CD2v protein nanoparticle based on African swine fever virus, fusion protein, preparation and application

Abstract

The invention provides CD2v protein nanoparticles based on African swine fever virus, fusion protein, and preparation and application of the CD2v protein nanoparticles and the fusion protein. The preparation method comprises the following steps: fusing an envelope protein CD2v extracellular domain on the surface of an African swine fever virus with Spaticher to obtain a recombinant fusion protein, then connecting the recombinant fusion protein with AP205-Spytag nanoparticles, and identifying and purifying the AP205 nanoparticles to finally obtain the AP205 nanoparticles connected with the CD2v extracellular domain. The immunogenicity of the CD2v is successfully improved by utilizing the AP205 nano-particle carrier, and meanwhile, the advantages of the AP205 nano-particles serving as the carrier for displaying exogenous antigens in the field of vaccines are disclosed.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to CD2v protein nanoparticles and fusion proteins based on African swine fever virus, as well as preparation and application thereof. Background Art

[0002] African swine fever virus (ASFV) is a double-stranded DNA (dsDNA) arbovirus and the only member of the genus Asfivirus in the Asfarviridae family. The virus causes African swine fever (ASF) after infecting pigs, which is a highly contagious hemorrhagic disease. The virus has an icosahedral symmetric shell (260-300nm), an envelope, and a large genome (170-194kbp), containing 150 open reading frames (ORFs). The virus particle structure is similar to that of the iridovirus, and the replication mode is similar to that of the poxvirus. It is the only member of the Asfarviridae family.

[0003] According to the 2019 classification report released by the International Committee of Taxonomy of Viruses (ICTV), the African swine fever virus family has been classified into the Pokkesviricetes, Asfuvirales. Mass spectrometry analysis of viral proteomics revealed that there are at least 40 different viral protein molecules in the five viral domains of the outer envelope, capsid, inner envelope, nucleocapsid and nucleolus of the virus particles. Among them, the ASFV structural proteins p30, p54, p72, pp62 and CD2v encoded by genes CP204L, E183L, B646L, CP530R and EP402R have become the main targets in vaccine design.

[0004] There is still no commercial vaccine for African swine fever, and no prototype vaccine for virus-like particles has appeared. However, in terms of protein subunit vaccines, Ruiz-Gonzalvo et al. used recombinant CD2v protein of ASFV expressed by baculovirus, immunized pigs three times with recombinant CD2v protein, and found that it could produce antibodies that recognize CD2v. Subsequently, the ASFV E75 strain was used for a virus attack test. Although two pigs died, one of the pigs showed neutralizing activity against the virus after the virus attack. This study shows that CD2v has poor immunogenicity and high doses of antigen may be required to induce good immune protection in the body.

[0005] In view of this, it is necessary to provide a CD2v protein nanoparticle, fusion protein, preparation and application based on African swine fever virus to solve or at least alleviate the technical defect of poor immunogenicity of the above-mentioned CD2v. Summary of the invention

[0006] The main purpose of the present invention is to provide CD2v protein nanoparticles, fusion proteins, and preparation and application based on African swine fever virus to solve the technical problem of poor immunogenicity of CD2v.

[0007] To achieve the above objectives, the present invention provides a CD2v-Spycatcher fusion protein based on African swine fever virus, and the amino acid sequence of the CD2v-Spycatcher fusion protein is shown in SEQ ID NO.1.

[0008] The present invention also provides a CD2v protein nanoparticle based on African swine fever virus, wherein the CD2v protein nanoparticle is formed by self-assembly of CD2v-Spycatcher fusion protein and AP205-Spytag nanoparticles;

[0009] The amino acid sequence of the CD2v-Spycatcher fusion protein is shown in SEQ ID NO.1, and the amino acid sequence of the AP205-Spytag nanoparticle is shown in SEQ ID NO.2.

[0010] The present invention also provides a method for preparing CD2v protein nanoparticles based on African swine fever virus, comprising the steps of:

[0011] S1, obtain CD2v-Spycatcher fusion protein and AP205-Spytag nanoparticles;

[0012] Wherein, the amino acid sequence of the CD2v-Spycatcher fusion protein is shown in SEQ ID NO.1, and the amino acid sequence of the AP205-Spytag nanoparticle is shown in SEQ ID NO.2;

[0013] S2, incubating the CD2v-Spycatcher fusion protein and the AP205-Spytag nanoparticles in a buffer solution to obtain the CD2v protein nanoparticles.

[0014] Furthermore, the nucleotide sequence encoding the CD2v-Spycatcher fusion protein is shown in SEQ ID NO.3, and the nucleotide sequence encoding the AP205-Spytag nanoparticle is shown in SEQ ID NO.4.

[0015] Furthermore, the molar concentration ratio of the CD2v-Spycatcher fusion protein and the AP205-Spytag nanoparticles in the buffer solution is 2:1-1:3.

[0016] The present invention also provides a use of any of the CD2v-Spycatcher fusion proteins described above in the preparation of an African swine fever vaccine.

[0017] The present invention also provides a use of any of the CD2v protein nanoparticles described above in the preparation of an African swine fever vaccine.

[0018] The present invention also provides an African swine fever vaccine, which includes the CD2v-Spycatcher fusion protein as described above, or the African swine fever vaccine includes the CD2v protein nanoparticles as described above.

[0019] The present invention also provides a use of any of the above-described CD2v-Spycatcher fusion proteins in the preparation of CD2v-specific antibodies.

[0020] The present invention also provides a use of any of the above-described CD2v protein nanoparticles in the preparation of CD2v-specific antibodies.

[0021] The beneficial effects of the present invention include at least:

[0022] The present invention provides a CD2v-Spycatcher fusion protein based on African swine fever virus, and the CD2v-Spycatcher fusion protein is immunogenic; in order to further enhance its immunogenicity, the present invention also provides a CD2v protein nanoparticle. The present invention connects the prepared AP205-Spytag nanoparticle carrier with the CD2v extracellular domain protein, and successfully improves the immunogenicity of CD2v by using the AP205 nanoparticle carrier, while revealing the advantages of AP205 nanoparticles as carriers for displaying exogenous antigens in the vaccine field. Specifically, in the present invention, 2-4 weeks after the first immunization, after the CD2v extracellular domain is displayed on the surface of the AP205 nanoparticles, the antibody level is 50-100 times that of the non-nanoparticle group. Although the difference in antibody levels is reduced after booster immunization, it is still about 50 times different. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0024] Figure 1 This is the SDS-PAGE identification diagram of the CD2v-Spycatcher fusion protein in Example 1 of the present invention;

[0025] Figure 2 This is the SDS-PAGE identification diagram of the AP205-Spytag nanoparticles in Example 1 of the present invention;

[0026] Figure 3 This is an SDS-PAGE identification diagram after the connection reaction between the CD2v-Spycatcher fusion protein and the AP205-Spytag nanoparticles in Example 1 of the present invention;

[0027] Figure 4 This is a UV absorption peak chromatogram of the CD2v-AP205 nanoparticle gel chromatography column identified in Example 1 of the present invention; along the extension direction of the horizontal axis, the tube numbers shown in the figure are 3, 4, 5, 6, 7, 8, 9, and 10 respectively;

[0028] Figure 5 This is the SDS-PAGE image of CD2v-AP205 nanoparticles in Example 1 of the present invention;

[0029] Figure 6 The transmission electron micrographs of the nanoparticles in Example 1 of the present invention are shown in FIG. 1 ; wherein (a) is the AP205-spytag nanoparticle; (b) is the CD2v-AP205 nanoparticle; the coordinate scales are all 100 nm;

[0030] Figure 7 This is a monitoring chart of the mouse CD2v-specific IgG antibody titer at different time points in Example 1 of the present invention.

[0031] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with the implementation methods and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Furthermore, the technical solutions between the various embodiments of the present invention may be combined with each other, but this must be based on the fact that they can be implemented by ordinary technicians in the field. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0034] The present invention provides a CD2v-Spycatcher fusion protein based on African swine fever virus, and the amino acid sequence of the CD2v-Spycatcher fusion protein is shown in SEQ ID NO.1. The CD2v-Spy catcher fusion protein is immunogenic and can induce the production of specific antibodies in the body. However, in order to obtain a higher performance African swine fever vaccine, it is necessary to further improve the immunogenicity of the CD2v-Spycatcher fusion protein.

[0035] It should be noted that there are many ways to solve the problem of insufficient protein immunogenicity, among which nanoparticles are the most effective. Nanoparticles are macromolecular complexes composed of multiple proteins. Their appearance and shape are similar to the corresponding real virus particles, but the particles do not have the complete genetic material of the virus. Therefore, they have the ability to enter cells, but will not replicate in the body. Since their components are all proteins, their biosafety is the same as that of protein subunit vaccines, and because their immunogenicity is close to that of real virus particles, they have become the focus of research in the field of vaccines.

[0036] The application of nanoparticles in vaccines is generally a new generation of vaccines that use nanoparticles as carriers or immunogens. Since nanoparticles are similar in shape to virus particles, they can more effectively stimulate the immune system, thereby stimulating the body to produce cellular and humoral immune responses more quickly. Among different types of immunogens, nanoparticles have stronger immunogenicity and immune stimulation capabilities than polymer proteins.

[0037] In the experiments on RSV vaccine design and development, mice and non-human primates were used as models, respectively. Nanoparticles displaying 20 DS-Cav1 trimers on their surface were used as immunogens to induce the body to produce neutralizing antibodies at a level 10 times higher than that of DS-Cav1 trimers.

[0038] However, in the research of nanoparticles, there are still different difficulties in preparing nanoparticles from different viral sources, such as the inability to assemble efficiently in vitro, low expression yield, low purification efficiency, high cost of large-scale industrial production and cost control when applied to economic animals. These problems need to be gradually resolved. Therefore, it is necessary to open up additional paths to provide new concepts and ideas for the research and development of nanoparticle vaccines.

[0039] To this end, the present invention provides a CD2v protein nanoparticle based on African swine fever virus, wherein the CD2v protein nanoparticle is self-assembled by CD2v-Spycatcher fusion protein and AP205-Spytag nanoparticles, that is, formed by spontaneous connection.

[0040] The amino acid sequence of the CD2v-Spycatcher fusion protein is shown in SEQ ID NO.1, and the amino acid sequence of the AP205-Spytag nanoparticle is shown in SEQ ID NO.2.

[0041] The amino acid sequence of SEQ ID NO.1 is: MIILIFLIFSNIVLSIDYWVSFNKTIILDSN ITNDNNDINGVSWNFFNNSFNTLATCGKAGNFCECSNYSTSIYNITNNCSLTIFPHNDVFDTTYQVVWNQIINYTIKLLTPATPPNITYNCTNFLITCKKNNGTNTNIYLNINDTFVKYTNESILEYNWNNSNINNFTATCIINNTIS TSNETTLINCTYLTLSSNYFYTFFKLYMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIHHHHHH;

[0042] The amino acid sequence of SEQ ID NO.2 is: MANKPMQPITSTANKIVWSDPTRLSTT FSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSI RTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTTA AHIVMVDAYKPTKHHHHHH.

[0043] It should be noted that when the CD2v protein nanoparticles were identified by electrophoresis, the molecular weight of the corresponding band was significantly higher than that of the CD2v-Spycatcher fusion protein. In molecular sieve identification, the ultraviolet absorption peaks of different components were at different positions, and in transmission electron microscopy observation, the surface of the CD2v protein nanoparticles showed obvious irregular protrusions, i.e., they had synaptic structures. The CD2v protein nanoparticles in the present invention can be understood as CD2v-AP205 nanoparticles.

[0044] In order to obtain the CD2v protein nanoparticles, a method for preparing CD2v protein nanoparticles based on African swine fever virus comprises the steps of:

[0045] S1, Obtain CD2v-Spycatcher fusion protein and AP205-Spytag nanoparticles.

[0046] Among them, the amino acid sequence of the CD2v-Spycatcher fusion protein is shown in SEQ ID NO.1, and the amino acid sequence of the AP205-Spytag nanoparticle is shown in SEQ ID NO.2.

[0047] The nucleotide sequence encoding the CD2v-Spycatcher fusion protein may be as shown in SEQ ID NO.3, and the nucleotide sequence encoding the AP205-Spytag nanoparticle may be as shown in SEQ ID NO.4.

[0048] SEQ ID NO.The nucleotide sequence of 3 is: ATGATCATCCTGATCTTCCTGATCTTCAGCAACATCGTGCTGAGCATCGACTACTGGGTGAGCTTCAACAAGACCATCATCCTGGACAGCAACATCACCAACGACAACAACGACATCAACGGCGTGAGCTGGAACTTCTTCAACAACAGCTTCAACACCCTGGCCACCTGCGGCAAGGCCGGCAACTTCTGCGAGTGCAGCAACTACAGCACCAGCATCTACAACATCACCAACAACTGCAGCCTGACCATCTTCCCCCACAACGACGTGTTCGACACCACCTACCAGGTGGTGTGGAACCAGATCATCAACTACACCATCAAGCTGCTGACCCCCGCCACCCCCCCCAACATCACCTACAACTGCACCAACTTCCTGATCACCTGCAAGAAGAACAACGGCACCAACACCAACATCTACCTGAACATCAACGACACCTTCGTGAAGTACACCAACGAGAGCATCCTGGAGTACAACTGGAACAACAGCAACATCAACAACTTCACCGCCACCTGCATCATCAACAACACCATCAGCACCAGCAACGAGACCACCCTGATCAACTGCACCTACCTGACCCTGAGCAGCAACTACTTCTACACCTTCTTCAAGCTGTACATGGTGGACACCCTGAGCGGCCTGAGCAGCGAGCAGGGCCAGAGCGGCGACATGACCATCGAGGAGGACAGCGCCACCCACATCAAGTTCAGCAAGAGGGACGAGGACGGCAAGGAGCTGGCCGGCGCCACCATGGAGCTGAGGGACAGCAGCGGCAAGACCATCAGCACCTGGATCAGCGACGGCCAGGTGAAGGACTTCTACCTGTACCCCGGCAAGTACACCTTCGTGGAGACCGCCGCCCCCGACGGCTACGAGGTGGCCACCGCCATCACCTTCACCGTGAACGAGCAGGGCCAGGTGACCGTGAACGGCAAGGCCACCAAGGGCGACGCCCACATCCACCACCATCACCACCATTGA;.

[0049] SEQ ID The nucleotide sequence of NO.4 is: ATGGCCAACAAGCCCATGCAGCCCATCACCAGCACCGCCAACAAGATCGTGTGGAGCGACCCCACCAGGCTGAGCACCACCTTCAGCGCCAGCTGCTGAGGCAGAGGGTGAAGGTGGGCATCGCCGAGCTGAACAACGTGAGCGGCCAGTACGTGAGCGTGTACAAGAGGCCCGCCCCCAAGCCCGAGGGCTGCGCCGACGCCTGCGTGATCATGCCCAA CGAGAACCAGAGCATCAGGACCGTGATCAGCGGCAGCGCCGAGAACCTGGCCACCCTGAAGGCCGAGTGGGAGACCCACAAGAGGAACGTGGACACCCTGTTCGCCAGCGGCAACGCCGGCCTGGGCTTCCTGGACCCCACCGCCGCCATCGTGAGCAGCGACACCACCGCCGCCCACATCGTGATGGTGGACGCCTACAAGCCCACCAAGCACCACCATCACCACCATTAA.

[0050] S2, incubating the CD2v-Spycatcher fusion protein and the AP205-Spytag nanoparticles in a buffer solution to obtain the CD2v protein nanoparticles; at this time, the CD2v protein nanoparticles exist in the buffer solution.

[0051] Wherein, before incubation, the CD2v-Spycatcher fusion protein and the AP205-Spytag nanoparticles can be fully mixed in a buffer solution, the incubation temperature can be 4-37° C., and the incubation time can be 1-72 h.

[0052] In order to ensure the preparation efficiency of the CD2v protein nanoparticles, as an exemplary description, the CD2v-Spycatcher fusion protein and the AP205-Spytag nanoparticles can be placed in a buffer solution to a fixed volume, the molar concentration of the CD2v-Spycatcher fusion protein can be 1-6 μM, and the molar concentration of the AP205-Spytag nanoparticles can be 1-2 μM.

[0053] The molar concentration ratio of the CD2v-Spycatcher fusion protein and the AP205-Spytag nanoparticles in the buffer solution may be 2:1-1:3; preferably, it may be 1:1.

[0054] In order to improve the purity of the CD2v protein nanoparticles, the step S2 may also include: after incubating the CD2v-Spycatcher fusion protein and the AP205-Spytag nanoparticles in a buffer solution, purifying the generated CD2v protein nanoparticles; the purification method may specifically be gel chromatography.

[0055] The CD2v protein nanoparticles in the present invention are prepared by any of the preparation methods described above.

[0056] The present invention also provides a use of any of the CD2v-Spycatcher fusion proteins described above in the preparation of an African swine fever vaccine.

[0057] The present invention also provides a use of any of the CD2v protein nanoparticles described above in the preparation of an African swine fever vaccine.

[0058] The present invention also provides an African swine fever vaccine, which includes the CD2v-Spycatcher fusion protein as described above, or the African swine fever vaccine includes the CD2v protein nanoparticles as described above; the African swine fever vaccine can generally also include an adjuvant for preparing the vaccine.

[0059] The present invention also provides a use of any of the above-described CD2v-Spycatcher fusion proteins in the preparation of CD2v-specific antibodies.

[0060] The present invention also provides a use of any of the above-mentioned CD2v protein nanoparticles in the preparation of CD2v-specific antibodies.

[0061] It should be noted that the present invention prepared nanoparticles fused with Spytag based on AP205, and after negative staining, particles with uniform morphology and size were observed under a transmission electron microscope, with a diameter of about 30nm. At the same time, the envelope protein CD2v extracellular domain on the surface of the African swine fever virus was fused with Spycatcher to obtain a recombinant fusion protein, which was connected to the AP205-Spytag nanoparticles, and identified and purified, and finally the AP205 nanoparticles connected to the CD2v extracellular domain were obtained. Under a transmission electron microscope, compared with the AP205 nanoparticles not connected to CD2v, there are obvious protrusions on its surface, and in the electrophoresis identification, the apparent molecular weight of the target band is significantly increased, indicating that the prepared AP205-Spytag nanoparticle carrier is successfully connected to the CD2v extracellular domain protein.

[0062] In subsequent mouse immunization experiments, the ability of different immunogens to stimulate the body to produce antibodies was evaluated. The results showed that 2-4 weeks after the first immunization, after the CD2v extracellular domain was displayed on the surface of AP205 nanoparticles, the antibody level was 50-100 times that of the non-nanoparticle group. Although the difference in antibody levels was reduced after booster immunization, it was still about 50 times. The present invention successfully improved the immunogenicity of CD2v using AP205 nanoparticle carriers, and revealed the advantages of AP205 nanoparticles as carriers to display exogenous antigens in the field of vaccines.

[0063] The present invention is now specifically described with reference to examples:

[0064] Example 1

[0065] 1. Plasmid construction

[0066] The sequence of ASFV serotype 8 Pig / HLJ / 18CD2v (GenBank number: QBH90546) was downloaded from the NCBI database, and the extracellular domain of CD2v was fused with the Spycatcher sequence (CD2v-Spycatc her), codon optimized and synthesized by the company (for obtaining CD2v-Spycatcher fusion protein), and subcloned into the pCDNA3 series eukaryotic expression vector.

[0067] The Spytag sequence was fused with the gene sequence of AP205 nanoparticles (AP205-Spytag), synthesized by the company (for obtaining AP205-Spytag nanoparticles) after codon optimization, and subcloned into the pET-28a prokaryotic expression vector.

[0068] In the present invention, the amino acid sequence of the CD2v-Spycatcher fusion protein is shown in SEQ ID NO. 1, and the amino acid sequence of the AP205-Spytag nanoparticle is shown in SEQ ID NO. 2. The nucleotide sequence encoding the CD2v-Spycatcher fusion protein is shown in SEQ ID NO. 3, and the nucleotide sequence encoding the AP205-Spytag nanoparticle is shown in SEQ ID NO. 4.

[0069] 2. Plasmid extraction

[0070] Take the constructed plasmid out of the -20℃ refrigerator, take 1μL of plasmid and mix it with 30μL of Escherichia coli competent cells DH5α, place it on ice for 30min, put it in a 42℃ water bath for 60s, immediately put it on ice for 5min, add 400μL of LB medium without antibiotics, culture it in a shaker at 37℃200rpm for 40min, take 20μL and evenly spread it on the LB culture containing antibiotics, and place it upside down in a 37℃ constant temperature incubator. After culturing for 12h, add 5mL of LB containing antibiotics to the test tube, pick a single colony from the culture dish into the test tube, culture it in a shaker at 37℃200rpm for 14h, and continue to expand the culture volume as needed, and then use a commercial extraction kit to extract the plasmid.

[0071] 3. Expression and purification of CD2v-Spycatcher fusion protein

[0072] Select 293F cells with good growth status, fresh SMM 293-TII medium, and adjust the cell density to 2×106 cells / mL. Transfect 293F cells with the plasmid of CD2v extracellular domain fused to Spycatcher, and culture them in a constant temperature shaker with 5% CO2 at 140 rpm and 37°C for 4-5 days.

[0073] Obtain the total protein in the supernatant expressing the recombinant protein (collect the supernatant by centrifugation).

[0074] After ultrafiltration through a 10 kDa membrane, the total protein in the supernatant was mixed with Ni-NTA filler and incubated for 1 h. The total protein in the supernatant was purified by nickel column, and the following operations were performed in sequence:

[0075] Collect the flow-through liquid F;

[0076] Wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) with a concentration of 20 mM imidazole was used to remove foreign proteins to obtain a washing solution W1 with a concentration of 20 mM imidazole;

[0077] Wash buffer (50 mM NaH2PO4, 300 mM NaCl, 30 mM imidazole, pH 8.0) with a concentration of 30 mM imidazole was used to remove foreign proteins to obtain a washing solution W2 with a concentration of 30 mM imidazole;

[0078] Wash buffer (50 mM NaH2PO4, 300 mM NaCl, 50 mM imidazole, pH 8.0) with a concentration of 50 mM imidazole was used to remove foreign proteins to obtain a washing solution W3 with a concentration of 50 mM imidazole;

[0079] The target protein was eluted using Elution buffer (50 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole, pH 8.0) and collected in order to obtain eluates E1-E4 containing the target protein.

[0080] The collected liquid was subjected to SDS-PAGE electrophoresis.

[0081] Experimental results:

[0082] The protein solutions collected before and after nickel column purification were identified by SDS-PAGE. Figure 1 As shown:

[0083] When the protein sample was identified by electrophoresis, the addition of β-me put the sample under reducing conditions, the disulfide bonds were destroyed, and an obvious diffuse band appeared near 70kDa. When β-me was not added during electrophoresis, two obvious diffuse bands were observed on the electrophoretogram, located near 70 and 180kDa, respectively, and the main band was located near 180kDa; the target protein (CD2v-Spycatcher fusion protein) was effectively purified and enriched.

[0084] The following is Figure 1 The specific meaning of each symbol in:

[0085] M: protein standard 26616; 27: CD2v-Spycatcher fusion protein control sample (eluent); F: flow-through; W1-W3 (W1, W2, W3): washing solutions with different imidazole concentrations; E1-E4 (E1, E2, E3, E4): eluent; +β-me means that a reducing agent was added during sample pretreatment; -β-me means that no reducing agent was added during sample pretreatment.

[0086] 4. Expression and purification of AP205-Spytag nanoparticles

[0087] Mix 1 μL AP205-Spytag plasmid with 100 μL E. coli competent cells BL21, place on ice for 30 minutes, place in a 42°C water bath for 60 seconds, immediately place on ice for 5 minutes, add 400 μL LB medium without antibiotics, resuscitate in a 37°C shaker for 60 minutes, take 100 μL and evenly spread on LB culture containing Kanamycin, and culture in a 37°C constant temperature incubator for 12 hours. Then add 10 mL LB containing Kanamycin to the test tube, pick a single colony from the culture dish into the test tube, culture at 37°C 200rpm for 12 hours, transfer to a large volume shake flask, culture at 37°C 200rpm for 2 hours, add the inducer IPTG, and culture at 28°C 180rpm for 14 hours. After centrifugation at 4,000 g for 10 min, the supernatant was discarded, the colony precipitate was collected, resuspended in lysis buffer (100 mM NaH2PO4, 300 mM NaCl, 20 mM Mimidazole, pH 8.0), disrupted in an ultrasonic disruptor, centrifuged at 20,000 g for 30 min at 4°C, and the total protein in the supernatant was collected.

[0088] The total protein in the supernatant was mixed with Ni-NTA filler and incubated for 1 hour. The total protein in the supernatant was purified by nickel column and the following operations were performed in sequence:

[0089] Collect the flow-through liquid F;

[0090] Use a low-concentration imidazole wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) to remove impurities and obtain a low-concentration imidazole wash buffer W1;

[0091] A high-concentration imidazole wash buffer (50 mM NaH2PO4, 300 mM NaCl, 30 mM imidazole, pH 8.0) was used to remove impurities and obtain a high-concentration imidazole wash buffer W2;

[0092] The target protein was eluted using Elution buffer (50 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole, pH 8.0) and collected in order to obtain eluates E1-E5 containing the target protein.

[0093] The quality of the collected fluid was checked by SDS-PAGE electrophoresis.

[0094] Experimental results:

[0095] The protein solutions of AP205-Spytag nanoparticles before and after purification were collected by using nickel column and identified by SDS-PAGE, as shown in Figure 2As shown:

[0096] After electrophoretic separation, a clear band was observed around 18 kDa, and its molecular weight was consistent with the expected molecular weight of AP205-Spytag nanoparticles, indicating that the target protein (AP205-Spytag nanoparticles) was effectively purified and enriched during the purification process, providing a basis for subsequent experiments.

[0097] The following is Figure 2 The specific meaning of each symbol in:

[0098] M: protein standard 26616; F: flow-through; W1, W2: washing solutions with different concentrations of imidazole; E1-E5 (E1, E2, E3, E4, E5): eluents.

[0099] 5. Ligation reaction between CD2v-Spycatcher and AP205-Spytag nanoparticles

[0100] CD2v-Spycatcher fusion protein and AP205-Spytag nanoparticles were mixed and incubated in 20 μL PBS at the following molar concentration ratios to spontaneously form binding products, which were then identified by SDS-PAGE electrophoresis.

[0101] 1: 0 μM AP205-Spytag nanoparticles and 6 μM CD2v-Spycatcher fusion protein;

[0102] 2: 2 μM AP205-Spytag nanoparticles and 0 μM CD2v-Spycatcher fusion protein;

[0103] 3: 2 μM AP205-Spytag nanoparticles and 1 μM CD2v-Spycatcher fusion protein, ratio 2:1;

[0104] 4: 2 μM AP205-Spytag nanoparticles and 2 μM CD2v-Spycatcher fusion protein, ratio 1:1;

[0105] 5: 2 μM AP205-Spytag nanoparticles and 4 μM CD2v-Spycatcher fusion protein, ratio 1:2;

[0106] 6: 2μM AP205-Spytag nanoparticles and 6μM CD2v-Spycatcher fusion protein, ratio 1:3.

[0107] Experimental results:

[0108] In the reactivity test experiment between CD2v-Spycatcher fusion protein and AP205-Spytag, the molar concentration of AP205-Spytag nanoparticles was 2μM, and the concentration of the added CD2v-Spyc atcher fusion protein was gradually increased from 0μM, 1μM, 2μM, 4μM and 6μM to react with AP205-Spytag nanoparticles respectively, and the results were analyzed by electrophoresis.

[0109] Reference Figure 3 As shown, when the molar concentration of CD2v-Spycatcher fusion protein is 2 μM, the molar concentration ratio of the two is 1:1 (Line 4); compared with Line 2, the band of AP205-Spytag nanoparticles in Line 4 is significantly weakened to disappear, and the band representing CD2v-Spycatcher fusion protein is significantly higher in Line 4 than in Line 1; this indicates that a connection reaction occurred between the CD2v-Spycatcher fusion protein and the AP205-Spyt ag nanoparticles.

[0110] Further observation revealed that when the molar concentration of the CD2v-Spycatcher fusion protein continued to increase and the molar concentration ratio of the two changed, the band representing the CD2v-Spycatcher fusion protein gradually moved downward, indicating that when the molar concentration ratio of the two was 1:1, the reaction had reached saturation.

[0111] The above experimental results show that the CD2v-Spycatcher fusion protein can successfully undergo a connection reaction with AP205-Spytag nanoparticles and reach saturation at a certain concentration ratio.

[0112] The following is Figure 3 The specific meaning of each symbol in:

[0113] M: protein standard 26616;

[0114] 1: 0 μM AP205-Spytag nanoparticles and 6 μM CD2v-Spycatcher fusion protein;

[0115] 2: 2 μM AP205-Spytag nanoparticles and 0 μM CD2v-Spycatcher fusion protein;

[0116] 3: 2 μM AP205-Spytag nanoparticles and 1 μM CD2v-Spycatcher fusion protein, ratio 2:1;

[0117] 4: 2 μM AP205-Spytag nanoparticles and 2 μM CD2v-Spycatcher fusion protein, ratio 1:1;

[0118] 5: 2 μM AP205-Spytag nanoparticles and 4 μM CD2v-Spycatcher fusion protein, ratio 1:2;

[0119] 6: 2μM AP205-Spytag nanoparticles and 6μM CD2v-Spycatcher fusion protein, ratio 1:3.

[0120] 6. Separation of Nanoparticles by Gel Chromatography

[0121] A chromatography column filled with gel chromatography filler Sepharose 4FF was installed in the AKTA Pure fast protein purification system. A Superdex 200 gel chromatography column was installed in the AKTA Pure fast protein purification system. The residual ethanol in the column was rinsed clean at a flow rate of 1 mL / min, and the chromatography column was balanced with PBS as the loading mobile phase at a flow rate of 1 mL / min. The purified and concentrated CD2v-Spycatcher fusion protein and AP205-Spytag nanoparticles were mixed at an optimal molar concentration ratio (1:1) to obtain a CD2v-AP205 solution, which was centrifuged at 4°C and 10,000g for 5 min in a centrifuge. 1.1 to 1.2 mL of the supernatant after centrifugation was taken with a 2.5 mL disposable syringe and pushed into a 1 mL sample loop. The sample was then separated at a flow rate of 0.5 mL / min. According to the ultraviolet absorption peak diagram, the flow-through obtained at different time points was collected, and each component was identified by SDS-PAGE electrophoresis.

[0122] Experimental results:

[0123] According to the optimal connection ratio, the CD2v-Spycatcher fusion protein and AP205-Spytag nanoparticle reaction system was amplified to expand the reaction volume. The prepared connection sample was identified by molecular sieve to obtain the ultraviolet absorption peak diagram of the flow-through component at different time points ( Figure 4 ) and the corresponding electrophoresis identification results ( Figure 5 ).

[0124] When the flow-through sample is the 6th tube, according to the electrophoresis diagram of the negative control CD2v-Spycatcher fusion protein, the corresponding sample is an excess of unconnected CD2v-Spycatcher fusion protein, and when the flow-through sample is the 5th tube, there are some excess unconnected CD2v-Spycatcher fusion protein and AP205-Spytag nanoparticles connected to CD2v, and when the flow-through samples are the 3rd and 4th tubes, the corresponding samples are purified and connected CD2v-AP205 nanoparticles. It is further explained that the present invention successfully prepares AP205-Spytag nanoparticles, connects CD2v-Spycatcher fusion protein to AP205-Spytag nanoparticles, and smoothly purifies to obtain CD2v-AP205 nanoparticles (CD2v protein nanoparticles prepared by the present invention).

[0125] The following is Figure 5 The specific meaning of each symbol in:

[0126] M: protein standard 26616; 27: CD2v-Spycatcher fusion protein control sample; 3-6: corresponding tube numbers after gel chromatography column flow-through.

[0127] 7. Nanoparticle morphology identification

[0128] According to the electrophoresis identification results, the component whose main component was CD2v-AP205 nanoparticles was selected, and 10 μL was taken for use; and a control group (10 μL AP205-Spytag nanoparticles) was set up at the same time.

[0129] The obtained CD2v-AP205 nanoparticles and AP205-Spytag nanoparticles were added to the copper grid of the electron microscope and allowed to stand for 1 min. The residual solution was removed with filter paper, and then 10 μL of uranyl acetate solution was added. After 30 seconds, the residual solution was removed to complete the negative staining step. The morphology of the AP205 surface after displaying the CD2v extracellular domain was observed using a 200KV transmission electron microscope.

[0130] Experimental results:

[0131] like Figure 6 As shown, the AP205-spytag nanoparticle sample was observed under an electron microscope to have a diameter of about 30nm, with regular morphology, high uniformity between particles, and a rounded surface. However, the surface of the CD2v-AP205 nanoparticles showed objects that were obviously similar to synapses, which was completely inconsistent with the smooth surface of the AP205-spytag nanoparticles, indicating that the CD2v-Spycatcher fusion protein was successfully displayed on the surface of the AP205-Spytag nanoparticles.

[0132] 8. Protein concentration determination

[0133] The protein concentration was determined using the BCA protein quantification kit. The main steps are as follows:

[0134] Dilute the 2 mg / mL BSA standard with normal saline to 1.5 mg / mL, 1 mg / mL, 0.75 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL and 0.025 mg / mL, respectively, to form BSA standard samples with different concentrations ranging from 0-2 mg / mL, and keep at 4°C for later use;

[0135] According to the number of protein samples to be tested and BSA standard samples of different concentrations, prepare the working solution of BCA Reagent A and B at a ratio of 50:1, mix thoroughly, and use it immediately after preparation;

[0136] Take a new 96-well plate, add 10 μL of BSA standard samples of different concentrations and protein samples to be tested, then add 200 μL of freshly prepared working solution to each sample well, and incubate in a 37°C incubator for 30 min;

[0137] The 96-well plate was taken out and the absorbance values ​​corresponding to different wells were measured with an ELISA reader at a wavelength of 562 nm;

[0138] According to the absorbance values ​​corresponding to the different concentrations of BSA standard samples, the calculation formula corresponding to the standard curve was obtained, and the linear relationship between the two was evaluated according to R2;

[0139] The protein concentration of each sample was calculated using the formula formed by the standard curve.

[0140] Then, according to the number of mice in each group, the total amount of immunogen required for each immunization was calculated, and the protein concentration and solution volume of each group were adjusted using PBS.

[0141] 9. Animal Immunization

[0142] The mice were randomly divided into three groups, namely, CD2v-Spycatcher fusion protein group, CD2v-AP205 protein nanoparticle group and control group, with 6 mice in each group.

[0143] The CD2v-Spycatcher fusion protein group used CD2v-Spycatcher fusion protein as the immunogen; the CD2v-AP205 protein nanoparticle group used CD2v-AP205 nanoparticles (CD2v protein nanoparticles) as the immunogen.

[0144] Each group was immunized twice subcutaneously (first immunization and second booster immunization), each time with the same immunogen, each dose of 5μg / mouse, and the control group was injected with an equal volume of PBS solution at the same time; the first immunization and the second booster immunization were separated by 4 weeks. A small amount of serum was collected every two weeks and stored at -20℃ for future use.

[0145] 10. Antibody titer assessment

[0146] On a 96-well plate, CD2v extracellular domain protein was used as an antigen, after coating and BSA blocking, mouse serum at different dilutions was added and incubated at 37°C for 1 hour. Then, the solution was removed and washed 5 times with PBST, followed by the addition of goat anti-mouse HRP secondary antibody diluted 1:7000 and incubation at 37°C for 30 minutes. Then, the solution was removed and washed 5 times with PBST, TMB colorimetric solution was added, and incubation was performed at 37°C for 10 minutes. Finally, 2M H2SO4 solution was added to terminate the color reaction, and the value at OD450nm was read using an enzyme reader.

[0147] According to the positive result detected at the highest dilution factor, the dilution factor value at this time is converted into Log 10 The changes in total IgG antibody levels were plotted on the ordinate with different collection time points as the horizontal axis to analyze the differences between different experimental groups.

[0148] Experimental results:

[0149] The serum of mice immunized at different time points in different experimental groups was collected and the CD2v-specific IgG was measured by ELISA. Figure 7 As shown:

[0150] 2-4 weeks after the first immunization, the level of CD2v-specific antibodies in the mouse serum of the CD2v-AP205 protein nanoparticle group was significantly higher than that of the CD2v-Spycatcher fusion protein group, with a difference of about 50 times.

[0151] After the second immunization in week 4, it was found that the titer of CD2v-specific antibodies in the group with CD2v-AP205 protein nanoparticles as the immunogen was 10 4.5 The CD2v-Spycatcher fusion protein group has a CD2v-specific antibody titer of 10 3.2 The CD2v antibody levels produced by each group tended to be stable from the 6th week to the 10th week, and no downward trend was observed.

[0152] This indicates that the antibody level produced after immunization with CD2v-AP205 protein nanoparticles is significantly higher than that of the CD2v-Spycatcher fusion protein group, and high-level antibodies are produced earlier. In summary, the present invention successfully utilizes nanoparticles to improve the immunogenicity of CD2v. Nanoparticle carriers can effectively improve the immunogenicity and antibody level of the vaccine, and have important potential when used in ASFV vaccines.

[0153] Figure 7 In the experiment, the IgG antibody titer of mouse sera collected from 0 to 10 weeks was detected by ELISA endpoint method and converted into Log value for statistical analysis. P value was calculated by two-tailed test. **p<0.01.

[0154] The above technical solutions of the present invention are only preferred embodiments of the present invention, and the patent scope of the present invention is not limited thereto. All equivalent structural changes made by using the contents of the present invention specification and drawings under the technical concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A CD2v-Spycatcher fusion protein based on African swine fever virus, characterized in that: The amino acid sequence of the CD2v-Spycatcher fusion protein is shown in SEQ ID NO.

1.

2. A CD2v protein nanoparticle based on African swine fever virus, characterized in that: The CD2v protein nanoparticles are formed by self-assembly of CD2v-Spycatcher fusion protein and AP205-Spytag nanoparticles; The amino acid sequence of the CD2v-Spycatcher fusion protein is shown in SEQ ID NO.1, and the amino acid sequence of the AP205-Spytag nanoparticle is shown in SEQ ID NO.

2.

3. A method for preparing CD2v protein nanoparticles based on African swine fever virus, characterized in that: Includes steps: S1, obtain CD2v-Spycatcher fusion protein and AP205-Spytag nanoparticles; Wherein, the amino acid sequence of the CD2v-Spycatcher fusion protein is shown in SEQ ID NO.1, and the amino acid sequence of the AP205-Spytag nanoparticle is shown in SEQ ID NO.2; S2, incubating the CD2v-Spycatcher fusion protein and the AP205-Spytag nanoparticles in a buffer solution to obtain the CD2v protein nanoparticles.

4. The preparation method according to claim 3, characterized in that: The nucleotide sequence encoding the CD2v-Spycat cher fusion protein is shown in SEQ ID NO.3, and the nucleotide sequence encoding the AP205-Spytag nanoparticle is shown in SEQ ID NO.

4.

5. The preparation method according to claim 3, characterized in that: The molar concentration ratio of the CD2v-Spycatcher fusion protein and the AP205-Spytag nanoparticles in the buffer solution is 2:1-1:

3.

6. Use of the CD2v-Spycatcher fusion protein as described in claim 1 in the preparation of African swine fever vaccine.

7. Use of the CD2v protein nanoparticles as described in claim 2 in the preparation of African swine fever vaccine.

8. An African swine fever vaccine, characterized in that: The African swine fever vaccine comprises the CD2v-Spycatcher fusion protein as described in claim 1, or the African swine fever vaccine comprises the CD2v protein nanoparticles as described in claim 2.

9. Use of the CD2v-Spycatcher fusion protein as claimed in claim 1 in preparing CD2v-specific antibodies.

10. Use of the CD2v protein nanoparticles according to claim 2 in preparing CD2v-specific antibodies.

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