTTCCCTGTAGGTCAAAGCCACATGCTGCTGGAACTGCGTACTGCTTTCTCTCAGGTTAAGACCTTCTTCCAGACCAAAGACCAGCTGGATAACATCCTGCTGACCGATAGCCTGATGCAGGATTTCAAAGGTTACCTGGGCTGTCAGGCTCTGTCCGAAATGATTCAGTTCTACCTGGTTGAAGTTATGCCGCAGGCAGAGAAACACGGCCCGGAAATTAAGGAACATCTGAACAGCCTGGGCGAGAAACTGAAGACCCTGCGTATGCGTCTGCGCCGCTGCCATCGCTTCCTGCCGTGCGAGAACAAATCCAAAGCCGTGGAACAGGTCAAATCCGACTTTAACAAACTGCAAGACCAGGGCGTGTACAAAGCGATGAATGAATTTGACATCTTTATCAACTGCATCGAAGCGTATATGATGATCAAAATGAAATCCTAA (SEQ ID NO:2).

[0061] 1.2 The amino acid sequence of SpyTag is: RGVPHIVMVDAYKRYK;

[0062] The nucleotide sequence obtained by codon optimization of the SpyTag coding sequence is as follows: CGTGGTGTTCCGCACATCGTTATGGTTGATGCGTACAAACGTTACAAA (SEQ ID NO:3).

[0063] 1.3 The amino acid sequence of the linker is: GGGGSGS (SEQ ID NO:4).

[0064] Among them, G (Gly) is glycine and S is serine (Ser).

[0065] The nucleotide sequence of the linker coding sequence is as follows: GGTGGTGGTTCTGGTAGC (SEQ ID NO:5).

[0066] 1.4 The nucleotide sequence of the Spytag-IL-10 fusion gene after codon optimization is as follows:

[0067] CGTGGTGTTCCGCACATCGTTATGGTTGATGCGTACAAACGTTACAAAGGTGGTGGTTCTGGTAGCTCTCGTGGTCAATATTCTCGTGAAGATAACAACTGCACGCACTTCCCTGTAGGTCAAAGCCACATGCTGCTGGAACTGCGTACTGCTTTCTCTCAGGTTAAGACCTTCTTCCAGACCAAAGACCAGCTGGATAACATCCTGCTGACCGATAGCCTGATGCAGGATTTCAAAGGTTACCTGGGCTGTCAGGCTCTGTCCGAAATGATTCAGTTCTACCTGGTTGAAGTTATGCCGCAGGCAGAGAAACACGGCCCGGAAATTAAGGAACATCTGAACAGCCTGGGCGAGAAACTGAAGACCCTGCGTATGCGTCTGCGCCGCTGCCATCGCTTCCTGCCGTGCGAGAACAAATCCAAAGCCGTGGAACAGGTCAAATCCGACTTTAACAAACTGCAAGACCAGGGCGTGTACAAAGCGATGAATGAATTTGACATCTTTATCAACTGCATCGAAGCGTATATGATGATCAAAATGAAATCCTAA(SEQ ID NO:6).

[0068] 2. Construction and transformation of recombinant plasmid

[0069] 2.1 Design primers and clone the IL-10 gene fragment with Spytag from the pThioHisA vector by PCR method (see Figure 1 ).

[0070] The primer sequences are as follows:

[0071] Forward primer: GGTATCGAAGGTAGGCATATGCGTGGTGTTCCGCAC(SEQ ID NO:7);

[0072] Reverse primer: CAGGTCGACAAGCTTGAATTCTTAGGATTTCATTTTGATC AT(SEQ ID NO:8).

[0073] 2.2 Clone the amplified IL-10 gene fragment with Spytag into pCold by homologous recombination TMThe SpyTag-IL10 recombinant plasmid was obtained between the multiple cloning sites NdeⅠ and EcoRI of the TF vector (see Figure 2 Middle A).

[0074] 2.3 The SpyTag-IL10 recombinant plasmid was transformed into BL21(DE3) competent cells, inoculated on LB plates containing ampicillin, and single clones were screened.

[0075] 2.4 Expand and culture the correct monoclonal strain at 37°C and 220 rpm. 600 When the concentration reached 0.5-0.6, 0.5 mmol / L IPTG was added and induced on a constant temperature shaker at 16°C for 12 h, and the bacterial solution was collected.

[0076] 2.5 Purification of SpyTag-IL10 recombinant protein

[0077] The collected bacterial solution was centrifuged at 8000 rpm for 10 minutes to collect the bacterial precipitate, which was resuspended in ice-cold PBS and then disrupted by ultrasonication.

[0078] The supernatant after disruption was centrifuged at 13,000 rpm for 20 minutes, the supernatant was collected, and filtered through a 0.45 μm filter to obtain a pure protein supernatant.

[0079] The protein supernatant was purified by nickel affinity chromatography using Ni Sepharose 6 Fast Flow medium, and the target protein was eluted by gradient elution (using 0.01 M PBS, 500 mM sodium chloride, 500 mM imidazole, pH 6.8 elution buffer).

[0080] The target protein was purified by nickel column, and the background solution was replaced with PBS to finally obtain the purified SpyTag-IL10 recombinant protein.

[0081] The Spytag-IL10 recombinant protein sample was separated using a 12% stain-free SDS-PAGE gel, and the target bands were photographed using ImageLab. The gel was then blotted onto a PVDF membrane using a mouse anti-IL-10 monoclonal antibody (1:1000) as the primary antibody and an HRP-conjugated anti-mouse IgG antibody (1:5000) as the secondary antibody. Western blotting (WB) was performed using standardized protocols and methods, and protein bands were visualized using ECL.

[0082] Experimental Results Figure 2 Center C shows that SpyTag-IL10 recombinant protein was successfully expressed in vitro.

[0083] Comparative Example 1

[0084] To explore methods for improving the expression level of SpyTag-IL10 recombinant protein, the Trx tag was linked to the SpyTag-IL10 protein sequence via a linker peptide. The resulting Ttx-SpyTag-IL10 protein coding sequence was subjected to in vitro recombinant expression and detection using the method of Example 1.

[0085] The nucleotide sequence of Trx is as follows:

[0086] TCTGATAAAATTATTCATCTGACTGATGATTCTTTTGATACTGATGTACTTAAGGCAGATGGTGCAATCCTGGTTGATTTCTGGGCACACTGGTGCGGTCCGTGCAAAATGATCGCTCCGATTCTGGATGAAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCACAACCCGGGCACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGCAACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCC(SEQ ID NO:9);

[0087] The amino acid sequence of Trx is as follows:

[0088] MSDKIIHLTDDSFDTDVLKADGAILVDFWAHWCGPCKMIAPILDEIADEYQGKLTVAKLNIDHNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA(SEQ ID NO:10);

[0089] The amino acid sequence of the linker peptide is as follows: GSGS(SEQ ID NO:11);

[0090] The nucleotide sequence of the linker peptide is as follows: GGCTCTGGATCC(SEQ ID NO:17).

[0091] The results are shown in Figure 2 In C, compared with the tag-free SpyTag-IL10 recombinant protein prepared in Example 1, the addition of the Trx tag increased the expression level of the SpyTag-IL10 recombinant protein. However, the optimal purification temperature for the Trx tag is 30 °C, and purification at too high a temperature affects protein assembly and expression.

[0092] Example 2

[0093] In order to further explore methods for improving the expression level of recombinant proteins, in this example, a TF tag was added to the N-terminus of the recombinant protein, and recombinant expression and detection were carried out using the method of Example 1.

[0094] The nucleotide sequence of TF is as follows:

[0095]

[0096] The amino acid sequence of TF is as follows:

[0097] MQVSVETTQGLGRRVTITIAADSIETAVKSELVNVAKKVRIDGFRKGKVPMNIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKINPAGAPTYVPGEYKLGEDFTYSVEFEVYPEVELQGLEAIEVEKPIVEVTDADVDGMLDTLRKQQATWKEKDGAVEAEDRVTIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIKGHKAGEEFTIDVTFPEEYHAENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDGSVEGLRAEVRKNMERELKSAIRNRVKSQAIEGLVKANDIDVPAALIDSEIDVLRRQAAQRFGGNEKQALELPRELFEEQAKRRVVVGLLLGEVIRTNELKADEERVKGLIEEMASAYEDPKEVIEFYSKNKELMDNMRNVALEEQAVEAVLAKAKVTEKETTFNELMNQQA (SEQ ID NO:13);

[0098] The amino acid sequence of the linker peptide is as follows: GSGS (SEQ ID NO:14);

[0099] The nucleotide sequence of the linker peptide is as follows: GGCTCTGGATCC (SEQ ID NO:15).

[0100] Figure 3 The results showed that the addition of the TF tag greatly improved the expression level of the recombinant protein. The optimal purification temperature of the TF tag is 16 °C. Therefore, considering the subsequent assembly of virus-like particles, the TF tag was selected for the expression of the recombinant protein.

[0101] Example 3

[0102] In vitro recombinant expression method of spytag-E7 antigen peptide

[0103] 1. The sequence of spytag-E7 antigen peptide is as follows: The antigen peptide sequence is RGVPHIVMVDAYKRYKGSG RAHYNIVTF (SEQ ID NO:16); The underlined part is the sequence of the E7 peptide, the non-underlined part is spytag, and the bold font is the linker arm. The spytag-E7 antigen peptide was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0104] Example 4

[0105] Preparation method and identification of virus-like particles (NC)

[0106] Preparation method of 1.4S-Spycatcher recombinant protein

[0107] The pThioHisA-4S-Spycatcher plasmid constructed in the laboratory was transformed into 1) BL21(DE3) competent cells, inoculated on an LB plate containing ampicillin, and monoclonal colonies were screened. The selected monoclonal colonies were cultured and then 1 mmol / L IPTG was added, followed by induction culture at 30 °C for 16 h. The bacterial cells were separated, and the protein supernatant was isolated from the bacterial cells. The obtained protein supernatant was purified by nickel column chromatography, and the experimental steps were the same as those described in step 2.5 of Example 1. The NC virus-like particle sample was separated using a 12% stain-free SDS-PAGE protein gel, and the pictures of the target bands were taken and recorded with ImageLab.

[0108] 2. Purification method of virus-like particles (NC)

[0109] To purify NC assembled into virus-like particles, gel filtration chromatography was performed using Sepharose 6 Fast Flow packing material (using an assembly buffer of 50 mM Tris, 200 mM sodium chloride, pH 7.6), and uniform virus-like particles NC were obtained. The morphology of the virus-like particles was observed using a transmission electron microscope.

[0110] The results are shown in Figure 4 . The purified NC particles were spherical and of uniform size, with a large number.

[0111] Comparative Example 2

[0112] To explore the effect of the assembly solution on the assembly effect of virus-like particles, virus-like particle purification was carried out according to the method of Example 4 using assembly solution 1 (20 mM PB, 500 mM sodium chloride, pH 6.0) and assembly solution 2 (20 mM PB, 200 mM sodium chloride, pH 7.0) respectively.

[0113] The results are shown in Figure 5 and Figure 6 . Under the above two assembly solution conditions, the ability to assemble into virus-like particles was poor, and most of them did not form particle structures.

[0114] Example 5

[0115] Preparation method of virus-like particles NC presenting Spytag-IL10

[0116] Incubate 10 μL of 1 mg / mL Spytag-IL10 with 20 μL of 1 mg / mL virus-like particle NC overnight at 4 °C to obtain a virus-like particle sample presenting IL-10 (NC-IL10).

[0117] Example 6

[0118] Preparation method of virus-like particle NC presenting Spytag-IL10 and Spytag-E7

[0119] Incubate 10 μL of 1 mg / mL Spytag-IL10, 5 μL of 1 mg / mL Spytag-E7 peptide with 20 μL of 1 mg / mL NC virus-like particles overnight at 4 °C to obtain a virus-like particle sample presenting Spytag-IL10 and Spytag-E7 peptide (NC-IL10+E7).

[0120] Example 7

[0121] Antitumor experiments of NC-IL10 and NC-IL10+E7

[0122] To evaluate the antitumor effects of NC-IL10 and NC-IL10+E7, TC-1 tumor cells were subcutaneously inoculated into the right side of C57BL / 6 female mice to establish a tumor-bearing mouse model. The vaccine was injected subcutaneously once every 7 days for a total of three times, and the injection dose was 100 μL / time. The vaccines included the vaccine prepared with NC-IL10 and the vaccine prepared with NC-IL10+E7. The preparation method of the vaccine was incubation at 4 °C. At the same time, injection of the same dose of PBS was used as a blank control, injection of the vaccine prepared with only E7 was used as positive control 1, and injection of the vaccine prepared with only IL-10 was used as positive control 2. The growth of tumors was regularly monitored, the tumor volume was measured, the secretion of IFN-γ in the tumors of mice after vaccination was detected by ELISPOT, and the proportions of NK cells and MDSC cells in the spleens of mice after vaccination were measured by flow cytometry. To exclude quality problems of the ELISPOT plate, a blank control was also set, specifically adding only complete medium. To more accurately calculate the number of spots in the experimental group by the machine through negative and positive controls when reading the spots on the ELISPOT, negative and positive controls were also set. The negative control was complete medium containing test cells, and the positive control was complete medium containing a positive stimulant (PMA) and test cells.

[0123] The effects of NC-IL10 and NC-IL10+E7 in inhibiting tumors in the TC-1 mouse tumor model are shown in Figures 7 to 10。The results of tumor volume measurement showed that, compared with the PBS group, the immune methods of the other 4 groups could effectively inhibit tumor growth. From the results of the measurement of IFN-γ secreted by the tumor, it could be seen that, compared with the PBS group, the E7 peptide group and the IL-10 group, NC-IL10 and NC-IL10+E7 could significantly increase the secretion of IFN-γ, and the stimulating activity of NC-IL10+E7 was higher. It can be seen that the vaccine prepared by the present invention kills tumors by increasing the secretion amount of IFN-γ. From the proportions of NK cells and MDSC cells in different treatment groups, it was known that after injecting the NC-IL10 vaccine and the NC-IL10+E7 vaccine, the proportion of NK cells in the spleens of mice increased significantly, and after injecting the NC-IL10 vaccine and the NC-IL10+E7 vaccine, the proportion of MDSC cells in the spleens of mice decreased significantly. It can be seen that the improved NC-IL10 and NC-IL10+E7 of the present invention play an immunotherapeutic role in tumors to varying degrees, and NC-IL10+E7 further improves the anti-tumor activity compared with NC-IL10.

[0124] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A virus-like particle presenting IL-10, characterized in that, Virus-like particles surface-modified with IL-10.

2. The virus-like particle presenting IL-10 according to claim 1, characterized in that, The virus-like particles include virus-like particles of norovirus S protein.

3. The virus-like particle presenting IL-10 according to claim 1 or 2, characterized in that, The surface of the virus-like particles presenting IL-10 further includes E7 antigen peptide surface-modified.

4. The virus-like particle presenting IL-10 according to claim 3, characterized in that, The modification between the IL-10 or E7 antigen peptide and the virus-like particles is connected through the Spycatcher-SpyTag system or the biotin-streptavidin system.

5. A method for preparing a virus-like particle presenting IL-10 according to any one of claims 1 to 4, characterized in that, Including the following steps: Recombinantly expressing IL-10 modified with a first handshake molecule by genetic engineering means or performing first handshake molecule modification after recombinantly expressing IL-10 by genetic engineering means to obtain IL-10 modified with a first handshake molecule; Recombinantly expressing a viral protein modified with a second handshake molecule by genetic engineering means and self-assembling to obtain virus-like particles modified with a second handshake molecule; Or recombinantly expressing a viral protein by genetic engineering means, self-assembling, and then performing second handshake molecule modification on the obtained virus-like particles to obtain virus-like particles modified with a second handshake molecule; Mixing IL-10 modified with a first handshake molecule and virus-like particles modified with a second handshake molecule to allow the first handshake molecule and the second handshake molecule to undergo a ligation reaction to obtain virus-like particles surface-modified with IL-10; Among them, the preparation of IL-10 modified with a first handshake molecule and virus-like particles modified with a second handshake molecule is not restricted by the time sequence.

6. The preparation method according to claim 5, wherein During the recombinant expression of the IL-10 modified with a first handshake molecule, the gene fragment encoding the IL-10 modified with a first handshake molecule includes a TF tag.

7. The preparation method according to claim 5, characterized in that, During the self-assembly of the virus-like particles modified with a second handshake molecule, the assembly solution used includes 45-55 mM Tris and 180-220 mM NaCl; the pH value of the assembly solution is 6-8.

8. The preparation method according to any one of claims 5 to 7, characterized in that, When the surface of the virus-like particles presenting IL-10 is further modified with E7 antigen peptide, recombinantly expressing E7 antigen peptide modified with a first handshake molecule by genetic engineering means or preparing E7 antigen peptide modified with a first handshake molecule by artificial synthesis method; Reacting the E7 antigen peptide modified with a first handshake molecule and the IL-10 modified with a first handshake molecule together with a second handshake molecule to obtain virus-like particles surface-modified with IL-10 and E7 antigen peptide.

9. Use of the virus-like particles presenting IL-10 according to any one of claims 1 to 4 or the virus-like particles presenting IL-10 prepared by the preparation method according to any one of claims 5 to 8 in the preparation of anti-tumor vaccines or drugs; The tumors include at least one of the following: HPV-related cancers, colon cancer, melanoma, breast cancer, non-small cell lung cancer, and laryngeal epidermoid carcinoma.

10. An anti-tumor vaccine or drug, characterized in that, The active ingredient includes the virus-like particles presenting IL-10 according to any one of claims 1 to 4 or the virus-like particles presenting IL-10 prepared by the preparation method according to any one of claims 5 to 7.

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