A dengue polypeptide vaccine based on spherical nucleic acid carriers, its preparation method and applications

Abstract

The present invention provides a dengue polypeptide vaccine based on spherical nucleic acid carriers, its preparation method and application, belonging to the technical field of vaccine adjuvants. The present invention provides a polypeptide TBB, whose amino acid sequence is shown in SEQ ID NO.1. The present invention constructs a dengue polypeptide vaccine based on spherical nucleic acid carriers by using the polypeptide TBB. This dengue polypeptide vaccine can effectively present antigens to BMDCs, stimulate the maturation and activation of BMDCs, effectively solve the problem of poor immunogenicity of polypeptide vaccines, and at the same time exhibit good biosafety.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine adjuvants, and particularly relates to a dengue polypeptide vaccine based on a spherical nucleic acid carrier, a preparation method thereof, and an application thereof. Background Art

[0002] Dengue virus (DENV) is an arthropod-borne single-stranded positive-sense RNA virus that can cause dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Although the diseases caused by DENV have a certain degree of self-limitation, the antibody-dependent enhancement (ADE) effect increases the severity and mortality rates after a second infection with a heterotypic virus. Since there is no specific therapeutic drug for dengue virus, there is an urgent need to develop safe and effective vaccines to prevent infection and disease progression. Currently, only one live attenuated vaccine (CyD-TDV) has obtained clinical use permission in some countries, but clinical trials of CyD-TDV have shown that it is only effective and safe for seropositive individuals who have previously been infected with dengue virus. For seronegative individuals who receive the vaccine for the first time, it increases the risk of developing severe dengue fever. Therefore, the research and continuous development of vaccines remain effective measures for preventing dengue virus infection.

[0003] Polypeptide vaccines can stimulate epitope-specific antibodies and immune responses, thereby eliminating the risk of adverse reactions associated with the vaccine. At the same time, connecting the polypeptide vaccine to nanoparticles for delivery can effectively enhance immunogenicity and metabolic stability. As an emerging nanomaterial, spherical nucleic acid can be used as a carrier for polypeptide or protein delivery. In antiviral research, spherical nucleic acid can not only carry the corresponding antigenic epitopes, but also the nucleic acid adsorbed on its surface is usually formed by immunostimulatory nucleic acids, greatly enhancing the immune response of the body. The dengue polypeptide vaccine based on a spherical nucleic acid carrier is expected to become a candidate vaccine for preventing dengue virus infection. Summary of the Invention

[0004] The purpose of the present invention is to provide a dengue polypeptide vaccine based on a spherical nucleic acid carrier, a preparation method thereof, and an application thereof. The present invention constructs a dengue polypeptide vaccine based on a spherical nucleic acid carrier using polypeptide TBB. This dengue polypeptide vaccine can effectively present antigens to BMDCs, stimulate the maturation and activation of BMDCs, effectively solve the problem of poor immunogenicity of polypeptide vaccines, and at the same time exhibit good biosafety.

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

[0006] The present invention provides a polypeptide TBB, and the amino acid sequence of the polypeptide TBB is as shown in SEQ ID NO.1.

[0007] The present invention also provides a dengue polypeptide vaccine based on a spherical nucleic acid vector, and the dengue polypeptide vaccine includes the above-mentioned polypeptide TBB.

[0008] The present invention also provides a preparation method of the above-mentioned dengue polypeptide vaccine, including the following steps:

[0009] (1) Functionalize the gold nanoparticles to obtain functionalized gold nanoparticles;

[0010] (2) Couple the polypeptide TBB with CpG to obtain TBB-CpG;

[0011] (3) Mix the functionalized gold nanoparticles with the TBB-CpG to obtain the dengue polypeptide vaccine.

[0012] Preferably, the functionalization method in step (1) is: mix the gold nanoparticles with CpG complement, freeze, thaw, and then centrifuge to obtain the functionalized gold nanoparticles.

[0013] Preferably, the nucleotide sequence of the CpG complement is as shown in SEQ ID NO.2; the molar ratio of the gold nanoparticles to the CpG complement is 1:(5000-15000); the freezing temperature is -40 to -20 °C, and the time is 1 to 3 h.

[0014] Preferably, the coupling method in step (2) is: mix CpG with tris(2-carboxyethyl)phosphine hydrochloride, then add 4,4'-dipyridyldisulfide for incubation, and after purification, obtain a CpG pretreatment; react the CpG pretreatment with the polypeptide TBB to obtain TBB-CpG.

[0015] Preferably, the nucleotide sequence of the CpG is as shown in SEQ ID NO.3; the molar ratio of the CpG to tris(2-carboxyethyl)phosphine hydrochloride is 1:(80-120); the mixing temperature is 20 to 25 °C, and the time is 1 to 3 h; the molar ratio of the CpG to the 4,4'-dipyridyldisulfide is 1:(800-1100); the incubation temperature is 20 to 25 °C, and the time is 20 to 40 min.

[0016] Preferably, the molar ratio of the CpG pretreatment to the polypeptide TBB is (0.5-2):(0.5-2); the reaction temperature is 20 to 25 °C, and the time is 20 to 40 min.

[0017] Preferably, the molar ratio of CpG complement on the functionalized gold nanoparticles to CpG on the TBB-CpG in step (3) is (0.5 - 2):(0.5 - 2); the temperature of the mixing is 35 - 40 °C, and the time is 15 - 40 min.

[0018] The present invention also provides an application of the above polypeptide TBB or the above dengue polypeptide vaccine in the preparation of an anti-dengue drug.

[0019] The present invention provides a dengue polypeptide vaccine based on a spherical nucleic acid carrier, its preparation method and application. The present invention constructs a dengue polypeptide vaccine based on a spherical nucleic acid carrier by using the polypeptide TBB and gold nanoparticles, and the preparation process is as Figure 1 shown. This dengue polypeptide vaccine can effectively present antigens to BMDCs, and can stimulate the maturation and activation of BMDCs, can effectively solve the problem of poor immunogenicity of polypeptide vaccines, has no cytotoxicity, and shows good biosafety. As a new type of third-generation vaccine technology, the dengue polypeptide vaccine of the present invention provides new ideas and research significance for the study of dengue vaccines. Description of the Drawings

[0020] Figure 1 It is a schematic diagram of the preparation process of the dengue polypeptide vaccine based on a spherical nucleic acid carrier of the present invention.

[0021] Figure 2 It is the ultraviolet spectrum diagram of AuNP and AuNP-CpG complement in Example 1.

[0022] Figure 3 It is the agarose gel electrophoresis diagram of CpG and TBB-CpG in Example 1.

[0023] Figure 4 It is the detection result diagram of the particle size and Zeta potential of AuNP and SNA-TBB in Experimental Example 1.

[0024] Figure 5 It is the TEM map of AuNP and SNA-TBB in Experimental Example 1.

[0025] Figure 6 It is the cell viability result diagram after treating BMDCs with TBB, TBB-CpG, and SNA-TBB in Experimental Example 2.

[0026] Figure 7 It is the fluorescence signal detection result diagram after treating BMDCs with PBS, TBB, TBB-CpG, and SNA-TBB at the same concentration in Experimental Example 3.

[0027] Figure 8The figure shows the detection results of fluorescence signals after treating BMDCs with different concentrations of SNA-TBB in Experimental Example 3.

[0028] Figure 9 The figure shows the detection results by flow cytometry after treating BMDCs with different concentrations of TBB, TBB-CpG, and SNA-TBB in Experimental Example 3.

[0029] Figure 10 The figure shows the results of the effects of PBS, TBB, TBB-CpG, and SNA-TBB on antibody expression in immature BMDCs in Experimental Example 4. Among them, A shows the expression of CD11c in BMDCs after treatment with PBS, TBB, TBB-CpG, and SNA-TBB, and B shows the expression of CD80 and CD86 in BMDCs after treatment with PBS, TBB, TBB-CpG, and SNA-TBB.

[0030] Figure 11 The figure shows the detection results of the secretion amount of IL-12p70 in BMDCs after treatment with PBS, TBB, TBB-CpG, and SNA-TBB in Experimental Example 5.

[0031] Figure 12 The figure shows the detection results of the specific IgG antibody level after immunizing mice with PBS, TBB, TBB-CpG, and SNA-TBB in Experimental Example 6. Detailed implementation manners

[0032] The present invention provides a polypeptide TBB, and the amino acid sequence of the polypeptide TBB is shown as SEQ ID NO.1: KYVKQNTLKLATGGVLGSQEGSMVDRGWGNGCGLFGKG.

[0033] The present invention also provides a dengue polypeptide vaccine based on a spherical nucleic acid carrier, and the dengue polypeptide vaccine preferably includes the above-mentioned polypeptide TBB.

[0034] The present invention also provides a preparation method of the above-mentioned dengue polypeptide vaccine, including the following steps:

[0035] (1) Functionalize the gold nanoparticles to obtain the functionalized gold nanoparticles;

[0036] (2) Couple the polypeptide TBB with CpG to obtain TBB-CpG;

[0037] (3) Mix the functionalized gold nanoparticles with TBB-CpG to obtain the dengue polypeptide vaccine.

[0038] The present invention first functionalizes the gold nanoparticles to obtain the functionalized gold nanoparticles.

[0039] In the present invention, the method of functional modification preferably is: mixing gold nanoparticles with CpG complement, freezing, thawing, and then centrifuging to obtain the functionalized gold nanoparticles.

[0040] In the present invention, the particle size of the gold nanoparticles preferably is 1 - 50 nm, and more preferably is 30 nm.

[0041] In the present invention, the nucleotide sequence of the CpG complement preferably is as shown in SEQ ID NO.2: AACGTCAGGAACGTCATGGA - SH.

[0042] In the present invention, the molar ratio of the gold nanoparticles to the CpG complement preferably is 1:(5000 - 15000), and more preferably is 1:10000.

[0043] In the present invention, the temperature of freezing preferably is - 40 to - 20 °C, and more preferably is - 20 °C.

[0044] In the present invention, the time of freezing preferably is 1 - 3 h, and more preferably is 2 h.

[0045] In the present invention, the temperature of thawing preferably is 20 - 25 °C, and more preferably is 25 °C.

[0046] In the present invention, the rotation speed of centrifuging preferably is 5000 - 8000 rpm, and more preferably is 7000 rpm.

[0047] In the present invention, the time of centrifuging preferably is 15 - 40 min, and more preferably is 30 min.

[0048] In the present invention, polypeptide TBB is coupled with CpG to obtain TBB - CpG.

[0049] In the present invention, the nucleotide sequence of the CpG preferably is as shown in SEQ ID NO.3: TCCATGACGTTCCTGACGTT - SH.

[0050] In the present invention, the method of coupling preferably is: mixing CpG with tris(2 - carboxyethyl)phosphine hydrochloride (TCEP), then adding 4,4'-dipyridyl disulfide (DTDP) for incubation, and after purification, obtaining a CpG pretreatment product; reacting the CpG pretreatment product with polypeptide TBB to obtain TBB - CpG.

[0051] In the present invention, the molar ratio of the CpG to the TCEP preferably is 1:(80 - 120), and more preferably is 1:100.

[0052] In the present invention, the temperature of the mixing is preferably 20 to 25 °C, more preferably 25 °C.

[0053] In the present invention, the time of the mixing is preferably 1 to 3 h, more preferably 2 h.

[0054] In the present invention, the molar ratio of the CpG to the DTDP is preferably 1:(800 - 1100), more preferably 1:1000.

[0055] In the present invention, the temperature of the incubation is preferably 20 to 25 °C, more preferably 25 °C.

[0056] In the present invention, the time of the incubation is preferably 20 to 40 min, more preferably 30 min.

[0057] In the present invention, the purification preferably uses a nucleic acid purification column.

[0058] In the present invention, the molar ratio of the CpG pretreatment to the polypeptide TBB is preferably (0.5 - 2):(0.5 - 2), more preferably 1:1.

[0059] In the present invention, the temperature of the reaction is preferably 20 to 25 °C, more preferably 25 °C.

[0060] In the present invention, the time of the reaction is preferably 20 to 40 min, more preferably 30 min.

[0061] The present invention mixes the functionalized gold nanoparticles with TBB-CpG to obtain the dengue polypeptide vaccine.

[0062] In the present invention, during the mixing, the molar ratio of the CpG complement on the functionalized gold nanoparticles to the CpG on the TBB-CpG is preferably (0.5 - 2):(0.5 - 2), more preferably 1:1.

[0063] In the present invention, the temperature of the mixing is preferably 35 to 40 °C, more preferably 37 °C.

[0064] In the present invention, the time of the mixing is preferably 15 to 40 min, more preferably 30 min.

[0065] The present invention also provides an application of the above-mentioned polypeptide TBB or the above-mentioned dengue polypeptide vaccine in the preparation of an anti-dengue drug.

[0066] The following combines examples to detail the technical solutions provided by the present invention, but they cannot be understood as limiting the protection scope of the present invention.

[0067] Example 1

[0068] This example provides a dengue polypeptide vaccine SNA-TBB based on spherical nucleic acid carriers, and the specific preparation process is as follows:

[0069] (1) Functional modification of gold nanoparticles (AuNP)

[0070] a. Mix AuNP (purchased from Beijing Xikai Innovation Technology Co., Ltd., 30 nm) and Cy3-labeled CpG complement at molar ratios of 1:5000, 1:10000, 1:15000, and 1:20000 respectively to make mixed solutions with different ratios, and place them in a -20 °C refrigerator for freezing for 2 h. After the freezing is completed, take out the mixed solutions of each ratio, slowly thaw them at room temperature, then centrifuge at 7000 rpm for 30 min, and discard the supernatant to remove the unconnected CpG complement.

[0071] b. Mix AuNP and Cy3-labeled CpG complement at molar ratios of 1:5000, 1:10000, 1:15000, and 1:20000 respectively, omit the freeze-thaw process, and make mixed solutions with different ratios.

[0072] Take 100 μL from each of the mixed solutions prepared in step a and step b and add them to a 96-well light-shielding plate to detect the fluorescence value. Calculate the total DNA concentration based on the fluorescence value of the mixed solution prepared in step b, calculate the residual DNA concentration based on the fluorescence value of the mixed solution prepared in step a, and calculate the loading rate of CpG complement at different mixing ratios according to formula 1. The calculation results are shown in Table 1.

[0073] Formula 1: Loading rate % = (total DNA concentration - residual DNA concentration) / total DNA concentration × 100%

[0074] Table 1 Loading rate of CpG complement at each ratio

[0075] AuNP:CpG complement Total concentration (pmol / μL) Residual concentration (pmol / μL) Loading rate % 1:5000 2.19 1.33 39％ 1:10000 3.75 2.25 40％ 1:15000 5.50 3.50 36％ 1:20000 6.6 4.65 30％

[0076] As can be seen from Table 1, when the molar ratios of AuNP to CpG complement are 1:5000, 1:10000, and 1:15000, the loading amounts of CpG complement are all above 35%. And when the ratio is 1:20000, the loading rate decreases significantly, indicating that in the low ratios, CpG complement has reached the maximum loading amount, and the CpG complement conjugated to AuNP has reached saturation. As the concentration of CpG complement increases, the loading rate no longer increases. When the molar ratio of AuNP:CpG complement is 1:10000, the loading rate is 40%, and the conjugation efficiency is the best. The functionalized gold nanoparticles (AuNP-CpG complement) obtained under this molar ratio are used for subsequent research.

[0077] The actual calculated values of the loading rates of AuNP and CpG complement are generally low, which may be related to the addition of excessive CpG complement. Because when conjugating AuNP and CpG complement by the freezing method, a large amount of free oligonucleotides are needed to protect AuNP from aggregating at low temperatures.

[0078] To further detect the functionalization of AuNP and CpG complement, 100 μL of each of AuNP and AuNP-CpG complement was taken and added to a 96-well plate respectively. The ultraviolet spectra of the two solutions were detected by an ultraviolet spectrophotometer. The wavelength setting condition of the ultraviolet spectrophotometer was between 300 nm and 1000 nm. Whether CpG complement was successfully modified on AuNP was judged by the ultraviolet spectrum. The ultraviolet spectrum is as Figure 2 shown. It can be seen that compared with AuNP, the strongest light absorption peak of AuNP-CpG complement has an obvious red shift, proving that CpG complement and AuNP have been successfully conjugated, forming a sphere with oligonucleotides attached to the surface of AuNP.

[0079] (2) Conjugation of CpG and polypeptide TBB: Thiolated CpG and TBB containing cysteine can be conjugated through a disulfide bond.

[0080] Preparation of 20 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride): Weigh 28.7 mg of TCEP powder in an analytical balance and dissolve it in 5 mL of ultrapure water to prepare a 20 mM TCEP solution. The molecular weight of TCEP is 247.1634. Aliquot and store at -20 °C in a refrigerator.

[0081] Preparation of 160 mM DTDP (4,4'-dipyridyl disulfide): Weigh 71 mg of DTDP powder on an analytical balance and dissolve it in 2 mL of DMSO to prepare a 160 mM DTDP solution. The molecular weight of DTDP is 220.31. Aliquot and store at -20 °C in a refrigerator.

[0082] Take 120 μL of CpG with a concentration of 100 μM and add it to a 1.5 mL centrifuge tube. Then take 60 μL of a 20 mM TCEP solution and add it to the above centrifuge tube and mix well. Incubate in a metal bath at 25 °C for 2 h to perform a disulfide bond pretreatment on the thiolated CpG sequence. After the pretreatment is completed, add 75 μL of 160 mM DTDP solution to the above centrifuge tube so that the molar ratio of CpG to DTDP is 1:1000. Place it in a metal bath and incubate at 25 °C for 30 min. After the incubation is completed, the obtained product is passed through a nucleic acid purification column (NAP-5 columns) for impurity removal treatment to obtain a CpG pretreatment product, which is stored in a refrigerator at 4 °C.

[0083] Mix the CpG pretreatment product and the polypeptide TBB evenly at a molar ratio of 1:1 and react in a metal bath at 25 °C for 30 min to obtain TBB-CpG.

[0084] To detect the coupling situation between TBB and CpG, agarose gel electrophoresis was used to detect CpG and TBB-CpG. The detection results are as Figure 3 shown. It can be seen that compared with the CpG group, the electrophoretic band of the TBB-CpG group is closer to the loading well position. The migration rate of the TBB-CpG group is significantly lower than that of the CpG group, indicating that after CpG and TBB are coupled, the molecular weight of TBB-CpG is higher than that of CpG, and the electrophoretic mobility of TBB-CpG decreases. At the same time, there is only one target band after electrophoresis in the TBB-CpG group, and no obvious band is seen at the position corresponding to CpG, indicating that when the molar ratio of CpG:TBB is 1:1, CpG and TBB are completely coupled.

[0085] (3) Preparation of dengue polypeptide vaccine SNA-TBB

[0086] Mix the AuNP-CpG complement in step (1) and the TBB-CpG in step (2) according to the molar ratio of the coupled nucleotides of 1:1, that is, the molar ratio of CpG complement on AuNP-CpG complement to CpG on the TBB-CpG is 1:1. After mixing, react in a metal bath at 37 °C for 30 min to obtain SNA-TBB. Store at room temperature.

[0087] Experimental Example 1

[0088] In this experimental example, the particle size and morphological characteristics of the SNA-TBB prepared in Example 1 were detected. The specific process is as follows:

[0089] (1) Particle size detection

[0090] The particle sizes and Zeta potentials of AuNP and SNA-TBB were determined by Nano-ZS. Take 0.5 mL each of AuNP and SNA-TBB, add deionized water to make up to 1 mL, and add them to the sample cell for particle size and Zeta potential tests respectively.

[0091] The detection results are as Figure 4 shown. It can be seen that the particle sizes of AuNP and SNA-TBB measured by dynamic light scattering method are about 30 nm and 50 nm respectively, and the particle size of the gold nanoparticles after binding nucleic acid and antigen increases significantly. The Zeta potentials of AuNP and SNA-TBB are about -12 mV and -31.5 mV respectively. It is found that the electroproperty of SNA-TBB modified with CpG and TBB is negatively charged in pure water, and compared with pure AuNP, its negative charge increases significantly, which is related to the strong negative charge of CpG. At the same time, it shows that SNA-TBB has good colloidal stability in aqueous solution.

[0092] (2) Morphological feature detection

[0093] The morphological features of AuNP and SNA-TBB were detected by transmission electron microscope. Dilute AuNP and SNA-TBB 5 times with distilled water and then ultrasonicate for 5 min. Place the sealing film on the glass slide, pick up the copper grid with forceps and place it on the sealing film. Use a pipette gun to take 10 μL of the sample and drop it on the copper grid, let it stand for 10 min, and use a small piece of filter paper to absorb the excess liquid on the copper grid. Let it stand at room temperature for 1 h to allow the copper grid to dry naturally. Use a transmission electron microscope (JEM1200EX, Japan) to detect the dispersibility and morphology of the two samples.

[0094] The detection results are as Figure 5 shown. It can be seen that SNA-TBB is spherical with a smooth surface and is evenly dispersed. Compared with pure AuNP, the particle size of SNA-TBB shows an increasing trend, about 50 nm, but the morphology changes little, and this result is consistent with the particle size measured by dynamic light scattering.

[0095] Experimental Example 2

[0096] The cytotoxicity of the SNA-TBB prepared in Example 1 was detected in this experimental example, and the specific process is as follows:

[0097] (1) Preparation of bone marrow-derived dendritic cells (BMDCs)

[0098] The BALB / c mice were sacrificed by cervical dislocation and soaked in 75% alcohol for 3 min. Under sterile conditions, the femurs and tibias of the mice were taken with scissors and forceps and soaked in a petri dish containing RPMI 1640. The muscle tissues of the femurs and tibias were removed with a sterile scalpel. The obtained femurs and tibias were soaked in PBS containing 1% antibiotics for 2 min. A 1 mL syringe was used to aspirate RPMI1640 and repeatedly rinse the bone marrow cavity until it turned white. The bone marrow cell suspension was filtered through a 200-mesh filter to remove tissue debris. The cell suspension was collected into a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, 2 mL of red blood cell lysate was added, and after gently pipetting evenly, it was left at room temperature for 5 min. 5 mL of 1640 complete medium was added to terminate the reaction, and it was centrifuged at 1000 rpm for 5 min. The supernatant was discarded. The cells were resuspended in 10 mL of 1640 complete medium and centrifuged at 1000 rpm for 5 min again for one wash.

[0099] The cells were resuspended and counted with 1640 complete medium containing 10 ng / mL GM-CSF and 20 ng / mL IL-4. The cells were cultured in a 6-well plate at a density of 3×10 6 cells / mL and cultured in a humidity incubator at 37℃ and 5% CO2. On the 2nd and 4th days, half of the medium was replaced with 1640 complete medium containing 10 ng / mL GM-CSF and 20 ng / mL IL-4. On the 6th day, the loosely attached clusters were collected as BMDCs.

[0100] (2) Detection of the effect of SNA-TBB on the activity of BMDCs by CCK8 method

[0101] The BMDCs were inoculated into a 96-well plate, and 1×10 4 cells were added to each well. A 100 μL cell suspension was prepared with 1640 complete medium and placed in a humidity incubator at 37℃ for culture in a 5% CO2 environment.

[0102] PBS, TBB, TBB-CpG in Example 1, and SNA-TBB in Example 1 were co-cultured with BMDCs at different concentrations (25, 50, 100, 200, 300, 400 nM) for 24 h. The cells with PBS added were used as the control group, and the untreated cells were used as the blank group. The cell viability was measured using a cell Counting Kit-8 kit (purchased from Beyotime, product number: C0037). 10 μL of CCK8 solution was added to each well and cultured at 37℃ for 2 h. The absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay reader. The cell viability (%) was calculated according to Formula 2:

[0103] Formula 2: Percentage of viable cells % = (measured value - blank value) / (control value - blank value) × 100%

[0104] The calculation results are as Figure 6 shown. It can be seen that TBB, TBB-CpG, and SNA-TBB at different concentrations did not show obvious cytotoxicity to BMDCs, indicating that the polypeptide vaccine SNA-TBB has good safety at the cellular level.

[0105] Experimental Example 3

[0106] This experimental example detected the uptake ability of BMDCs to SNA-TBB. The specific process is as follows:

[0107] (1) First, place the cell slide at the bottom of a 24-well plate. Inoculate BMDCs cells into the 24-well culture plate with the cell slide. Add 1×10 5 BMDCs cells into each well, and configure them into a 500 μL cell suspension with complete 1640 medium. Place it in a humidified incubator at 37 °C and culture it in an environment of 5% CO2.

[0108] (2) Incubate Cy5-labeled PBS (Blank), TBB, TBB-CpG, and SNA-TBB at different concentrations (calculated by TBB concentration, 50 nM, 100 nM, 200 nM, 300 nM) with BMDCs for 2 h. After incubation, wash the BMDCs three times with PBS, fix them with 4% paraformaldehyde, and stain the cell nuclei with DAPI for 15 min, then wash them three times again with PBS.

[0109] (3) Gently take out the cell slide from the 24-well plate with forceps and place it on a glass slide with anti-fluorescence quencher dropped on it. Carefully fix it around the cell slide with nail polish.

[0110] (4) Use a laser confocal microscope and a flow cytometer to observe the uptake of polypeptides by BMDCs cells respectively.

[0111] Among them, when the TBB concentration is 100 nM, the detection results of different components are as Figure 7 shown. It can be seen that when the TBB concentration remains the same, obvious red fluorescence signals can be observed in the cells after co-incubation of the SNA-TBB group with BMDCs, while weak fluorescence signals can be detected in the TBB-CpG group, and almost no fluorescence signal can be detected in the TBB group.

[0112] Among them, the detection results of SNA-TBB at different concentrations are as Figure 8 shown. It can be seen that as the polypeptide concentration increases, the fluorescence signal gradually increases.

[0113] Among them, the detection results of the flow cytometer are as Figure 9As shown, it can be seen that the average fluorescence intensity of SNA-TBB is significantly higher than that of other groups and increases with the increase of TBB concentration. This is consistent with the results observed by laser confocal microscopy.

[0114] The above results indicate that SNA-TBB can significantly improve the antigen uptake of BMDCs, and its mechanism is mainly the formation of spherical nucleic acid nanostructures that are beneficial to cell uptake.

[0115] Experimental Example 4

[0116] This experimental example detected the promoting effect of SNA-TBB on the maturation of BMDCs cells. The specific process is as follows:

[0117] (1) Incubate 1 μg / mL lipopolysaccharide, 300 nM TBB, 300 nM TBB-CpG, and 300 nM SNA-TBB with immature BMDCs at 37 °C for 24 h respectively. Lipopolysaccharide is used as the positive control.

[0118] (2) After treatment, collect the cell supernatant and store it at -20 °C. Digest the cells with trypsin and collect them in a 1.5 mL Ep tube. Wash the cells three times with PBS, centrifuge at 1000 rpm for 5 min each time, and discard the supernatant.

[0119] (3) Stain BMDCs with 15 μL perCP-Cy5.5-labeled anti-CD11c antibody, 4 μL PE-labeled anti-CD80 antibody, and 6.5 μL FITC-labeled anti-CD86 antibody. Incubate in the dark for 30 min, centrifuge at 1000 rpm for 5 min, and discard the supernatant.

[0120] (4) Wash again with PBS three times, centrifuge at 1000 rpm for 5 min each time. Resuspend the cells with 200 μL PBS and transfer them to a flow tube.

[0121] (5) Analyze the labeling of each antibody on the cells by flow cytometry.

[0122] The detection results are as Figure 10 shown. As Figure 10 can be seen from A, the expression of CD11c in each group is about 75%, indicating that BMDCs have a high purity. As Figure 10As can be seen from Figure B, taking BMDCs treated with an equal amount of TBB as the control group, PBS treatment as the negative control, and lipopolysaccharide treatment as the positive control, the expression levels of CD80+ and CD86+ in the PBS group, TBB group, and TBB-CpG group were 15.4%, 26.2%, and 28.9% respectively. The expression levels of CD80+ and CD86+ in the SNA-TBB group were 46.9%. The above results indicate that SNA-TBB can increase the expression of co-stimulatory molecules CD80 and CD86 on the surface of BMDCs and promote the maturation of BMDCs.

[0123] Experimental Example 5

[0124] This experimental example detected the effect of SNA-TBB on the secretion of IL-12p70 by BMDCs. The specific process is as follows:

[0125] IL-12p70 is a key cytokine that initiates the Th1 response. DCs can secrete biologically active IL-12p70 in the late stage of maturation and the early stage of antigen presentation to T cells. IL-12p70 can induce the differentiation of Th0 into Th1 and initiate cellular immunity. Therefore, IL-12p70 plays an irreplaceable role in immune activation. The secretion of IL12p70 in the supernatant after co-incubation of PBS, TBB, TBB-CpG, and SNA-TBB with BMDCs for 24 h was detected by enzyme-linked immunosorbent assay.

[0126] The operation was carried out according to the instructions of the IL-12p70 ELISAkit kit (Linkage Biotechnology, China):

[0127] (1) Take out the cell supernatant frozen in step (2) of Experimental Example 4 and equilibrate it to room temperature.

[0128] (2) Soak the enzyme-linked immunosorbent assay (ELISA) plate: Add 300 μL of 1× washing solution and let it stand for 30 s. After discarding the washing solution, pat the microplate dry on the absorbent paper.

[0129] (3) Add the standard product: Add 100 μL of the standard product diluted 2-fold in a serial dilution to the standard product wells. Add 100 μL of RPMI1640 medium to the blank well.

[0130] (4) Add the sample: Add 100 μL of cell culture supernatant to each sample well. Ensure continuous sample addition in steps (3) and (4), and complete the sample addition process within 15 min.

[0131] (5) Incubation: Seal the plate with a sealing film. Oscillate on a shaker at 200 rpm / min (ensure that the solution in each well does not spill out and can be fully mixed), and incubate at room temperature for 1.5 h.

[0132] (6) Washing: Discard the liquid and wash each well orifice 6 times with 300 μL of washing solution. After each wash, pat dry on absorbent paper to completely remove the residual liquid.

[0133] (7) Adding detection antibody: Add 100 μL of diluted detection antibody to each well. Seal the plate with a sealing film. Incubate on a shaker at 100 - 300 rpm (ensuring that the solution in each well does not spill out and can be fully mixed), and incubate at room temperature for 30 min. Wash 6 times with 300 μL of washing solution.

[0134] (8) Adding enzyme and incubating: Add 100 μL of streptavidin - horseradish peroxidase conjugate. Seal it with a new film. Incubate in the same way as above. Wash 6 times with 300 μL of washing solution

[0135] (9) Adding signal enhancer and incubating: Add 100 μL of diluted signal enhancer (diluted 1:100) to each well. Seal the plate with a new sealing film. Continue to incubate for 15 min. Wash 6 times with 300 μL of washing solution.

[0136] (10) Adding enzyme again and incubating: Add 100 μL of diluted streptavidin - horseradish peroxidase conjugate (diluted 1:100) to each well. Incubate in the same way as above and continue to incubate for 15 min.

[0137] (11) Adding substrate for color development: Add 100 μL of chromogenic substrate TMB to each well, protect from light, and incubate at room temperature for 30 min.

[0138] (12) Adding stop solution: Add 100 μL of stop solution to each well. The color changes from blue to yellow. Wash 6 times with 300 μL of washing solution.

[0139] (13) Measuring the reading: Within 30 min, using the enzyme - labeled method, when the maximum absorption wavelength is 450 nm and the reference wavelength is 570 nm, measure the OD value. After calibration, the OD value is the result measured at 450 nm minus the result measured at 570 nm. Only using the measurement method at 450 nm will cause the OD value to be too high and the accuracy to decrease.

[0140] The detection results are as Figure 11 shown. It can be seen that the IL - 12p70 levels in the SNA - TBB group are 8.6 - fold, 9.0 - fold, and 4.4 - fold those of PBS, TBB, and TBB - CpG, respectively. The results indicate that SNA - TBB can effectively promote BMDCs to secrete IL - 12p70 and initiate cellular immunity.

[0141] Experimental Example 6

[0142] This experimental example detected the immunogenicity of SNA - TBB, and the specific process is as follows:

[0143] (1) Six- to eight-week-old SPF-grade female BABL / c mice (purchased from Chongqing Tengxin Bill Experimental Animal Sales Co., Ltd. and raised in the Animal Center of Army Medical University) were randomly divided into 4 groups, with 3 mice in each group, corresponding to the PBS group, the TBB group, the TBB-CpG group, and the SNA-TBB group, respectively.

[0144] (2) All samples were replaced with PBS buffer, and the TBB mass of each group of samples was adjusted to 30 μg.

[0145] (3) After 3 days of adaptive feeding, each mouse was immunized by subcutaneous injection on days 0, 14, and 28, with an injection volume of 30 μg / mouse. Blood was collected from the tail vein 7 days after the last immunization.

[0146] (4) All blood samples were allowed to stand overnight at 4°C. Centrifuged at 3000 rpm for 10 min, and all sera were collected and stored at -80°C.

[0147] (5) The content of specific antibody IgG against TBB in mouse serum was determined by ELISA method:

[0148] ① Coating buffer (pH 9.6 0.05M carbonate buffer): Weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them fully with distilled water, and make up the volume to 1000 mL.

[0149] Washing buffer (pH 7.4 PBST 0.15M): Weigh 0.2 g of KH2PO4, 2.9 g of Na2HPO4·12H2O, 8.0 g of NaCl and 0.2 g of KCl, pipette 0.5 mL of Tween-20, and add distilled water to 1000 mL.

[0150] Blocking solution: Weigh 5 g of bovine serum albumin (BSA), add washing buffer to 100 mL, and prepare a 5% BSA solution for standby.

[0151] Stop solution (2M H2SO4): Take 178.3 mL of distilled water, and add 21.7 mL of concentrated sulfuric acid with a concentration of 98% drop by drop.

[0152] Substrate buffer (pH 5.0 phosphate citrate): Weigh 9.2 g of Na2HPO4·12H2O and 2.35 g of citric acid, dissolve them fully with distilled water, and make up the volume to 500 mL.

[0153] 0.75% H2O2: Pipette 1 mL of 30% H2O2, dilute it in 39 mL of ultrapure water, and store it at 4°C.

[0154] TMB mother liquor: Weigh 10 mg and dissolve it in 5 mL of absolute ethanol.

[0155] Tetramethylbenzidine (TMB) working solution: Weigh 10 mg of tetramethylbenzidine and dissolve it in 5 mL of absolute ethanol to prepare a 2 mg / mL TMB stock solution. Take 0.5 mL of the TMB stock solution, 10 mL of substrate buffer, and 32 μL of 0.75% H2O2, and mix them thoroughly to prepare the TMB working solution.

[0156] Antibody / serum diluent: Take 10 mL of 5% BSA solution and mix it thoroughly with 40 mL of washing buffer to prepare a 1% BSA solution for standby.

[0157] ② Experimental steps:

[0158] Antigen coating: Dilute the TBB polypeptide to 15 μg / mL with coating buffer, and coat 100 μL per well on a 96-well ELISA plate overnight at 4°C.

[0159] Blocking: Discard the coating buffer, wash 5 times with PBST solution to remove the uncoated antigen, add 200 μL of blocking solution (PBST containing 5% BSA) to each well, and incubate at 37°C for 2 h.

[0160] Binding of primary antibody: After blocking, discard the blocking solution, wash 3 times with PBST solution, then add 100 μL of diluted serum to each well, and add PBS solution as a blank control, and incubate at 37°C for 2 h.

[0161] Binding of secondary antibody: After incubation, wash 5 times with PBST solution, dilute HRP-goat anti mouse IgG with antibody diluent (1% BSA solution) at a ratio of 1:5000, add 100 μL of the diluted solution to each well, and incubate at 37°C for 1 h.

[0162] Color development: Discard the incubated antibody, wash 5 times with PBST solution, add 100 μL of chromogenic solution to each well, place it in the dark for color development for 10 min, and then add 100 μL of stop solution to each well to terminate the color development.

[0163] Detection: Read the absorbance value at 450 nm through an ELISA reader.

[0164] The detection results are as Figure 12 shown. It can be seen that 7 days after the last immunization, the IgG antibody titer produced by SNA-TBB immunized mice was significantly higher than that of the PBS group and the TBB group, and higher than that of the TBB-CpG group. This indicates that SNA-TBB can induce the production of higher antigen-specific IgG antibodies.

[0165] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, 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 method for preparing a dengue polypeptide vaccine based on a spherical nucleic acid carrier, characterized in that, The dengue polypeptide vaccine includes polypeptide TBB, and the amino acid sequence of the polypeptide TBB is as shown in SEQ ID NO.1; The preparation method of the dengue polypeptide vaccine comprises the following steps: (1) Functionalize the gold nanoparticles to obtain functionalized gold nanoparticles; (2) Couple polypeptide TBB with CpG to obtain TBB-CpG; (3) Mix the functionalized gold nanoparticles with the TBB-CpG to obtain the dengue polypeptide vaccine; The functionalization method in step (1) is: mix the gold nanoparticles with CpG complement, after freezing and thawing, centrifuge to obtain the functionalized gold nanoparticles; The nucleotide sequence of the CpG complement is as shown in SEQ ID NO.2; The nucleotide sequence of the CpG is as shown in SEQ ID NO.

3.

2. The preparation method according to claim 1, characterized in that, The molar ratio of the gold nanoparticles to the CpG complement is 1:(5000 - 15000); the temperature of the freezing is -40 to -20 °C, and the time is 1 to 3 h.

3. The preparation method according to claim 2, characterized in that, The coupling method in step (2) is: mix CpG with tris(2-carboxyethyl)phosphine hydrochloride, then add 4,4'-dipyridyl disulfide for incubation, after purification, obtain the CpG pre-treatment; react the CpG pre-treatment with polypeptide TBB to obtain TBB-CpG.

4. The preparation method according to claim 3, characterized in that, The molar ratio of the CpG to tris(2-carboxyethyl)phosphine hydrochloride is 1:(80 - 120); the temperature of the mixing is 20 to 25 °C, and the time is 1 to 3 h; the molar ratio of the CpG to the 4,4'-dipyridyl disulfide is 1:(800 - 1100); the temperature of the incubation is 20 to 25 °C, and the time is 20 to 40 min.

5. The preparation method according to claim 4, characterized in that, The molar ratio of the CpG pre-treatment to the polypeptide TBB is (0.5 - 2):(0.5 - 2); the temperature of the reaction is 20 to 25 °C, and the time is 20 to 40 min.

6. The preparation method according to claim 5, characterized in that, The molar ratio of the CpG complement on the functionalized gold nanoparticles in step (3) to the CpG on the TBB-CpG is (0.5 - 2):(0.5 - 2); the temperature of the mixing is 35 to 40 °C, and the time is 15 to 40 min.

7. A dengue polypeptide vaccine based on spherical nucleic acid carriers prepared by the preparation method according to any one of claims 1 to 6.

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