Silica-based hiv virus mper polypeptide nanovaccine and preparation method and application thereof

Abstract

The application discloses an HIV virus MPER polypeptide nanovaccine based on a silicon dioxide carrier and a preparation method and application thereof. The application uses special silicon dioxide nanoparticles as a carrier, and based on the atomic crystal characteristics (including a large specific surface area and certain thermal stability) of the silicon dioxide nanoparticles, links the MPER polypeptide to the surface of the silicon dioxide nanoparticles, reduces the oligomerization of the polypeptide, and improves the effective antigen immunity. In order to further realize the enrichment of the antigen, the MPER polypeptide is treated in a tetra-limb peptide mode in the application, so that a more stable system is formed after the coupling of the MPER polypeptide and the nanoparticles. Based on the stable polypeptide structure and the chemical properties, the MPER polypeptide has better rigidity and lower variability, guarantees the antigen quality, and improves the antigen activity. The nanovaccine obtained finally can effectively enter the body, induces high-titer neutralizing antibodies, and has great significance for controlling the HIV-1 transmission.

Description

Technical Field

[0001] This invention relates to the field of nanovaccine technology, and in particular to HIV MPER peptide nanovaccine based on silica carrier, its preparation method and application. Background Technology

[0002] HIV-1 causes an immune system deficiency disorder and continues to spread in all countries worldwide. Currently, there are no suitable prevention or treatment methods, making it a significant challenge to global public health. The highly variable nucleic acid sequence of HIV-1 leads to frequent mutations in its expressed proteins, and these mutations occur faster than vaccine development. Furthermore, there is currently no suitable antigen to induce broad-spectrum HIV-neutralizing antibodies; therefore, there are no commercially available antibodies that can effectively inhibit HIV-1. Current treatments for HIV-1 mainly include gene therapy and antiretroviral drugs, but these methods have limited efficacy. Therefore, antiretroviral therapy for HIV remains a historical challenge in scientific research.

[0003] Furthermore, the high variability and drug resistance of HIV are among the major obstacles to current HIV drugs. Most current research focuses only on short HIV peptides because their gene sequences are relatively short, resulting in a relatively low probability of mutation and thus less susceptibility to high variability.

[0004] The HIV env gene encodes an envelope protein. gp160 is a precursor to the HIV envelope glycoprotein, which is cleaved by proteases into the outer membrane protein gp120 and the transmembrane protein gp41. The HIV outer membrane protein gp120 and the transmembrane protein gp41 nonvalently bind to form the gp160 protein. Within the gp41 proximal outer region is a relatively conserved sequence called the MPER region, which is short, containing only about 22 amino acid residues. Its main function is to promote membrane fusion, thereby facilitating viral entry into host cells and achieving viral infection. However, the expression and purification of the MPER region protein is relatively difficult, making effective research difficult with current techniques. Furthermore, its tendency to oligomerize due to membrane proteins limits its usability in immunological studies, resulting in poor immunization efficacy and hindering its effective application. Some researchers have attempted to use peptide synthesis technology to synthesize the MPER sequence of gp41 for animal immunization experiments, thereby improving the antigen yield. However, due to the inherent variability of HIV, the MPER sequence cut from the solid-phase resin still exhibits oligodendrical characteristics, making it difficult to obtain effective immunization.

[0005] Therefore, overcoming the problems in the existing technologies mentioned above and obtaining a broad-spectrum HIV vaccine that does not suffer from antigen oligopoly and thus affect immune activity is a critical challenge that urgently needs to be addressed. Summary of the Invention

[0006] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the objective of this invention is to provide an HIV MPER peptide vaccine based on a silica carrier, its preparation method, and its application. This invention utilizes special silica nanoparticles as a carrier, leveraging their atomic crystal properties (including a large specific surface area and certain thermal stability) to link MPER peptides to their surface, reducing peptide oligomerization and improving effective antigenic immunogenicity. To further enrich the antigen, this invention treats the MPER peptides using a tetrapeptide method, thereby forming a more stable system after coupling with nanoparticles. Based on the stable peptide structure and its chemical properties, it exhibits better rigidity and reduced variability, ensuring antigen quality and improving antigen activity. The resulting nanoantigen can effectively enter the body, inducing high-titer neutralizing antibodies, which is of great significance for controlling the spread of HIV-1.

[0007] In a first aspect, the present invention provides an HIV artificial antigen comprising: HIV virus MPER or an active fragment thereof loaded on a silicon-based vector.

[0008] In this invention, "comprising," "containing," "having," or "including" means that at least the specified substance, component, element, or method step is present in the product, article, or method, but does not exclude the presence of other substances, components, elements, or method steps, even if the other such substances, components, elements, or method steps have the same function as the specified ones.

[0009] In this invention, the term "active fragment" refers to a truncated fragment that has the same function as the original sequence. In this invention, the length and position of the truncation are not limited.

[0010] In this invention, the term "same function" means that the difference from the original sequence in at least one function does not exceed 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In this invention, the function refers to immunogenicity against HIV.

[0011] In some embodiments of the present invention, the HIV virus MPER has a structure as shown in Formula I:

[0012]

[0013] In some embodiments of the present invention, the particle size of the HIV artificial antigen is less than 200 nm.

[0014] In some embodiments of the present invention, the particle size of the HIV artificial antigen is less than 180 nm, 170 nm or 165 nm.

[0015] In some embodiments of the present invention, the branch ends of the HIV virus MPER or its active fragment are connected to the silicon-based carrier.

[0016] In this invention, the term "branch end" refers to the branch end of a branched peptide, which consists of a main chain and branch chains.

[0017] In some embodiments of the present invention, the main chain end of the HIV virus MPER or its active fragment is not connected to the silicon-based carrier.

[0018] In some embodiments of the present invention, at least one branch end of the HIV virus MPER or its active fragment is connected to the silicon-based carrier.

[0019] In some embodiments of the present invention, 2-4 branch ends of the HIV virus MPER or its active fragment are connected to the silicon-based carrier.

[0020] In some embodiments of the present invention, all four branches of the HIV virus MPER or its active fragment are connected to the silicon-based carrier.

[0021] In some embodiments of the present invention, the silicon-based carrier is linked to at least one HIV virus MPER or an active fragment thereof.

[0022] In some embodiments of the present invention, the silicon-based carrier is linked to at least two HIV virus MPERs or their active fragments.

[0023] In some embodiments of the present invention, the silicon-based carrier includes amorphous silicon dioxide, surface-aminated silicon dioxide, and surface-carboxylated silicon dioxide.

[0024] In some embodiments of the present invention, the silicon dioxide is silicon dioxide nanoparticles.

[0025] Silica nanocarriers have been widely used in biomedicine, tissue engineering, chemical engineering and other fields. They are a relatively common and mature carrier. Therefore, using silica nanoparticles to couple peptides to form nanoantigens is a feasible vaccine delivery system.

[0026] In some embodiments of the present invention, the surface of the silica nanoparticles is modified with carboxyl groups.

[0027] In some embodiments of the present invention, the method for preparing the HIV virus MPER or its active fragment is not limited, and may include, but is not limited to, conventional peptide preparation methods in the art such as solid-phase synthesis and biosynthesis.

[0028] In some embodiments of the present invention, the HIV virus MPER or its active fragment is obtained by solid-phase synthesis.

[0029] In some embodiments of the present invention, the HIV virus MPER or its active fragment may further contain chemically modified or modified sequences to further enhance its function in at least one aspect, wherein the function includes, but is not limited to: stability, immunogenicity, binding, specificity, resistance, etc.

[0030] A second aspect of the present invention provides a method for preparing the HIV artificial antigen described above, comprising the following steps:

[0031] Amino-modified silica nanoparticles were prepared, and the surface of the amino-modified silica nanoparticles was modified by carboxylation. HIV virus MPER or its active fragment was coupled onto the carboxylated silica nanoparticles to obtain HIV artificial antigen.

[0032] In some embodiments of the present invention, the amino-modified silica nanoparticles are prepared by catalytic hydrolysis reaction.

[0033] In some embodiments of the present invention, the modified amino silica nanoparticles are prepared by catalytic hydrolysis of orthosilicate and 3-aminopropyltrimethoxysilane.

[0034] In some embodiments of the present invention, the volume ratio of the orthosilicate to 3-aminopropyltrimethoxysilane is 3-5:1.

[0035] In some embodiments of the present invention, the volume ratio of the orthosilicate to 3-aminopropyltrimethoxysilane is 4:1.

[0036] In some embodiments of the present invention, the catalytic hydrolysis reaction specifically comprises:

[0037] Mixture A and mixture B are combined and refluxed, centrifuged, and the supernatant is discarded. After washing, ammonia is added and refluxed again to obtain amino-modified silica nanoparticles. Mixture A includes orthosilicate, 3-aminopropyltrimethoxysilane, and ethanol; mixture B includes ammonia, ethanol, and water.

[0038] In some embodiments of the present invention, the reflux temperature is 90-110°C.

[0039] In some embodiments of the present invention, the carboxylation modification includes: using succinic anhydride to carboxylate modified amino-modified silica nanoparticles.

[0040] In some embodiments of the present invention, the carboxylation modification specifically involves reacting amino-modified silica nanoparticles and succinic anhydride in a solvent to obtain carboxylated silica nanoparticles.

[0041] In some embodiments of the present invention, the carboxylation modification reaction is carried out under protection, wherein the protection is provided by an inert gas.

[0042] In some embodiments of the present invention, the inert gas includes nitrogen.

[0043] In some embodiments of the present invention, the solvent includes DMF.

[0044] In some embodiments of the present invention, the carboxylation modification further includes steps such as centrifugation, washing, and drying after the reaction is completed.

[0045] In some embodiments of the present invention, the HIV virus MPER or its active fragment is obtained by solid-phase synthesis.

[0046] In some embodiments of the present invention, the carrier for solid-phase synthesis comprises a resin, wherein the resin comprises royal jelly resin.

[0047] In some embodiments of the present invention, after solid-phase synthesis, HIV virus MPER or its active fragments are stripped from the vector.

[0048] In some embodiments of the present invention, the stripping is performed using a peptide cutting fluid.

[0049] In some embodiments of the present invention, the polypeptide cleavage solution includes trifluoroacetic acid.

[0050] In some embodiments of the present invention, the trifluoroacetic acid content (wt%) in the polypeptide cleavage solution is greater than or equal to 90%.

[0051] In some embodiments of the present invention, the polypeptide cutting fluid further includes water, anisole, phenol, and ethylenedithiol.

[0052] In some embodiments of the present invention, the volume ratio of trifluoroacetic acid, water, anisole, phenol and ethylenedithiol is 9:0.3:0.3:0.3:0.1.

[0053] In some embodiments of the present invention, the stripping method includes: mixing and reacting solid-phase synthesized HIV virus MPER or its active fragment with a peptide cleavage solution, and then removing the carrier precipitate to obtain HIV virus MPER or its active fragment.

[0054] In some embodiments of the present invention, the stripping method further includes: drying, reconstituted and centrifuging the HIV virus MPER or its active fragments.

[0055] In some embodiments of the present invention, the stripping method specifically involves: mixing and reacting solid-phase synthesized HIV virus MPER or its active fragment with peptide cleavage solution under ice bath conditions, removing the carrier precipitate, drying, adding ice-cold ether, and centrifuging to obtain HIV virus MPER or its active fragment precipitate.

[0056] In some embodiments of the present invention, the coupling is carried out by a condensation reaction.

[0057] In some embodiments of the present invention, after coupling is completed, cleaning is performed using DMF and DCM.

[0058] A third aspect of the present invention provides the use of the HIV artificial antigen described above in the preparation of medicaments for the prevention and / or treatment of HIV infection.

[0059] In some embodiments of the present invention, the medicament further includes pharmaceutically acceptable excipients.

[0060] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of the following: antioxidants, preservatives, coating materials, solvents, lyophilization protectants, or carriers.

[0061] In this invention, the pharmaceutically acceptable excipients used do not affect the functionality of the HIV artificial antigen itself.

[0062] In some embodiments of the present invention, the drug includes at least one of a vaccine and an antibody.

[0063] In some embodiments of the present invention, the medicament further includes a second active substance.

[0064] In some embodiments of the present invention, the selected second active substance does not affect the functionality of the HIV artificial antigen itself, and has an additive or synergistic effect on the HIV artificial antigen.

[0065] In some embodiments of the present invention, the second active substance is an anti-HIV infection drug or an adjunct to HIV treatment.

[0066] In some embodiments of the present invention, the anti-HIV infection drug includes at least one of nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), integrase inhibitors (INSTIs), fusion inhibitors (FIs), and CCR5 antagonists.

[0067] In some embodiments of the present invention, the nucleoside reverse transcriptase inhibitor (NRTI) includes, but is not limited to, zidovudine (AZT), lamivudine (3TC), tenofovir (TDF), and emtricitabine (FTC).

[0068] In some embodiments of the present invention, the non-nucleoside reverse transcriptase inhibitor (NNRTI) includes, but is not limited to, efavirenz (EFV), nevirapine (NVP), and rilpivirine (RPV).

[0069] In some embodiments of the present invention, the protease inhibitor (PI) includes, but is not limited to, lopinavir (LPV), ritonavir (RTV), and darunavir (DRV).

[0070] In some embodiments of the present invention, the integrase inhibitor (INSTI) includes, but is not limited to, raltelapvir (RAL), dolutegravir (DTG), and bicitlapvir (BIC).

[0071] In some embodiments of the present invention, the fusion inhibitor (FI) includes, but is not limited to, enfuvirtide (T-20) and albuvirtide.

[0072] In some embodiments of the present invention, the CCR5 antagonist includes, but is not limited to, malaviro (MVC).

[0073] In some embodiments of the present invention, the HIV treatment adjuvant includes: an synergist, or a reagent for increasing compliance or reducing HIV treatment side effects.

[0074] In some embodiments of the present invention, the synergist includes, but is not limited to, ritonavir.

[0075] A third aspect of the present invention provides the use of the HIV artificial antigen described above in the preparation of HIV diagnostic products.

[0076] In some embodiments of the present invention, the HIV diagnostic product includes at least one of diagnostic reagents, diagnostic kits, detection chips, and test strips.

[0077] In some embodiments of the present invention, the HIV diagnostic product can be used to diagnose HIV based on, but not limited to, the following methods: enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, chemiluminescence immunoassay, time-resolved fluorescence immunoassay, agglutination reaction, immunodiffusion, colloidal gold immunochromatography, immunosensors, etc.

[0078] The beneficial effects of this invention are:

[0079] This invention successfully prepared an artificial antigen based on HIV MPER, which avoids the oligomerization problem of MPER peptides in existing technologies, thereby improving its immunogenicity. Furthermore, the obtained artificial antigen has no significant toxicity to cells and animals, exhibiting good biosafety and biocompatibility, and can be used in practical clinical medicine.

[0080] The method for preparing artificial antigens based on HIV virus MPER in this invention involves coupling MPER tetrapeptides to the surface of specially treated silica nanoparticles, thereby enhancing the rigidity of MPER and preventing peptide interactions that could lead to a decrease in antigen potency. Furthermore, this nano-silica-based carrier enables efficient antigen delivery, making it easier for cells to absorb, reducing the amount of unsuitable vaccines used, and promoting the absorption of antigen drugs. Attached Figure Description

[0081] Figure 1 The liquid chromatogram (A) and mass spectrum (B) of the MPER tetrapeptide are shown.

[0082] Figure 2 This is a schematic diagram of the structure of SiO2-COOH@MPER.

[0083] Figure 3 The particle size distribution of SiO2-COOH@MPER is shown in Figure A, and its potential is compared with that of intermediates such as SiO2-NH2 and SiO2-COOH in Figure B.

[0084] Figure 4 Electron micrographs of SiO2-COOH and SiO2-COOH@MPER.

[0085] Figure 5 The plasmid map for the GST@MPER expression plasmid.

[0086] Figure 6 The liquid chromatogram (A), electrophoresis (B), and mass spectrum (C) of the corresponding fraction of GST@MPER protein are shown.

[0087] Figure 7 The results of toxicity experiments on different concentrations of SiO2-COOH@MPER on fibroblast L929 (A) and monkey kidney embryonic cells MA104 (B) are presented.

[0088] Figure 8 The results are from the drug hemolysis experiment of SiO2-COOH@MPER.

[0089] Figure 9 This is a flowchart of an animal immunization experiment.

[0090] Figure 10 The images show changes in mouse body weight (A), changes in serum total IgG levels (B), serum antibody specificity (C), and organ H&E staining (D) in an animal immunization experiment. Detailed Implementation

[0091] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0092] Example 1

[0093] This embodiment provides a method for synthesizing tetrapod peptides as shown in Formula I.

[0094] The synthetic route is as follows:

[0095]

[0096] The specific steps are as follows:

[0097] (1) Weigh 0.025 mmol of royal jewel resin and add it to 5 mL of dichloromethane (DCM) for activation overnight. Drain the dichloromethane and wash the royal jewel resin 5 times with 8 mL of N,N-dimethylformamide (DMF).

[0098] (2) Dissolve 10 molar amounts of each of the amino acids, HATU and HOBt in 3 mL of DMF, then add them to a solid-phase reactor, introduce N2 and stir, and react at 60 °C for 5 min. Then add 2 mL of DMF solution containing 12.5% ​​(v / v) DIEA and continue the reaction for 55 min to complete the condensation reaction.

[0099] (3) After the condensation reaction is complete, drain the liquid and wash with 8 mL of DMF 5 times.

[0100] (4) First Fmoc protection removal: Add 8 mL of DMF solution containing 20% ​​(v / v) piperidine, react for 5 min, and drain the liquid.

[0101] (5) Second Fmoc protection removal: Add 8 mL of DMF solution containing 20% ​​(v / v) piperidine again, react for 20 min, and drain the liquid.

[0102] (6) Wash five times with 8 mL of DMF solution. Then repeat steps (2)-(6) to complete the synthesis of the amino acid sequence shown in Formula I.

[0103] (7) After the synthesis of the last amino acid is completed, wash with 8 mL DMF 10 times, and then wash with 8 mL DCM 10 times to obtain the resin immobilized with the tetrapod peptide shown in Formula I. Vacuum dry and store the resin immobilized with the tetrapod peptide shown in Formula I at -20℃.

[0104] Tetrapod peptides were detected using HPLC and mass spectrometry, and the results are as follows: Figure 1 As shown.

[0105] As can be seen from the spectrum, the tetrapod peptide synthesized in this embodiment has high purity. Further mass spectrometry analysis revealed that the synthesized tetrapod peptide, after mass spectrometry electrospray ionization, showed a single polypeptide chain (due to the easy breakage of the polylysine end during electrospray ionization) with a molecular weight of 2912.231, while the theoretical value of a single MPER polypeptide chain is 2912.3. This proves that the target MPER was successfully synthesized in this embodiment with high purity, which can meet the requirements of subsequent experiments.

[0106] Example 2

[0107] This embodiment provides a method for preparing SiO2-NH2, the specific steps of which are as follows:

[0108] (1) Prepare the premixed solution:

[0109] Mixture A: Mix 10 mL of orthosilicate, 2.5 mL of 3-aminopropyltrimethoxysilane and 95.5 mL of anhydrous ethanol until homogeneous to obtain mixture A.

[0110] Mixture B: Mix 6 mL of ammonia water, 32.5 mL of anhydrous ethanol and 49.5 mL of water until homogeneous to obtain mixture B.

[0111] (2) Place mixture B in a round-bottom flask and stir magnetically at a stirring speed of 1000 rpm. Then quickly add mixture A and reflux at 100°C for 3 hours.

[0112] (3) After reflux for 3 hours, add 3 mL of ammonia water to the round-bottom flask and reflux at 100 °C overnight. After reflux, centrifuge at 8000 rpm for 5 minutes at room temperature, discard the supernatant, wash the precipitate three times with anhydrous ethanol, and dry under vacuum to obtain SiO2-NH2.

[0113] The SiO2-NH2 obtained in this example was resuspended in anhydrous ethanol, and its particle size and potential were measured after ultrasonic dispersion for 30 min.

[0114] Example 3

[0115] This embodiment provides a method for preparing SiO2-COOH, the specific steps of which are as follows:

[0116] (1) Dissolve 200 mg of SiO2-NH2 prepared in the above example and 27.968 g of succinic anhydride in 100 mL of DMF and react under N2 protection with magnetic stirring at 600 rpm for 5 h.

[0117] (2) After the reaction is complete, centrifuge at 8000 rpm for 5 min at room temperature, discard the supernatant, wash the precipitate 3 times with anhydrous ethanol, and dry it under vacuum to obtain SiO2-COOH.

[0118] Example 4

[0119] This embodiment provides a method for preparing an HIV MPER peptide vaccine based on a silica carrier, the specific steps of which are as follows:

[0120] The king resin with tetrapod peptides of Formula I, obtained in Example 1 above, was condensed with 7 mg of SiO2-COOH prepared in Example 3 at 60 °C for 6 h. After the reaction was complete, the mixture was washed 10 times with 8 mL of DMF and then 10 times with 8 mL of DCM. After vacuum drying, king resin@MPER@SiO2-COOH was obtained.

[0121] The resin in the resin@MPER@SiO2-COOH was removed using conventional methods in the art. The solution was dialyzed with guanidine hydrochloride solution for 24 hours, and then dialyzed with water to replace the guanidine hydrochloride. The solution was then freeze-dried to obtain purified SiO2-COOH@MPER powder.

[0122] Among them, a peptide cutting fluid was used to remove the resin. The peptide cutting fluid was composed of trifluoroacetic acid, water, benzyl sulfide, phenol, and ethylenedithiol in a volume ratio of 9:0.3:0.3:0.3:0.1.

[0123] The specific exfoliation method is as follows: Wang resin@MPER@SiO2-COOH was placed in a round-bottom flask, and 6 mL of peptide cleavage solution was added under ice bath conditions. The reaction was allowed to proceed for 2 hours. The liquid was then poured into a solid-phase reactor, and the round-bottom flask was washed three times with 2 mL of trifluoroacetic acid. The washings were also combined with the washings in the solid-phase reactor. The liquid in the solid-phase reactor was dried with N2 to obtain a viscous SiO2-COOH@MPER. 20 mL of ice-cold diethyl ether was added to the viscous SiO2-COOH@MPER, and the mixture was centrifuged at 8000 rpm for 15 minutes at 4°C. The supernatant was discarded, and the SiO2-COOH@MPER precipitate was obtained. This precipitate was washed three times with 20 mL of ice-cold diethyl ether, and finally dried under vacuum to obtain SiO2-COOH@MPER powder.

[0124] The purified SiO2-COOH@MPER powder and the SiO2-COOH prepared in Example 3 were resuspended in anhydrous ethanol, and their particle size and potential were measured after ultrasonic dispersion for 30 min. Simultaneously, the morphology of the purified SiO2-COOH@MPER and the SiO2-COOH prepared in Example 3 were observed using transmission electron microscopy.

[0125] The structural diagram of SiO2-COOH@MPER is shown below. Figure 2 As shown.

[0126] The results are as follows Figure 3 and Figure 4 As shown.

[0127] It can be observed that the unloaded SiO2-COOH nanoparticles are smooth, while those loaded with peptides become rougher. Further potentiometric analysis reveals a significant change in the potential of the SiO2-COOH nanoparticles after peptide loading, shifting from negative to positive, further confirming the successful coupling of the peptides to the negatively charged nanocarrier. Particle size measurement of the SiO2-COOH@MPER nanoparticles successfully loaded with peptides on carboxylated silica surfaces revealed an average particle size of 160.1 nm, meeting the standard requirements for nanoantigen size. Therefore, this invention successfully yields a SiO2-COOH@MPER nanoantigen with suitable size, suitable for immunological research, and is expected to increase immunogen absorption, achieve efficient drug delivery, provide a stable nanoenvironment, and reduce antigen degradation.

[0128] Example 5

[0129] This embodiment provides a method for preparing a fusion protein containing HIV virus MPER, the specific steps of which are as follows:

[0130] The GST@MPER expression plasmid (hereinafter referred to as pGEX-4T-1-MPER, plasmid map shown) was constructed based on the commercially available pGEX-4T-1 vector. Figure 5 As shown, the sequence of the inserted fragment is the encoded sequence of the sequence shown in SEQ ID NO:1, ELLELDKWASLWNWFNITNWLWYIKIF(SEQ ID NO:1)).

[0131] The constructed pGEX-4T-1-MPER (dry powder form) was dissolved in 20 μL of sterile ultrapure water. Then, 1 μL of the plasmid solution was transferred into 50 μL of DH5α competent cells and incubated on ice for 30 min. Immediately afterwards, the temperature was raised to 42℃ and incubated for 1 min. It was then incubated on ice again for 1 min to complete the cold-heat shock cission. 200 μL of SOC medium was added, and the cells were incubated at 37℃ on a shaker for 30 min. The bacterial culture was then spread onto solid LB agar plates containing 0.1 mg / mL ampicillin, and the plates were incubated overnight at 37℃ in a floating incubator.

[0132] After incubation, single clones were picked and placed in centrifuge tubes containing 10 mL of LB liquid medium containing ampicillin, and incubated overnight at 37°C in a shaker. Plasmids were then extracted from the cultured clones using a commercially available plasmid extraction kit.

[0133] Take 1 μL of the extracted plasmid and transfer it into 50 μL of BL21(DE3) competent cells. Incubate on ice for 30 min, then immediately incubate at 42°C for 1 min. Incubate again on ice for 1 min to complete the cold-heat shock cistransfer. Add 200 μL of SOC medium and incubate at 37°C on a shaker for 30 min. Then, take the bacterial culture and spread it onto a solid LB agar plate containing 0.1 mg / mL ampicillin. Finally, incubate the plate overnight at 37°C in a floating incubator. After incubation, pick single colonies and place them in centrifuge tubes containing 10 mL of LB liquid medium containing ampicillin. Incubate overnight at 37°C on a shaker. The next day, transfer them to 1 L shake flasks containing ampicillin and expand the culture at 37°C. When the OD600 reaches 0.6, add 0.15 mL of 1 M IPTG to induce expression, and then incubate overnight at 16°C on a shaker.

[0134] After culturing, the bacterial culture was centrifuged at 4200 rpm for 1 h at 4°C. The supernatant was discarded, and the bacterial pellet was retained. The pellet was resuspended in 20 mL of PBS, and 100 mg / mL lysozyme stock solution (purchased from Beijing Solarbio Science & Technology Co., Ltd.) was added to bring the final concentration to 1 mg / mL. The mixture was incubated on ice for 30 min, and then sonicated for 15 min using a cell disruptor. The sonicated bacterial culture was centrifuged at 13000 rpm for 1 h at 4°C. The supernatant was retained and filtered through a 0.45 μm needle filter. The filtered supernatant was bound to GST-tagged purification resin on a 4°C rotating plate for 2 h, and then transferred to a chromatography column for purification. After the bacterial supernatant was passed through the chromatography column, the column was washed 5 times with 10 volumes of 50 mM Tris-HCl pH 8.0 buffer containing 150 mM NaCl to remove impurities. The resin was then washed 10 times with 10 volumes of PBS buffer. The column was then equilibrated 3 times with 50 mM Tris-HCl pH 8.0. Finally, the protein was eluted with 10 mM reduced glutathione prepared with 50 mM Tris-HCl to obtain the GST@MPER fusion protein eluent. The GST@MPER fusion protein eluent was concentrated and purified by SEC-HPLC, and the fraction was collected. The fraction marked with * (see [link to chromatogram]) was used to purify the protein. Figure 6 SDS-PAGE gel electrophoresis was performed, followed by mass spectrometry analysis.

[0135] The results are as follows Figure 6 As shown.

[0136] The electrophoresis results show that the target protein in this embodiment has a molecular weight greater than 25 kDa and no other impurities, proving that the GST@MPER protein after molecular sieve filtration has high purity. Furthermore, the mass spectrum shows that the main peak has a molecular weight of 29384.994, which is consistent with the theoretical molecular weight of the fused GST@MPER protein. Therefore, it can be confirmed that the target protein obtained in this embodiment is indeed the GST@MPER protein.

[0137] Test Example 1

[0138] This test case evaluated the safety of the SiO2-COOH@MPER prepared in the above examples using mouse fibroblast L929 cells and monkey kidney embryonic cells MA-104, respectively. The specific test steps are as follows:

[0139] After resuscitating the cells and passaged 2-3 times until the cells reach a confluence greater than 90%, discard the culture medium, wash the cells with 2-3 mL of PBS, add 2 mL of 0.25% trypsin and digest in a 37°C incubator for 2 min, then add 4 mL of DMEM complete culture medium to stop the digestion, obtaining a cell suspension. Transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 900 g for 3 min, and discard the supernatant. Then, resuspend the cells in 2 mL of complete culture medium and perform cell counting. Based on the cell count results, dilute the cells to 60,000 cells / mL, and then seed 100 μL into 96-well plates (i.e., 6,000 cells / well) and culture for 24 h. After culturing, discard the cell culture medium in the 96-well plate and add 100 μL of different concentrations of SiO2-COOH@MPER prepared in the above examples (prepared with incomplete culture medium (without fetal bovine serum) at concentrations of 0.0156 mg / mL, 0.0313 mg / mL, 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL). Three parallel wells were used for each concentration. Control wells (no drug, cells + culture medium) and blank wells (no drug, culture medium only) were also set up. The 96-well plate was then incubated in an incubator for 16 h.

[0140] After culture, the liquid in the 96-well plate was aspirated, and 100 μL of 5 mg / mL MTT solution was added. The plate was incubated at 37°C in the dark for 4 hours. The liquid was then aspirated, and 150 μL of DMSO was added. The plate was then reacted on a shaker for 10 minutes to fully dissolve the formazan. After the reaction, the absorbance at 490 nm was measured using a microplate reader. Cell viability was calculated based on the absorbance values ​​using the following formula.

[0141]

[0142] The results are as follows Figure 7 As shown.

[0143] It can be observed that, based on the cytotoxicity results, the SiO2-COOH@MPER prepared in the above examples did not exhibit significant cytotoxicity to normal fibroblasts (L929) and monkey embryonic kidney cells (MA104). At the maximum dosage concentration (1 mg / mL), the survival rate of both cell types exceeded 90%, and this dosage concentration is actually much higher than the actual in vivo dosage concentration. Therefore, it can be determined that SiO2-COOH@MPER has high cellular safety.

[0144] Test Example 2

[0145] This test case further verifies the safety of SiO2-COOH@MPER based on in vitro hemolysis. The specific test steps are as follows:

[0146] (1) Preparation of 2% red blood cells:

[0147] Take 2 mL of fresh rat blood into an anticoagulant tube containing sodium citrate, dilute it 10-fold with PBS (pH 7.4, 0.01 M, containing 0.89% NaCl), shake well, centrifuge at 750 rpm for 10 min, discard the supernatant, and obtain the cell pellet. Wash the obtained cells 2-3 times with PBS until colorless, and then resuspend the cells in PBS to form a 2% red blood cell suspension.

[0148] (2) Detection of hemolysis rate:

[0149] Different concentrations of SiO2-COOH@MPER (prepared with 0.8 mL of PBS to concentrations of 0.0156 mg / mL, 0.0313 mg / mL, 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL) were added to 0.2 mL of 2% erythrocyte suspension. Ultrapure water (0.2 mL 2% erythrocytes + 0.8 mL H2O) was used as the positive control, and PBS (0.2 mL 2% erythrocytes + 0.8 mL PBS) was used as the negative control.

[0150] After incubation at 37℃ for 2 hours, all groups were centrifuged at 10050 rpm for 15 minutes, and the supernatant was collected and allowed to stand at room temperature for 30 minutes to allow the hemoglobin contained therein to be fully oxidized to oxygen and hemoglobin. Then, the negative control group was used as a blank, and the absorbance value of the supernatant at 540 nm was measured by UV-Vis spectrophotometer, and the hemolysis rate (HR) was calculated according to the following formula.

[0151]

[0152] The results are as follows Figure 8 As shown.

[0153] It was found that no hemolysis occurred in any of the groups treated with SiO2-COOH@MPER, demonstrating that SiO2-COOH@MPER has good biocompatibility and can be further applied in vivo.

[0154] Test Example 3

[0155] This test case is based on an animal immunization experiment to detect the immunomodulatory activity of SiO2-COOH@MPER.

[0156] Five- to six-week-old female Balb / c mice were used as experimental animals. They were randomly divided into three groups (Control, MPER, and SiO2-COOH@MPER groups), with five mice in each group. Immunization was performed on days 0, 14, 28, 42, 56, and 70 (denoted as D0, D14, D28, D42, D56, and D70), using the corresponding substances. The total experimental period was 84 days. The experimental flowchart is shown below. Figure 9 As shown.

[0157] In this study, the Control group received a subcutaneous injection of 200 μL PBS, while the MPER and SiO2-COOH@MPER groups received 200 μL of 0.5 mg / mL MPER and SiO2-COOH@MPER, respectively. During immunization, mouse body weight was recorded at each time point. Blood samples were collected at D0, D42, D56, D70, and D84, and serum was separated for IgG antibody detection.

[0158] After the experiment, mice were euthanized by cervical dislocation, and their heart, liver, spleen, lungs, and kidneys were harvested. The attached fat was removed, and the organs were washed with PBS and stored in 4% paraformaldehyde. The organs were stained with eosin and hematoxylin and eosin and then H&E staining was performed to observe the effect of immunogens on the organs during the experimental period. The changes in serum IgG content were detected using a commercially available total IgG ELISA kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd.).

[0159] Then, the GST@MPER protein prepared in Example 5 above was used to determine the specificity of the antibody in serum. The specific steps are as follows:

[0160] (1) Add 1.59g Na2CO3 and 1.465g NaHCO3 to 1000mL of distilled water to prepare a 0.05M carbonate buffer with pH 9.6 as a coating buffer.

[0161] (2) Prepare an antigen solution of 15 μg / mL using coating buffer with the GST@MPER protein prepared in Example 5 above, and then add it to a 96-well ELISA plate (100 μL / well) so that each well contains 1.5 μg of antigen. Place the 96-well ELISA plate with the antigen in 4°C overnight.

[0162] (3) Remove the 96-well ELISA plate and wash it 5 times with PBST buffer containing 0.05% Triton-X100. After the last wash, pat the plate dry.

[0163] (4) Add 200 μL of 2% BSA to block the ELISA plate, and then place it at 37°C for 2 hours. After blocking, remove the ELISA plate and wash it 5 times with PBST. After the last wash, pat the plate dry.

[0164] (5) Add primary antibody: Add 100 μL of serum as primary antibody according to the serum concentrations of 100×, 900×, 2700×, 8100×, 24300× and 729000×. Repeat each concentration in 3 wells. Then incubate at 37℃ for 2 h. After taking it out, wash the plate 3 times with PBST. After the last wash, pat the plate dry.

[0165] (6) Add secondary antibody: horseradish peroxidase-labeled goat anti-mouse IgG (H+L) (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) was selected as the secondary antibody. The amount added was 100 μL / well. After adding the secondary antibody, the plate was incubated at 37℃ for 1 h. After taking it out, the plate was washed 3 times with PBST. After the last wash, the plate was patted dry.

[0166] (7) Add colorimetric solution: The colorimetric solution is trypan blue (purchased from Kingclone (Beijing) Biotechnology Co., Ltd.), and the amount added is 100 μL / well. The color is developed at 37℃ for 15 min.

[0167] (8) Add the stop solution (purchased from Kingclone (Beijing) Biotechnology Co., Ltd.) (50 μL / well), and then immediately measure the OD value at a wavelength of 450 nm using a microplate reader.

[0168] The results are as follows Figure 10 As shown.

[0169] The weight data and organ H&E staining results of the experimental mice showed that SiO2-COOH@MPER did not have a significant impact on the health of the mice throughout the experimental period, and no obvious pathological changes were found. Therefore, SiO2-COOH@MPER will not cause any burden on the body, at least throughout the experimental period, thus ensuring its safety as a drug.

[0170] Secondly, it was observed that after six immunizations, the serum IgG levels in the SiO2-COOH@MPER group mice were significantly higher than those in the Control and MPER groups. Furthermore, ELISA results using serum antibodies against MPER showed that even after 84 days of immunization and a 2700-fold dilution, the serum from the SiO2-COOH@MPER group still exhibited a stronger specific binding reaction than that of MPER, indicating that SiO2-COOH@MPER can induce an immune response to some extent. In contrast, MPER alone, when used for immunization, has weak immunogenicity and cannot effectively induce an immune response.

[0171] Furthermore, since HIV MPER (661-681) is an unstable membrane protein that is prone to oligomerization and difficult to purify, the presence of specific antibodies in serum can be measured using GST@MPER fusion protein to effectively confirm whether SiO2-COOH@MPER in this invention undergoes oligomerization (GST does not have non-specific adsorption for IgG). The results show that SiO2-COOH@MPER does not oligomerize. Therefore, it can be confirmed that the conjugation of MPER with SiO2-COOH to form a nanoimmunogen successfully overcomes the inherent limitations of MPER in HIV antibody development, demonstrating that immobilized MPER can also induce specific antibodies, and further achieving the effective development of broad-spectrum HIV antibodies.

[0172] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An HIV artificial antigen, characterized in that, The HIV artificial antigen includes: HIV virus MPER loaded on a silicon-based vector; The HIV virus MPER has the structure shown in Formula I: Formula I The branch ends of the HIV virus MPER are connected to the silicon-based carrier; the main chain end of the HIV virus MPER is not connected to the silicon-based carrier. The silicon-based carrier is surface-carboxylated silicon dioxide.

2. The HIV artificial antigen according to claim 1, characterized in that, The particle size of the HIV artificial antigen is less than 200 nm.

3. The HIV artificial antigen according to claim 1, characterized in that, The silicon dioxide is silicon dioxide nanoparticles.

4. The method for preparing the HIV artificial antigen according to any one of claims 1-3, comprising the following steps: Amino-modified silica nanoparticles were prepared, and the surface of the amino-modified silica nanoparticles was modified by carboxylation. HIV virus MPER was then coupled onto the carboxylated silica nanoparticles to obtain HIV artificial antigen.

5. The preparation method according to claim 4, characterized in that, The modified amino silica nanoparticles were prepared by catalytic hydrolysis of orthosilicate and 3-aminopropyltrimethoxysilane.

6. The preparation method according to claim 5, characterized in that, The volume ratio of the orthosilicate to 3-aminopropyltrimethoxysilane is 3-5:

1.

7. The use of the HIV artificial antigen according to any one of claims 1-3 in the preparation of medicaments for the prevention and / or treatment of HIV infection.

8. The application according to claim 7, characterized in that, The drug also includes pharmaceutically acceptable excipients; The pharmaceutically acceptable excipients include at least one of the following: antioxidants, preservatives, coating materials, solvents, lyophilization protectants, or carriers.

9. The application according to claim 8, characterized in that, The drug includes at least one of a vaccine and a serum antibody.

10. The application according to claim 7, characterized in that, The drug also includes a second active substance; The second active substance is an anti-HIV infection drug or an adjunct to HIV treatment.

11. The application according to claim 10, characterized in that, The anti-HIV infection drugs include at least one of the following: nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), integrase inhibitors (INSTIs), fusion inhibitors (FIs), and CCR5 antagonists; The HIV treatment adjuvants include: potentiators, and agents for increasing adherence or reducing HIV treatment side effects.

12. The use of the HIV artificial antigen according to any one of claims 1-3 in the preparation of HIV diagnostic products; The HIV diagnostic products include at least one of diagnostic reagents, diagnostic kits, detection chips, and test strips.

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