Preparation method and application of sCAR1 mutant nanoparticles for resisting CV-B3 infection

Abstract

The present invention provides a preparation method and application of sCAR1 mutant nanoparticles for CV-B3 infection, belonging to the field of biomedicine technology. The present invention solves the problem that there are currently no preventive vaccines and specific therapeutic drugs for CV-B3 infection. The present invention constructs nanoparticles sCAR1 Mu+Ferritin that fully display sCAR1 Mu, and the IC50 for blocking CV-B3 infection in Vero cells and SH-SY5Y cells is 413.05 ng / ml and 140.93 ng / ml, respectively. When 200 μg sCAR1 Mu+Ferritin is intraperitoneally injected 6 hours in advance, followed by intraperitoneal injection of a lethal infection dose of CV-B3, the viral load in the liver, spleen, brain and heart tissues can be significantly reduced, and the inflammatory pathology in the heart and brain tissues can be effectively alleviated, and a protection rate of 68.75% can be provided in terms of survival rate.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a preparation method and application of sCAR1 mutant nanoparticles for resisting CV-B3 infection. Background Art

[0002] CV-B3 (Coxsackievirus B3) is a regular icosahedral symmetrical particle without an envelope structure in terms of viral morphology, with a diameter of about 30 nm. The viral capsid is composed of four structural proteins: VP1 (Virus protein 1), VP2 (Virus protein 2), VP3 (Virus protein 3) and VP4 (Virus protein 4). VP1, VP2 and VP3 are displayed on the outside of the viral capsid, while VP4 is wrapped inside the viral capsid. The Canyon region composed of VP1, VP2 and VP3 is an important region for CV-B3 virus particles to bind to the CAR receptor. CV-B3 infection can cause clinical symptoms such as hand, foot and mouth disease-like rash, blisters, viral myocarditis and viral meningitis. Among them, viral meningitis and myocarditis usually cause more serious pathological symptoms in the body, and in some patients, especially infants and young children, death cases will occur. Currently, there is no vaccine or specific therapeutic drug for CV-B3 infection, so CV-B3 infection can only be treated with supportive treatment in clinic. The research on antiviral drugs is mainly focused on the main biochemical and molecular biological events that occur during the viral infection cycle (adsorption and binding of the virus, release of the viral genome, replication of the viral genome, formation of the viral replication microenvironment, translation and synthesis of viral proteins, enzymatic properties of viral non-structural proteins, assembly and release of viral particles, etc.).

[0003] When a virus becomes pandemic or a virus strain mutates frequently, the dual time lags in vaccine development and vaccine effectiveness make it difficult to provide effective response measures for epidemic prevention and control in the first place. The development of specific neutralizing antibodies may face the dilemma of high technical barriers and reduced antibody neutralization titer or even ineffectiveness due to virus mutations. Small molecule interfering drugs have problems such as difficult-to-find mechanisms, difficult screening, low delivery efficiency and complex pharmacokinetics. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method and application of sCAR1 mutant nanoparticles for resisting CV-B3 infection, which solves the problem that there is currently no preventive vaccine and specific therapeutic drug for CV-B3 infection.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides the use of sCAR1 protein [a splice variant member CAR1 protein of Coxsackievirus-adenovirus receptor CAR containing a complete extracellular segment (D1 and D2 domains)] in the preparation of an anti-CV-B3 virus drug, wherein the amino acid sequence of the sCAR1 protein is shown in SEQ ID NO.2.

[0007] The present invention provides a modified mutant sCAR1 Mu protein, wherein the sCAR1 Mu protein is modified based on the sCAR1 protein whose amino acid sequence is shown in SEQ ID NO.2;

[0008] The amino acid sequence of the modified mutant sCAR1 Mu protein is shown in SEQ ID NO.5.

[0009] The present invention provides a mutant nanoparticle, which takes ferritin nanoparticles as a matrix and displays the mutant sCAR1 Mu protein on its surface.

[0010] The present invention provides a mutant nanoparticle, the amino acid sequence of the mutant nanoparticle is shown as SEQ ID NO.1.

[0011] The present invention provides a recombinant expression vector for expressing the mutant nanoparticles, characterized in that the basic vector used by the recombinant expression vector is PTT5.

[0012] The present invention provides use of the modified mutant sCAR1 Mu protein, the mutant nanoparticle or the recombinant expression vector in the preparation of a drug for resisting CV-B3 virus.

[0013] The present invention provides a modified mutant CAR3 Mu protein, wherein the modified mutant CAR3 Mu protein is obtained by modification based on the CAR protein;

[0014] The amino acid sequence of the modified mutant CAR3 Mu protein is shown in SEQ ID NO.6.

[0015] In the present invention, both the modified mutant CAR3 Mu protein and the modified mutant sCAR1 Mu protein are based on CAR protein modification, retaining amino acid residues that strongly interact with CV-B3, and mutating sites that strongly interact with CAR itself and JAML (Junctional Adhesion Molecule-Like), based on the same inventive concept, and are both used to resist CV-B3 virus.

[0016] The present invention provides a mutant nanoparticle, characterized in that it uses ferritin nanoparticles as a matrix and displays the mutant CAR3 Mu protein on its surface.

[0017] The present invention provides a mutant nanoparticle, the amino acid sequence of the mutant nanoparticle is shown as SEQ ID NO.7.

[0018] The present invention provides the use of the modified mutant sCAR1 Mu protein or the mutant nanoparticle in preparing a drug for resisting CV-B3 virus.

[0019] Beneficial effects of the present invention:

[0020] 1. Based on the antiviral principle of receptor analogs, the present invention discovered that the splice variant member CAR1 protein of Coxsackievirus and Adenovirus Receptor (CAR) containing a complete extracellular segment (D1 and D2 domains) has an anti-CV-B3 infection effect in vitro, and limits its infection ability by binding to CV-B3 to uncapsidate and release the viral genome.

[0021] 2. CAR protein has the characteristic of forming homodimers inside the molecule, and can bind to the cell membrane surface molecules CAR and JAML, which has the possible risk of promoting cell proliferation. To overcome this risk, the present invention designs a sCAR1 Mu mutant, which has the characteristics of no longer forming homodimers inside the molecule and no longer binding to the cell membrane surface molecules CAR and JAML, which significantly improves the effectiveness and safety of sCAR1 Mu against CV-B3 infection.

[0022] 3. Based on Ferrtin protein, nanoparticles that fully display CAR1 Mu - CAR1 Mu + Ferritin were constructed. The IC50 for blocking CV-B3 infection in Vero cells and SH-SY5Y cells were 413.05 ng / ml and 140.93 ng / ml, respectively.

[0023] 4. The efficacy and safety of sCAR1 Mu+Ferritin against CV-B3 infection were further evaluated in 3-day-old Balb / c mice. The present invention found that 200 μg sCAR1 Mu+Ferritin was injected intraperitoneally 6 h in advance, followed by intraperitoneal injection of a lethal infection dose of CV-B3 (10 3.5 CCID50), it can significantly reduce the viral load in the liver, spleen, brain and heart tissues, effectively alleviate the inflammatory pathology in the heart and brain tissues, and provide a 68.75% protection rate in terms of survival rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The SWISS MODLE structure prediction map for sCAR1 / CAR3 expression construction, in the figure UniProt: Universal Protein Resource.

[0025] Figure 2 Figure 2 shows the analysis of sCAR1 / CAR3 eukaryotic expression and purification. (A) Coomassie Brilliant Blue staining analysis of sCAR1 / CAR3 expression after plasmid transfection into 293T cells; (B) Western Blot identification after sCAR1 / CAR3 expression and purification.

[0026] Figure 3 sCAR1 / CAR3 binds to CV-B3 and causes CV-B3 to uncapsidate and release viral genome. (A) Ultracentrifuge CV-B3 virus stock solution to obtain CV-B3 solid virus particles. (BC) sCAR1 / CAR3 and CV-B3 are immunoprecipitated. (D) sCAR1 / CAR3 and CV-B3 are co-incubated in the chamber to release the genome of CV-B3 into hollow particles. (E) High concentration (500μg / ml) to low concentration (16μg / ml) of sCAR1 / CAR3 co-incubated with CV-B3 causes CV-B3 to release viral genome into the supernatant.

[0027] Figure 4 Analysis of potential safety issues of sCAR1 / CAR3. (A) sCAR1 / CAR3 undergoes internal dimerization. (B) sCAR1 / CAR3 undergoes immunoprecipitation with cell membrane surface adhesion molecules CAR and JAML. (C) High concentration (400μg / ml) sCAR1 / CAR3 promotes abnormal cell proliferation. (D) Analysis of the effect of high concentration (400μg / ml) sCAR1 / CAR3 on cell adhesion and pro-inflammatory cytokines. ns: not significant.

[0028] Figure 5 Design information for the amino acid residues and mutation sites of CAR binding to cell membrane surface adhesion molecules (CAR and JAML) and CV-B.

[0029] Figure 6 Figure 1 shows the expression and purification analysis of mutant sCAR1 Mu / CAR3 Mu. (A) Coomassie blue staining analysis of the supernatant after eukaryotic expression in 293T cells, the flow-through sample after purification, the wash solution, and the eluate. (B) Western Blot identification of sCAR1 Mu / CAR3 Mu.

[0030] Figure 7 Safety analysis of mutant sCAR1 Mu / CAR3 Mu (A) sCAR1 Mu and CAR3 Mu BS 3 Detection of intramolecular dimerization after cross-linking. (B) Immunoprecipitation detection of sCAR1 Mu / CAR3 Mu and cell membrane surface adhesion molecules CAR and JAML. (C) CCK8 assay to detect the effect of gradient concentrations of sCAR1 Mu / CAR3 Mu on the proliferation of Vero cells and SH-SY5Y cells. (D) Effect of high concentration (400 μg / ml) sCAR1 Mu / CAR3 Mu on cell proliferation and pro-inflammatory cytokines at the mRNA level in 293T cells.

[0031] Figure 8 To detect and compare the anti-CV-B3 infection ability of sCAR1 / CAR3 and mutant sCAR1 Mu / CAR3 Mu at the cellular level.

[0032] Fig. 9 Figure 1 shows the SWISS MODLE structure prediction and expression purification analysis of sCAR1 Mu+Ferritin / CAR3 Mu+Ferritin. (A) Schematic diagram of the construction of the sCAR1 Mu+Ferritin / CAR3 Mu+Ferritin expression plasmid and the SWISS MODLE structure prediction diagram. (B) Coomassie Brilliant Blue staining analysis of the expression supernatant, purified flow-through sample, wash solution and eluate of sCAR1 Mu+Ferritin / CAR3 Mu+Ferritin. (C) Western Blot identification of purified sCAR1 Mu+Ferritin / CAR3 Mu+Ferritin.

[0033] Fig.10 The morphological characterization of sCAR1 Mu+Ferritin / CAR3 Mu+Ferritin. (A) Native PAGE (Native Polyacrylamide Gel Electrophoresis) Coomassie Brilliant Blue staining of sCAR1 Mu+Ferritin / CAR3 Mu+Ferritin. (B) Transmission electron microscopy observation of sCAR1 Mu+Ferritin / CAR3Mu+Ferritin. (C) sCAR1 Mu+Ferritin / CAR3 Mu+FerritinBS 3 Intramolecular dimerization detection after cross-linking.

[0034] Fig.11To detect and compare the anti-CV-B3 infection ability of sCAR1 Mu / CAR3 Mu and sCAR1 Mu+Ferritin / CAR3 Mu+Ferritin at the cellular level.

[0035] Fig.12 The antiviral effect of sCAR1 Mu+Ferritin in mice. (A) The survival rate of mice infected with CV-B3 at different doses. (B) The weight change of mice infected with CV-B3 at different doses of sCAR1 Mu+Ferritin. (C) The survival curve of mice infected with CV-B3 at different doses of sCAR1 Mu+Ferritin. (DK) Comparison of the changes in viral load in various tissues and organs when sCAR1Mu+Ferritin intervened in CV-B3 infection. (L) Inflammatory pathology of heart tissue when sCAR1Mu+Ferritin intervened in CV-B3 infection. (M) Inflammatory pathology of brain tissue when sCAR1Mu+Ferritin intervened in CV-B3 infection.

[0036] Fig.13 The expression and purification of CAR3-6His-PTT5 plasmid. (A) The 6His purification tag is connected to the C-terminus of CAR3 through the 4GS flexible linker, as well as the sequence information of CAR3's own signal peptide. (B) Hydrophobicity analysis of CAR3's own signal peptide. (C) Coomassie brilliant blue staining of the supernatant purification sample collected after CAR3-6His-PTT5 plasmid expression. (D) Western Blot identification of cell lysate and supernatant purification samples collected after CAR3-6His-PTT5 plasmid expression.

[0037] Fig.14 The expression and purification of CAR3 (higher hydrophilic signal peptide replaces self-signal peptide)-10His-PTT5 plasmid. (A) The 10His tag is connected to the C-terminus of CAR3 through the 4GS flexible linker, and the sequence information of the higher hydrophilic signal peptide. (B) Hydrophobicity analysis of the higher hydrophilic signal peptide. (C) Coomassie brilliant blue staining of the collected samples of CAR3 (higher hydrophilic signal peptide replaces self-signal peptide)-10His-PTT5 plasmid expression and purification. (D) Western Blot identification of the collected samples of CAR3 (higher hydrophilic signal peptide replaces self-signal peptide)-10His-PTT5 plasmid expression and purification. DETAILED DESCRIPTION

[0038] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] The sequence information involved in the embodiments of the present invention is as follows:

[0040] Recombinant protein name: sCAR1 Mu+Ferritin

[0041] Coxsackie virus and adenovirus receptor [Mus musculus (house mouse)]: Gene ID: 13052

[0042] Human Ferritin Heavy chain Gene ID: 2495;

[0043] Recombinant expression plasmid: PTT5;

[0044] Modified sequence (sCAR1 Mu+Ferritin):

[0045] MGWSCIILFLVATGVHSLSITTPEQRIEKAKGETAYLPCKFTLSPEDQGPLAIAWLISPSDNQIVDQAIIAYSGDKIYDNAAPDLKGRVHFTSNDVKSGDASIQVTNLQLSDIG TYQCAVKKAPGVANKKFLLTVLVKPSGTRCFVDGSEEIGNDFKLKCEPKEGSLPLQFEWQKLSDSQTMPTPWLAEMTSPVISVKQASSEYSGTYSCTVQNRVGSDQCMLRLDVV PGGGGSGGGGSGGGGSMTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGL NAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNESGGGGSGGGGSGGGGSHHHHHHHHHH, as in SEQ Shown as ID NO.1;

[0046] Extracellular sequence before mutation (sCAR1):

[0047] MARLLCFVLLCGIADFTSGLSITTPEQRIEKAKGETAYLPCKFTLSPEDQGPLDIEWLISPSDNQIVDQVIILYSGDKIYDNYYPDLKGRVHFTSNDVKSGDASINVTNLQLSDIGTY QCKVKKAPGVANKKFLLTVLVKPSGTRCFVDGSEEIGNDFKLKCEPKEGSLPLQFEWQKLSDSQTMPTPWLAEMTSPVISVKNASSEYSGTYSCTVQNRVGSDQCMLRLDVVP, such as SEQ Shown as ID NO.2;

[0048] Signal peptide MARLLCFVLLCGIADFTSG, as shown in SEQ ID NO.3;

[0049] Signal peptide MGWSCIILFLVATGVHS, as shown in SEQ ID NO.4;

[0050] Sequence after signal peptide modification and amino acid point mutation:

[0051] MGWSCIILFLVATGVHSLSITTPEQRIEKAKGETAYLPCKFTLSPEDQGPLAIAWLISPSDNQIVDQAIIAYSGDKIYDNAAPDLKGRVHFTSNDVKSGDASIQVTNLQLSDIGTYQ CAVKKAPGVANKKFLLTVLVKPSGTRCFVDGSEEIGNDFKLKCEPKEGSLPLQFEWQKLSDSQTMPTPWLAEMTSPVISVKQASSEYSGTYSCTVQNRVGSDQCMLRLDVVP, such as SEQ Shown as ID NO.5.

[0052] Amino acid sequence of CAR3 Mu protein:

[0053] MGWSCIILFLVATGVHSLSITTPEQRIEKAKGETAYLPCKFTLSPEDQGPLAIAWLISPSDNQIVDQAIIAYSGDKIYDNAAPDLKGRVHFTSNDVKSGDASIQVTNLQLSDIGTYQCAVKKAPGVANKKFLLTVLGKSSFLLSTGVEWGGGAELQGGREGG, as shown in SEQ ID NO.6.

[0054] Amino acid sequence of CAR3 Mu+Ferritin:

[0055] MGWSCIILFLVATGVHSLSITTPEQRIEKAKGETAYLPCKFTLSPEDQGPLAIAWLISPSDNQIVDQAIIAYSGDKIYDNAAPDLKGRVHFTSNDVKSGDASIQVTNLQLSDIGTYQCAVKKAPGVANKKFLLTVLGKSSFLLSTGVEWGGGAELQGGREGGGGGGSGGGGSGGGGSMTTASTSQVRQNYHQDSE AAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNESGGGGSGGGGSGGGGSHHHHHHHHHH, as in SEQ ID Shown in NO.7.

[0056] Example

[0057] 1. sCAR1 binds to CV-B3 and releases the viral genome to form non-infectious hollow particles

[0058] The binding region of CV-B3 to the CAR receptor is mainly the D1 domain. The plasmid designed in the present invention connects the 10His tag to the C-terminus of sCAR1 and CAR3 through the 4GS flexible linker. Through SWISS MODLE structure prediction, it can be seen that the recombinant sCAR1 and CAR3 can well form their own D1, D2 and D1 domains (Figure 1).

[0059] After transfection and expression in 293T cells, the target protein was purified by nickel column and then subjected to SDS-PAGE and Western Blot. The results showed that clear bands matching the theoretical molecular weights of sCAR1 (28KDa) and CAR3 (18KDa) were obtained in the supernatant (Figure 2A). Subsequently, CAR antibody and His antibody were used to perform Western Blot specificity verification. The experimental results showed that the proteins at these bands were the target proteins recombinantly expressed in the present invention ( Figure 2 B).

[0060] CV-B3 virus particles ( Figure 3A) Immunoprecipitation experiments with sCAR1 / CAR3 showed that sCAR1 / CAR3 and CV-B3 were immunoprecipitated (Figure 3B, C), suggesting that sCAR1 / CAR3 can bind to CV-B3 under neutral conditions at room temperature. Electron microscopy showed that sCAR1 could convert most of the virus solid particles into non-infectious hollow particles (Figure 3D). TaqMan probe qPCR detection results showed that sCAR1 / CAR3 and CAR3 from high concentrations (500μg / ml) to low concentrations (16μg / ml) could significantly induce CV-B3 to release the viral genome into the supernatant (Figure 3E).

[0061] 2. The mutant sCAR1 Mu does not form homodimerization within the molecule, thus avoiding abnormal cell proliferation caused by heterodimerization with cell adhesion molecules

[0062] As a potential molecule for anti-CV-B3 infection, the development of sCAR1 must focus on its safety and efficacy. However, the phenomenon of intramolecular homodimerization after sCAR1 expression may affect their ability to bind to CV-B3 due to certain steric hindrances ( Figure 4 AB), and secondly, the ability of sCAR1 monomer to bind to the cell membrane surface molecules CAR and JAML may bring potential safety risks such as promoting cell proliferation (Figure 4C-D). The present invention found that the binding of CAR to CAR and the binding of CAR to JAML are mainly formed through ionic bonds formed by amino acids D54, E56, and K121, and hydrogen bonds formed by V70, L73, Y83, and Y84, while the binding of CAR to CV-B3 is mainly formed through hydrogen bonds and ionic bonds formed by T24, E26, T44, and Q50 with VP1, VP2, and VP3 of the virus particles ( Figure 5 sCAR1 / CAR3 was modified into mutants, that is, the amino acid residues on the CAR molecule that are involved in the strong interaction with CV-B were retained, while the amino acid sites that are involved in the strong interaction with CAR itself and JAML were subjected to nonsense mutations (the amino acids involved in ionic bonds and hydrogen bonds were mutated to alanine).

[0063] The present invention constructed mutant sCAR1 Mu (sCAR1 Mutation) / CAR3 Mu (CAR3 Mutation) for plasmid construction, expression purification and specificity verification. These results firstly showed that the introduction of mutations at 9 amino acid sites in sCAR1 and CAR3 would not affect their normal expression and purification (Fig. 6A, B).

[0064] Will the introduction of nonsense mutations at the 9 amino acid sites affect the dimerization of the recombinant protein? In previous experiments, it has been determined that the dimerization of the recombinant protein is formed by ionic bonds, hydrogen bonds and other forces. Therefore, the present invention directly detects BS 3 Dimerization of mutant sCAR1 Mu and CAR3 Mu under the action of cross-linking agent. The bands in the experimental results only appear at the theoretical molecular weight of each monomer, that is, mutant sCAR1 Mu and CAR3 Mu do not form homodimers ( Figure 7 A), but both exist in monomeric form. Further observation of whether sCAR1 Mu can bind to the cell membrane surface molecules CAR and JAML showed that sCAR1 Mu (28KDa) did not undergo immunoprecipitation with CAR (46KDa) and JMAL (54KDa) (Figure 7B), suggesting that the introduction of nonsense mutations at 9 amino acid sites eliminated the binding of sCAR1 Mu to the cell membrane surface molecules CAR and JMAL. The results of the CCK8 experiment showed that the addition of low concentrations (25μg / ml) to high concentrations (400μg / ml) of sCAR1 Mu did not show cytotoxicity to Vero cells and SH-SY5Y cells, and did not show significant effects on cell proliferation (Figure 7C, D).

[0065] Ferritin particles display anti-CV-B3 activity of sCAR1 Mu

[0066] The mutants sCAR1 Mu and CAR3 Mu obtained by the present invention through the previous point mutation experiment have been improved in both safety and efficacy ( Figure 8 AH), which strengthens the confidence of the present invention in further studying its anti-CV-B3 infection related research. In order to further improve its effectiveness and take into account the subsequent evaluation at the animal level, the present invention intends to use Ferritin, a nanoparticle with high safety, strong display and certain delivery ability, to display sCAR1 Mu and CAR3Mu on its surface; thus, the present invention constructs sCAR1 Mu / CAR3 Mu—4GS Linker—Human Ferritin Heavy Chain—10His plasmid ( Fig. 9 A), transfection eukaryotic expression and purification, and anti-CV-B3 infection related research and evaluation ( Fig. 9 B, C).

[0067] Since Ferritin can be assembled into 24 subunit nanoparticles (theoretical molecular weight is 450KDa), the present invention also conducted Native PAGE experiments on sCAR1 Mu+Ferritin and CAR3 Mu+Ferritin. The Coomassie Brilliant Blue results showed that the bands that appeared were roughly equivalent to the theoretical molecular weights of sCAR1 Mu+Ferritin (1650KDa) and CAR3 Mu+Ferritin (1410KDa). Fig.10 A), indicating that the fused recombinant protein of the present invention can also be assembled into nanoparticles. Under a transmission electron microscope, the present invention can observe uniform particles with a diameter of about 15 nm, and the surface of the particles has burr-like protrusions showing sCAR1 Mu and CAR3 Mu on the surface of ferritin nanoparticles (Figure 10B). In addition, the present invention also uses BS 3 Cross-linking experiments found that sCAR1 Mu+Ferritin and CAR3 Mu+Ferritin did not form dimerization ( Fig.10 C).

[0068] The results of TaqMan probe qPCR detection of viral load showed that the IC50 of sCAR1 Mu+Ferritin in Vero cells and SH-SY5Y cells to block CV-B3 infection was 413.05 ng / ml and 140.93 ng / ml, respectively (Figure 11A-H). Compared with monomeric sCAR1 Mu, the IC50 of sCAR1 Mu+Ferritin in Vero cells and SH-SY5Y cells was 22.3 times and 26.2 times higher than that of sCAR1 Mu, respectively.

[0069] 4. sCAR1 Mu+Ferritin can effectively alleviate the outcome of mice and tissue inflammatory pathology after CV-B3 infection

[0070] The sCAR1Mu+Ferritin was injected intraperitoneally 6 hours in advance, followed by an intraperitoneal injection of a 100% lethal dose of CV-B3 (10 3.5CCID50), first observed the weight change and survival rate (Figure 12A, B). The present invention found that the weight change of the high-dose injection group (200μg) was comparable to that of the NC group, and the weight continued to rise; the weight of the medium-dose group (100μg) rose slowly, while the low-dose group (50μg) and the Blank group showed a continuous decrease in weight since the intraperitoneal injection of the virus. In the survival rate statistics, the survival rates of the high, medium and low-dose groups were 68.75%, 18.75% and 0%, respectively. Judging from the weight and survival rate data, high and medium doses of sCAR1 Mu+Ferritin had a certain protective effect when intervening in mice infected with a lethal dose of CV-B3, while the low-dose group did not show a protective effect. In terms of pathological evaluation, the high-dose group can effectively alleviate the inflammatory pathology in the heart and brain tissues (Figure 12A-M).

[0071] 5. Exploration of expression and purification conditions of CAR receptor analogs

[0072] In order to more effectively express and purify CAR receptor analogs in the future, the present invention uses CAR3-PTT5 plasmid to explore relevant expression and purification conditions, such as Fig.13 As shown in AB, the C-terminus of the CAR3 full sequence (including its own signal peptide) was first connected to the 6His purification tag through the 4GS flexible linker and loaded onto the PTT5 vector for expression and purification. The present invention found that the expressed target protein CAR3 ( Fig.13 D), but the purification efficiency is extremely low ( Fig.13 C, D).

[0073] Since the purification effect was not ideal when the 6His purification tag was added, in order to improve the binding ability of the expressed target protein to the nickel column, the 6His tag was replaced with a 10His tag ( Fig.14 A). At the same time, the present invention found that when the expressed target protein carries its own signal peptide, the target protein is distributed both in the cell and in the supernatant, and the target protein in the cell needs to be released and extracted through complicated processes such as non-denaturing lysis and ultrasonic fragmentation. In order to simplify the extraction process of the target protein and improve its purification efficiency, the present invention replaces the target protein's own signal peptide (MARLLCFVLLCGIADFTSG, as shown in SEQ ID NO.3, hydrophilicity evaluation value: 1.563) with a more hydrophilic signal peptide (MGWSCIILFLVATGVHS, as shown in SEQ ID NO.4, hydrophilicity evaluation value: 1.576) ( Fig.14A, B), and then expression purification was performed. At the same time, gradient concentrations of imidazole elution were performed during purification (E1: 50mM Imidazole, E2: 100mM Imidazole, E3: 150mM Imidazole, E4: 250mM Imidazole, E5: 300mM Imidazole, E6: 350mM Imidazole, E7: 400mM Imidazole, E8: 450mM Imidazole) to find the most efficient imidazole concentration for eluting the target protein. Purification results ( Fig.14 C) It shows that the 10His tag can significantly improve the purification efficiency. When the imidazole gradient concentration elution is performed, it is found that when the imidazole concentration in the elution solution reaches 250mM, the target protein can be effectively eluted; in addition, the present invention finds that the target protein with a more hydrophilic signal peptide can basically be secreted into the cell supernatant, while the content in the cell is relatively small, and the yield after purification is extremely low. ( Fig.14 C, 14D) Therefore, combined with the conditions of the above experiments, 10His can be selected as the purification tag in the subsequent expression of CAR receptor analogs; the self-signal peptide is replaced with the more hydrophilic MGWSCIILFLVATGVHS signal peptide (as shown in SEQ ID NO.4) so ​​that most of the target protein is secreted into the cell supernatant. At the same time, when eluting the target protein, an elution solution with an imidazole concentration of 250mM can be directly selected.

[0074] It can be seen from the above embodiments that the nanoparticles provided by the present invention have the following advantages: 1. Highly effective antiviral activity, which can significantly reduce the intracellular viral load. 2. Good safety, non-toxic to cells, and does not affect cell proliferation. 3. The nanoparticles are highly stable and easy to store and transport. 4. The display efficiency and delivery capacity of recombinant proteins are improved through the Ferritin display system. 5. It is suitable for a variety of cell types, including Vero cells and SH-SY5Y cells, and shows a wide antiviral spectrum. 6. It shows a protective effect in animal models and can effectively alleviate pathological changes caused by viral infection.

[0075] In summary, the sCAR1 Mu+Ferritin nanoparticles of the present invention not only exhibited strong anti-CV-B3 activity in in vitro experiments, but also showed good protective effects in animal experiments, providing new strategies and tools for antiviral treatment.

[0076] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A modified mutant sCAR1 Mu protein, characterized in that: The sCAR1 Mu protein is obtained by modification based on the sCAR1 protein whose amino acid sequence is shown in SEQ ID NO.2; The amino acid sequence of the modified mutant sCAR1 Mu protein is shown in SEQ ID NO.

5.

2. A mutant nanoparticle, characterized in that: Ferritin nanoparticles are used as a matrix, and the mutant sCAR1 Mu protein described in claim 1 is displayed on its surface.

3. A mutant nanoparticle, characterized in that: The amino acid sequence of the mutant nanoparticle is shown in SEQ ID NO.

1.

4. A recombinant expression vector for expressing the mutant nanoparticles according to claim 3, characterized in that: The basic vector used by the recombinant expression vector is PTT5.

5. Use of the modified mutant sCAR1 Mu protein according to claim 1, the mutant nanoparticle according to claim 2 or 3, or the recombinant expression vector according to claim 4 in the preparation of drugs against CV-B3 virus.

6. A modified mutant CAR3 Mu protein, characterized in that: The modified mutant CAR3 Mu protein is obtained by modification based on the CAR protein; The amino acid sequence of the modified mutant CAR3 Mu protein is shown in SEQ ID NO.

6.

7. A mutant nanoparticle, characterized in that: Ferritin nanoparticles are used as a matrix, and the mutant CAR3 Mu protein described in claim 6 is displayed on its surface.

8. A mutant nanoparticle, characterized in that: The amino acid sequence of the mutant nanoparticle is shown in SEQ ID NO.

7.

9. Use of the modified mutant CAR3 Mu protein according to claim 6 or the mutant nanoparticles according to claim 7 or 8 in the preparation of drugs against CV-B3 virus.

Images