C type lectin protein nanoparticle based on African swine fever virus, fusion protein, preparation and application

Abstract

The invention provides a C type lectin protein nanoparticle based on African swine fever virus, a fusion protein, and preparation and application of the C type lectin protein nanoparticle and the fusion protein. According to the present invention, Ferritin nanoparticles inserted with a Spytag label are adopted as an exogenous antigen display carrier, covalent linkage is performed on the Ferritin nanoparticles and a recombinant protein obtained after fusion of an African swine fever virus C type lectin extracellular domain and a Spypatcher, and the linked nanoparticles are successfully purified by means of gel chromatography and the like; in animal immune evaluation experiments, it is found that after the C type lectin extracellular domain is connected with the Ferritin nano-particles, the level of a C type lectin specific antibody in an organism is obviously improved, and the T cell immune response of the organism can be increased.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to C type lectin 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. Currently, ASFV is divided into eight serotypes based on the sequence of CD2v (EP402R) or C typelectin (EP153R) proteins associated with the hemagglutination inhibition test. In addition, based on the sequence of p72 (B646L), ASFV has 23 different genotypes. The antibodies formed after infection between different strains do not have complete protection, which is also a key factor in the difficulty of prevention and control.

[0003] African swine fever (ASF), caused by infection of domestic pigs, is an acute infectious disease that threatens the global pig farming industry. It is mainly transmitted through direct contact with infected objects, eating raw infected pork, bites, insect bites, and indirect contact with infected areas. In the meat and products of dead pigs, the ASF virus can survive for several months and can withstand extreme high and low temperature environments. It has been more than a hundred years since it was first reported and is widely distributed in Eurasia. In 2018, ASFV was first reported in northern China and quickly spread from north to south in pig farms across the country, causing huge economic losses to the pig farming industry. The strains that infect Chinese pigs are mainly highly virulent strains, which are highly contagious and cause clinical symptoms including high fever, bleeding, ataxia and severe depression, with a mortality rate of nearly 100%. Therefore, China attaches great importance to the research and development of ASFV vaccines, but there is still no approved commercial vaccine available on the Chinese market.

[0004] The genome of ASFV is very complex and is a highly variable virus with high adaptability and harmfulness. It consists of at least 150 ORFs, many of which encode proteins whose functions and structures are unknown. This complexity makes ASFV very challenging for vaccine developers. Currently, no methods or drugs have been developed that can completely prevent and cure ASFV.

[0005] Many large animal health companies in my country are involved in the development of African swine fever vaccines. From the currently public information, it can be seen that my country focuses more on basic research on viruses and the development of new vaccines such as subunits, live vectors, and mRNA, but there are still challenges in their safety, effectiveness, and mass production. Vietnam is currently the only country in the world that produces commercial African swine fever vaccines. In 2022, Vietnam launched two African swine fever vaccine products, namely the NAVET-ASFVAC attenuated live vaccine of the Central Animal Pharmaceutical Joint Stock Company (Navetco) and the AVAC ASF attenuated live vaccine of Vietnam AVAC Joint Stock Company. However, during the application process, Vietnam temporarily suspended the use of the vaccine due to adverse phenomena and biological problems after the use of the vaccine. The development of ASFV vaccines needs to ensure that the vaccine is safe and effective, and will not have a negative impact on vaccinated animals or humans, but this problem has not yet been solved.

[0006] In view of this, it is necessary to provide a C type lectin protein nanoparticle, fusion protein, preparation and application based on African swine fever virus to solve or at least alleviate the technical defects of the above-mentioned African swine fever vaccine, such as poor effectiveness and biosafety. Summary of the invention

[0007] The main purpose of the present invention is to provide a C type lectin protein nanoparticle, fusion protein, preparation and application based on African swine fever virus to solve the technical problems of poor effectiveness and biosafety of the above-mentioned African swine fever vaccine.

[0008] To achieve the above objectives, the present invention provides a C type lectin-Spycatcher fusion protein based on African swine fever virus, and the amino acid sequence of the C type lectin-Spycatcher fusion protein is shown in SEQ ID NO.1.

[0009] The present invention also provides a C type lectin protein nanoparticle based on African swine fever virus, characterized in that the C type lectin protein nanoparticle is formed by self-assembly of C type lectin-Spycatcher fusion protein and Ferritin-Spytag nanoparticles;

[0010] The amino acid sequence of the C type lectin-Spycatcher fusion protein is shown in SEQ ID NO.1, and the amino acid sequence of the Ferritin-Spytag nanoparticle is shown in SEQ ID NO.2.

[0011] The present invention also provides a method for preparing C type lectin protein nanoparticles based on African swine fever virus, comprising the steps of:

[0012] S1, obtain C type lectin-Spycatcher fusion protein and Ferritin-Spytag nanoparticles;

[0013] Wherein, the amino acid sequence of the C type lectin-Spycatcher fusion protein is shown in SEQ ID NO.1, and the amino acid sequence of the Ferritin-Spytag nanoparticle is shown in SEQ ID NO.2;

[0014] S2, incubating the C type lectin-Spycatcher fusion protein and the Ferritin-Spytag nanoparticles in a buffer solution to obtain the C type lectin protein nanoparticles.

[0015] Furthermore, the nucleotide sequence encoding the C type lectin-Spycatcher fusion protein is shown in SEQ ID NO.3, and the nucleotide sequence encoding the Ferritin-Spytag nanoparticle is shown in SEQ ID NO.4.

[0016] Furthermore, the molar concentration ratio of the C type lectin-Spycatcher fusion protein and the Ferritin-Spytag nanoparticles in the buffer solution is 2:1-1:3.

[0017] The present invention also provides a use of any of the above-described C type lectin-Spycatcher fusion proteins in the preparation of an African swine fever vaccine.

[0018] The present invention also provides a use of any of the C type lectin protein nanoparticles described above in the preparation of an African swine fever vaccine.

[0019] The present invention also provides an African swine fever vaccine, characterized in that the African swine fever vaccine includes any of the C type lectin-Spycatcher fusion proteins described above, or the African swine fever vaccine includes any of the C type lectin protein nanoparticles described above.

[0020] The present invention also provides a use of any of the above-mentioned C type lectin-Spycatcher fusion proteins in the preparation of C type lectin-specific antibodies.

[0021] The present invention also provides a use of any of the above-mentioned C type lectin protein nanoparticles in the preparation of C type lectin-specific antibodies.

[0022] The beneficial effects of the present invention include at least:

[0023] The present invention provides a C type lectin-Spycatcher fusion protein based on African swine fever virus, and the C type lectin-Spycatcher fusion protein has immunogenicity; further, the present invention also provides a C type lectin protein nanoparticle, which is based on Ferritin nanoparticles inserted with Spytag tags as exogenous antigen display carriers, and is covalently linked to the recombinant protein obtained by fusion of the C type lectin extracellular domain of African swine fever virus and Spycatcher, and the linked nanoparticles are successfully purified by means such as gel chromatography. In animal immune evaluation experiments, it was found that after the C type lectin extracellular domain was linked to the Ferritin nanoparticles, the level of C type lectin-specific antibodies in the body was significantly improved.

[0024] Although after booster immunization, the titer of IgG antibodies stimulated by the C type lectin group not connected to nanoparticles was very similar, analysis of the titers of IgG antibody subtypes (IgG1 and IgG2a) revealed that the antibody titer of IgG2a in the nanoparticle group was significantly increased, indicating that the immune response caused by the nanoparticle group was mainly Th1-tropic, which is beneficial to increase the body's T cell immune response.

[0025] The present invention successfully improves the immunogenicity of C type lectin by using ferritin nanoparticles, provides a strong theoretical basis for solving the problem of insufficient T cell immune response in the design of African swine fever virus vaccine, and clarifies the application potential of C type lectin protein nanoparticles in the field of vaccines. In addition, the present invention also solves the problem of vaccine safety, because the protein does not involve live viruses, does not belong to inactivated vaccines and attenuated vaccines, and only has protein components, which are safe and harmless, thus ensuring the biological safety of the vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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.

[0027] Figure 1 This is the SDS-PAGE identification diagram of the C type lectin-Spycatcher fusion protein in Example 1 of the present invention;

[0028] Figure 2 This is the SDS-PAGE identification diagram of the Ferritin-Spytag nanoparticles in Example 1 of the present invention;

[0029] Figure 3 This is a transmission electron microscopy identification image of the Ferritin-Spytag nanoparticles in Example 1 of the present invention, with a coordinate scale of 100 nm;

[0030] Figure 4 This is an SDS-PAGE identification diagram of the product after the connection reaction between the C type lectin-Spycatcher fusion protein and the Ferritin-Spytag nanoparticles in Example 1 of the present invention;

[0031] Figure 5 This is a chromatogram of the ultraviolet absorption peak after identification of the C type lectin-ferritin nanoparticle gel chromatography column in Example 1 of the present invention; along the extension direction of the horizontal axis, the tube numbers are 2, 3, 4, 5, 6, 7, 8, and 9 in sequence;

[0032] Figure 6 This is the SDS-PAGE identification diagram of the C type lectin-Ferritin nanoparticles in Example 1 of the present invention;

[0033] Figure 7 This is a monitoring graph of the mouse C type lectin-specific IgG antibody titer at different time points in Example 1 of the present invention;

[0034] Figure 8 This is a titer distribution diagram of C type lectin-specific IgG1 and IgG2a antibody subtypes in the mouse serum at the tenth week in Example 1 of the present invention.

[0035] 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

[0036] 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.

[0037] 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.

[0038] It should be noted that in the process of vaccine development, the glycoprotein on the surface of enveloped virus particles is generally responsible for the binding of the virus to the host cell-specific receptors, mediating the membrane fusion and invasion of the virus and host cells, and is also one of the key antigenic proteins that induce the body to produce neutralizing antibodies. The C type lectin of ASFV also plays an important role in the interaction between ASFV and the host. This lectin can recognize specific carbohydrates on the surface of host cells and guide ASFV to invade host cells in a directional manner. Therefore, the C type lectin in ASFV is of great significance in the study of the infection mechanism of ASFV.

[0039] Experiments conducted on ASFV chimeric viruses in pigs have shown that serotype-specific CD2v and Ctype lectin proteins in ASFV chimeric viruses are essential for immune protection against homologous ASFV attacks, and C type lectin has the potential to be a candidate target for ASFV vaccines. Due to its rapid spread and high mortality rate, ASFV is highly valued by all countries. Therefore, strictly implementing various prevention and control measures and developing safe and effective vaccines have become urgent challenges to ensure the healthy development of the pig industry.

[0040] To this end, the present invention provides a C type lectin-Spycatcher fusion protein based on African swine fever virus, and the amino acid sequence of the C type lectin-Spycatcher fusion protein is shown in SEQ ID NO.1.

[0041] The present invention also provides a C type lectin protein nanoparticle based on African swine fever virus, wherein the C type lectin protein nanoparticle is formed by self-assembly of C type lectin-Spycatcher fusion protein and Ferritin-Spytag nanoparticles, that is, formed by spontaneous connection.

[0042] The amino acid sequence of the C type lectin-Spycatcher fusion protein is shown in SEQ ID NO.1, and the amino acid sequence of the Ferritin-Spytag nanoparticle is shown in SEQ ID NO.2.

[0043] The amino acid sequence of SEQ ID NO.1 is: MGEINKPICYQNDDKIFYCPKDWVGYN NVCYYFGNEEKNYNNASNYCKQLNSTLTNNNTILVNLTKTLNLTKTYNHESNYWVNYSLIKNESVLLRDSGYYKKQKHVSLLYICSKMVDTLSGLSSEQGQSGD MTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIHHHHHH;

[0044] SEQ ID The amino acid sequence of NO.2 is: MLSKDIIKLLNEQVNKEMNSSNLYMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLVDQYIKGIAKSRKSAHIVMVDAYKPTKHHHHHH.

[0045] It should be noted that, during electrophoresis, the molecular weight of the band corresponding to the C type lectin protein nanoparticles is significantly higher than that of the C type lectin-Spycatcher fusion protein, and in molecular sieve identification, the ultraviolet absorption peaks of different components have different peak positions. The C type lectin protein nanoparticles in the present invention can be understood as C type lectin-Ferritin nanoparticles or C type lectin-Ferritin protein nanoparticles.

[0046] In order to obtain the C type lectin protein nanoparticles, the present invention also provides a method for preparing C type lectin protein nanoparticles based on African swine fever virus, comprising the steps of:

[0047] S1, obtaining C type lectin-Spycatcher fusion protein and Ferritin-Spytag nanoparticles; the C type lectin-Spycatcher fusion protein and the Ferritin-Spytag nanoparticles are obtained by plasmid construction, plasmid extraction and protein expression purification.

[0048] The amino acid sequence of the C type lectin-Spycatcher fusion protein is shown in SEQ ID NO.1, and the amino acid sequence of the Ferritin-Spytag nanoparticle is shown in SEQ ID NO.2.

[0049] In the present invention, the nucleotide sequence encoding the C type lectin-Spycatcher fusion protein may be as shown in SEQ ID NO.3, and the nucleotide sequence encoding the Ferritin-Spytag nanoparticle may be as shown in SEQ ID NO.4.

[0050] The nucleotide sequence of SEQ ID NO.3 is: ATGGGCGAGATCAACAAGCCCATCTG CTACCAGAACGACGACAAGATCTTCTACTGCCCCAAGGACTGGGTGGGCTACAACAACGTGTGCTACTACTTCGGCAACGAGGAGAAGAACTACAACAACGCCAGCAACTACTGCAAGCAGCTGAACAGCACCCTGACCAACAACAACACCATCCTGGTGAACCTGACCAAGACCCTGAACCTGACCAAGACCTACAACCACGAGAGCAACTACTGGGTGAACTACAGCCTGATCAAGAACGAGAGCGTGCTGCTGAGGGACAGCGGCTACTACAAGAAGCAGAAGCACGTGAGCCTGCTGTACATCTGCAGCAAGATGGTGGACACCCTGAGCGGCCTGAGCAGCGAGCAGGGCCAGAGCGGCGACATGACCATCGAGGAGGACAGCGCCACCCACATCAAGTTCAGCAAGAGGGACGAGGACGGCAAGGAGCTGGCCGGCGCCACCATGGAGCTGAGGGACAGCAGCGGCAAGACCATCAGCACCTGGATCAGCGACGGCCAGGTGAAGGACTTCTACCTGTACCCCGGCAAGTACACCTTCGTGGAGACCGCCGCCCCCGACGGCTACGAGGTGGCCACCGCCATCACCTTCACCGTGAACGAGCAGGGCCAGGTGACCGTGAACGGCAAGGCCACCAAGGGCGACGCCCACATCCACCACCATCACCACCATTAG;

[0051] The nucleotide sequence of SEQ ID NO.4 is: ATGCTGAGCAAGGACATCATCAAGCT.

[0052] S2, incubating the C type lectin-Spycatcher fusion protein and the Ferritin-Spytag nanoparticles in a buffer solution to obtain the C type lectin protein nanoparticles; at this time, the C type lectin protein nanoparticles exist in the buffer solution.

[0053] Wherein, before incubation, the C type lectin-Spycatcher fusion protein and the Ferritin-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.

[0054] In order to ensure the preparation efficiency of the C type lectin protein nanoparticles, as an exemplary description, the C type lectin-Spycatcher fusion protein and the Ferritin-Spytag nanoparticles can be placed in a buffer solution to a fixed volume, the molar concentration of the C type lectin-Spycatcher fusion protein can be 1-6 μM, and the molar concentration of the Ferritin-Spytag nanoparticles can be 1-2 μM.

[0055] The molar concentration ratio of the C type lectin-Spycatcher fusion protein and the Ferritin-Spytag nanoparticles in the buffer solution may be 2:1-1:3, preferably 1:2.

[0056] In order to improve the purity of the C type lectin protein nanoparticles, the step S2 may also include: after incubating the C type lectin-Spycatcher fusion protein and the Ferritin-Spytag nanoparticles in a buffer solution, purifying the generated C type lectin protein nanoparticles; the purification method may specifically be gel chromatography.

[0057] It should be noted that the present invention is based on Ferritin nanoparticles inserted with Spytag tags as exogenous antigen display carriers, which are covalently linked to the recombinant protein obtained by fusing the extracellular domain of African swine fever virus C type lectin and Spycatcher, and the connected nanoparticles are successfully purified by gel chromatography and other means. In the animal immune evaluation experiment, it was found that after the extracellular domain of C type lectin was connected to Ferritin nanoparticles, the level of C type lectin-specific antibodies in the body was significantly improved, and it was beneficial to increase the body's T cell immune response.

[0058] The C type lectin protein nanoparticles in the present invention are prepared by any of the preparation methods described above.

[0059] The present invention also provides a use of any of the above-described C type lectin-Spycatcher fusion proteins in the preparation of an African swine fever vaccine.

[0060] The present invention also provides a use of any of the C type lectin protein nanoparticles described above in the preparation of an African swine fever vaccine.

[0061] The present invention also provides an African swine fever vaccine, which includes any of the Ctype lectin-Spycatcher fusion proteins described above, or the African swine fever vaccine includes any of the Ctype lectin protein nanoparticles described above; in addition, the African swine fever vaccine can generally also include an adjuvant for preparing the vaccine.

[0062] The present invention also provides a use of any of the above-mentioned C type lectin-Spycatcher fusion proteins in the preparation of C type lectin-specific antibodies.

[0063] The present invention also provides a use of any of the above-mentioned C type lectin protein nanoparticles in the preparation of C type lectin-specific antibodies.

[0064] The present invention is now specifically described with reference to examples:

[0065] Example 1

[0066] 1. Plasmid construction

[0067] The C type lectin sequence of ASFV serotype 8 Pig / HLJ / 18 (GenBank number: QBH90545) was downloaded from the NCBI database, and its extracellular domain sequence was obtained after screening. The extracellular domain sequence was fused with the Spycatcher sequence to generate the C type lectin-Spycatcher sequence. After codon optimization, the sequence will be synthesized by the company and subcloned into the pCDNA3 series eukaryotic expression vector (for obtaining the C type lectin-Spycatcher fusion protein).

[0068] In addition, in order to construct a vaccine based on Ferritin nanoparticles using the Spytag-Spycatcher system, the Spytag sequence was fused with the gene sequence of Ferritin nanoparticles to generate the Ferritin-Spytag sequence. It was also codon optimized and synthesized by the company and then subcloned into the prokaryotic expression vector pET-28a (for obtaining Ferritin-Spytag nanoparticles) for prokaryotic expression in vitro.

[0069] In the present invention, the amino acid sequence of the C type lectin-Spycatcher fusion protein is shown in SEQ ID NO.1, and the amino acid sequence of the Ferritin-Spytag nanoparticle is shown in SEQ ID NO.2. The nucleotide sequence encoding the C type lectin-Spycatcher fusion protein is shown in SEQ ID NO.3, and the nucleotide sequence encoding the Ferritin-Spytag nanoparticle is shown in SEQ ID NO.4.

[0070] 2. Plasmid extraction

[0071] Take out the plasmid stored in the refrigerator at -20℃, and mix 1μL of the plasmid with 30μL of competent E. coli DH5α. After standing on ice for 30 minutes, put it in a 42℃ water bath for 60s of heat shock, and then put the mixture back on ice for 5 minutes. Then add 400μL of LB medium without antibiotics and culture it on a shaker at 37℃, 200rpm for 40 minutes. Take 20μL of the mixture and evenly spread it on the LB medium containing antibiotics, and place it upside down in a constant temperature incubator at 37℃. After 12 hours, add 5mL of LB containing antibiotics to the test tube, select a single colony from the LB medium and put it into the test tube, culture it on a shaker at 37℃, 200rpm for 14 hours, and continue to expand the culture scale as needed. Finally, use a commercial plasmid extraction kit for plasmid extraction.

[0072] 3、C type Expression and purification of lectin-Spycatcher fusion protein

[0073] 2×10^6 293F cells in good growth condition were inoculated into a cell culture flask containing fresh SMM 293-TII medium, and the plasmid fused with the extracellular domain of C type lectin and Spycatcher was transfected into the cells, and cultured in a constant temperature shaker at 5% CO2, 37°C, and 140rpm for 4-5 days.

[0074] The total protein in the supernatant of the cells was obtained (the supernatant was collected by centrifugation), and after ultrafiltration through a 10 kDa membrane package, the total protein in the supernatant was mixed with Ni-NTA filler and incubated for 1 hour.

[0075] The total protein in the supernatant was purified by nickel column, and the following steps were performed in sequence:

[0076] Collect the flow-through liquid F;

[0077] 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 impurities and obtain a low-concentration imidazole washing solution W1;

[0078] 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 high-concentration imidazole washing solution W2;

[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-E5 containing the target protein.

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

[0081] Experimental results:

[0082] The protein solution before and after nickel column purification was identified by SDS-PAGE, referring to Figure 1 As shown: In the protein solution before purification, there are many impurity proteins, which makes the bands unclear.

[0083] After nickel column purification, the target protein (C type lectin-Spycatcher fusion protein) was effectively enriched and purified, and its bands showed obvious enhancement and diffusion. In terms of molecular weight, it was observed that the target protein was glycosylated, so its bands were mainly distributed in the range of 40-55kDa and appeared as diffuse bands.

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

[0085] M: protein standard 26616; S: ​​total protein in supernatant; F: flow-through; W1-W2: washing solutions with different imidazole concentrations; E1-E5 (E1, E2, E3, E4, E5): eluents.

[0086] 4. Ferritin-Spytag Nanoparticle Expression and Purification

[0087] Add 1 μL of Ferritin-Spytag plasmid to 100 μL of competent E. coli BL21, mix well and place on ice for 30 minutes, then place in a 42°C water bath for 60 seconds, immediately place on ice for 5 minutes, add 400 μL of LB medium without antibiotics, and culture in a 37°C shaker for 60 minutes. Take 100 μL of bacterial solution and evenly spread it on LB medium containing Kanamycin, and culture it in a 37°C constant temperature incubator for 12 hours.

[0088] Then, 10 mL of LB containing Kanamycin was added to the test tube, and a single colony was selected and placed in the test tube, cultured at 37°C and 200 rpm for 12 hours, transferred to a large-capacity shake flask, and cultured at 37°C and 200 rpm for 2 hours. The inducer IPTG was added, and the culture was continued at 28°C and 180 rpm for 14 hours. The bacterial solution was centrifuged at 4,000g, the supernatant was discarded, the colony precipitate was collected, and the lysis solution (100mM NaH2PO4, 300mM NaCl, 20mM imidazole, pH 8.0) was used for resuspending, ultrasonically disrupted, and centrifuged at 4°C and 20,000g for 30 minutes, and the total protein in the supernatant after centrifugation was collected.

[0089] 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:

[0090] Collect the flow-through liquid F;

[0091] 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 impurities and obtain a low-concentration imidazole washing solution W1;

[0092] 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 high-concentration imidazole washing solution W2;

[0093] 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.

[0094] The collected liquid was subjected to SDS-PAGE electrophoresis.

[0095] Experimental results:

[0096] Ferritin-Spytag nanoparticles were purified by using a nickel column, and their purity and molecular weight were determined by SDS-PAGE. Figure 2 It was shown that after electrophoresis identification, a distinct band could be clearly observed with a molecular weight of approximately 23 kDa, which was consistent with the expected molecular weight of Ferritin-Spytag nanoparticles, indicating that the target protein (Ferritin-Spytag nanoparticles) was successfully purified.

[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 imidazole concentrations; E1-E5 (E1, E2, E3, E4, E5): eluents.

[0099] 5. Nanoparticle morphology identification

[0100] Take 10 μL of Ferritin-Spytag nanoparticle sample, drop it onto the electron microscope copper grid and let it stand for 1 min, then use filter paper to absorb the remaining solution and keep the particles adsorbed on the grid.

[0101] Next, add 10 μL of uranyl acetate solution for negative staining. After 30 seconds, use filter paper to absorb the residual uranyl acetate solution to complete the negative staining step.

[0102] Finally, the morphology of Ferritin-Spytag nanoparticles was observed using a transmission electron microscope at an accelerating voltage of 200KV to gain a deeper understanding of the morphology and structure of the nanoparticles.

[0103] Experimental results:

[0104] See also Figure 3 As shown, the Ferritin-spytag nanoparticle sample was observed by transmission electron microscopy and was found to have a uniform spherical structure with a diameter of about 10 nm, and the particles were evenly distributed and had a regular morphology.

[0105] The observation results show that the Ferritin-spytag nanoparticle samples have high uniformity and morphological stability, which provides an important structural basis for its application in the biomedical field. In addition, transmission electron microscopy observation can also provide valuable microstructural information for subsequent further analysis.

[0106] 6、C type Ligation reaction of lectin-Spycatcher and Ferritin-Spytag nanoparticles

[0107] C type lectin-Spycatcher fusion protein and Ferritin-Spytag nanoparticles were mixed and incubated in 20 μL PBS at the following molar concentration ratios, thereby spontaneously forming a binding product.

[0108] 1: 0 μM Ferritin-Spytag nanoparticles and 6 μM C ​​type lectin-Spycatcher fusion protein;

[0109] 2: 2 μM Ferritin-Spytag nanoparticles and 0 μM C ​​type lectin-Spycatcher fusion protein;

[0110] 3: 2 μM Ferritin-Spytag nanoparticles and 1 μM C ​​type lectin-Spycatcher fusion protein, ratio 2:1;

[0111] 4: 2 μM Ferritin-Spytag nanoparticles and 2 μM C ​​type lectin-Spycatcher fusion protein, ratio 1:1;

[0112] 5: 2 μM Ferritin-Spytag nanoparticles and 4 μM C ​​type lectin-Spycatcher fusion protein, ratio 1:2;

[0113] 6: 2μM Ferritin-Spytag nanoparticles and 6μM C type lectin-Spycatcher fusion protein, ratio 1:3.

[0114] In order to detect the binding status, SDS-PAGE electrophoresis was used for identification, the charged properties and relative molecular mass of the product were observed, the binding effect of different concentrations of C type lectin-Spycatcher and Ferritin-Spytag nanoparticles was determined, and the appropriate molar concentration was selected for subsequent experiments.

[0115] Experimental results:

[0116] The reactivity of C type lectin-Spycatcher fusion protein with Ferritin-Spytag nanoparticles was tested. Figure 4As shown, when the molar concentration of Ferritin-Spytag nanoparticles was maintained at 2 μM, the molar concentration of Ctype lectin-Spycatcher was changed from 0 μM, 1 μM, 2 μM, 4 μM and 6 μM to react with Ferritin-Spytag nanoparticles respectively, and judgment was made based on the changes in the molar concentrations of the two.

[0117] When the molar concentration ratio of the two is 2:1, the band of Ferritin-Spytag in Line 3 is significantly weakened compared to Line 2, and the band representing C type lectin-Spycatcher in Line 3 is significantly higher than that in Line 1, indicating that C type lectin-Spycatcher and Ferritin-Spytag have undergone a connection reaction. If the molar concentration of C type lectin-Spycatcher continues to increase from Line 3 to Line 6, it is found that the band representing C type lectin-Spycatcher moves downward, indicating that the reaction reaches saturation when the molar concentration ratio of Ferritin-Spytag nanoparticles and C type lectin-Spycatcher fusion protein is 2:1. The subsequent scale-up is carried out in proportion to expand the connection volume.

[0118] The following is Figure 4 The specific meaning of each symbol in:

[0119] M: protein standard 26616;

[0120] 1: 0 μM Ferritin-Spytag nanoparticles and 6 μM C ​​type lectin-Spycatcher fusion protein;

[0121] 2: 2 μM Ferritin-Spytag nanoparticles and 0 μM C ​​type lectin-Spycatcher fusion protein;

[0122] 3: 2 μM Ferritin-Spytag nanoparticles and 1 μM C ​​type lectin-Spycatcher fusion protein, ratio 2:1;

[0123] 4: 2 μM Ferritin-Spytag nanoparticles and 2 μM C ​​type lectin-Spycatcher fusion protein, ratio 1:1;

[0124] 5: 2 μM Ferritin-Spytag nanoparticles and 4 μM C ​​type lectin-Spycatcher fusion protein, ratio 1:2;

[0125] 6: 2μM Ferritin-Spytag nanoparticles and 6μM C type lectin-Spycatcher fusion protein, ratio 1:3.

[0126] 7. Gel chromatography separation C type Lecturin protein nanoparticles

[0127] The AKTA Pure fast protein purification system was used, based on a chromatography column with gel chromatography filler Sepharose 4FF. The residual ethanol in the column was rinsed clean at a flow rate of 1 mL / min, and PBS was used as the loading mobile phase to balance the chromatography column at a flow rate of 1 mL / min.

[0128] Next, the purified and concentrated C type lectin-Spycatcher fusion protein and Ferritin-Spytag nanoparticles were mixed at the optimal molar concentration ratio (1:2) to obtain the C type lectin-Ferritin solution (C type lectin protein nanoparticle solution), which was placed in a centrifuge at 4°C 10,000g for 5 minutes, and 1.1 to 1.2 mL of the supernatant was taken out and pushed into a 1 mL sample loop with a 2.5 mL disposable syringe, and then the sample was separated at a flow rate of 0.5 mL / min. Finally, according to the UV absorption peak diagram, the flow-through liquid obtained at different time points was collected, and each component was identified by SDS-PAGE electrophoresis.

[0129] Experimental results:

[0130] The C type lectin-Ferritin nanoparticle samples prepared according to the optimal connection ratio were identified by molecular sieve, and the ultraviolet absorption peak diagram of the flow-through components at different time points was obtained ( Figure 5 ) and the corresponding electrophoresis identification results ( Figure 6 ), when the flow-through samples are tubes 5 to 9, according to the electrophoresis diagram of negative control Line 1, the corresponding samples are excess unconnected C typelectin-Spycatcher fusion protein, and when the flow-through volume is tubes 2 to 4, the corresponding samples are nanoparticles connected with C type lectin-Spycatcher fusion protein after purification. This shows that the connection reaction is effective, the C typelectin-Spycatcher fusion protein is successfully connected to the Ferritin-Spytag nanoparticles, and the purified sample is obtained.

[0131] The following is Figure 6 The specific meaning of each symbol in:

[0132] M: protein standard 26616; 1: C type lectin-Spycatcher fusion protein; 2-9: the corresponding tube number after the gel chromatography column flows through.

[0133] 8. Animal Immunization

[0134] In order to evaluate the immunogen of C type lectin-Spycatcher fusion protein and C type lectin-Ferritin protein nanoparticles (C type lectin protein nanoparticles), this example conducted a mouse immunization experiment.

[0135] First, the mice were randomly divided into three groups, with 6 mice in each group; namely, C type lectin-Spycatcher fusion protein group, C type lectin-Ferritin protein nanoparticle group and control group.

[0136] The C type lectin-Spycatcher fusion protein group was injected with C type lectin-Spycatcher fusion protein, and the C type lectin-Ferritin protein nanoparticle group was injected with C type lectin-Ferritin protein nanoparticles; the dose of each injection was 5 μg / mouse, and each immunization dose was mixed with an equal amount of adjuvant. A total of two immunizations were performed, with an interval of 4 weeks between the two immunizations; the control group was injected with an equal volume of PBS solution.

[0137] Serum was collected every two weeks and stored at -20°C for subsequent experiments. The production of specific antibodies in mouse serum and the subtype of antibodies were detected by ELISA to evaluate the immunogenic properties of C type lectin-S pycatcher fusion protein and C type lectin-Ferritin protein nanoparticles.

[0138] 9. Antibody titer assessment

[0139] The C type lectin extracellular domain protein was coated on a 96-well plate and blocked with BSA. Then, gradiently diluted mouse serum was added and incubated at 37°C for 1 hour. After discarding the solution, the plate was washed 5 times with PBST solution, and a 1:7000 diluted goat anti-mouse HRP secondary antibody was added. The plate was incubated at 37°C for 30 minutes. After discarding the solution, the plate was washed 5 times with PBST solution, TMB colorimetric solution was added and incubated at 37°C for 10 minutes. 2M H2SO4 solution was immediately added to terminate the color development reaction, and the value at OD450 nm was read with an enzyme reader to analyze the antibody titer between different experimental groups.

[0140] Experimental results:

[0141] In this example, sera from mice in different experimental groups after immunization at different time points were collected, and Ctype lectin-specific IgG was measured using the ELISA method.

[0142] Reference Figure 7 The results showed that 2-4 weeks after the first immunization, the level of C type lectin-specific antibodies in the serum of mice in the C type lectin-Ferritin protein nanoparticle group was significantly higher than that in the C type lectin-Spycatcher fusion protein group, with a difference of about 40 times.

[0143] Two weeks after the second immunization, that is, starting from the sixth week, the titer of Ctype lectin-specific antibodies in the Ctype lectin-ferritin protein nanoparticle group was 10 5 Floating left and right.

[0144] In comparison, the titer of C type lectin-specific antibodies in the C type lectin-Spycatcher fusion protein group can also reach 10 5 The C type lectin antibody levels produced by each group from week 6 to week 10 were similar in different experimental groups. However, the C type lectin-Ferritin protein nanoparticle group produced high levels of antibodies earlier, so the nanoparticle carrier has more potential in vaccine applications.

[0145] In summary, the results of this experiment show that both the C type lectin-Ferritin protein nanoparticle group and the C typelectin-Spycatcher fusion protein group can produce stable C type lectin-specific antibodies, but the advantage of the nanoparticle carrier is that it increases the speed of antibody production.

[0146] Figure 7In 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.

[0147] 10. Antibody subtype analysis

[0148] In order to detect the levels of IgG antibody subtypes IgG1 and IgG2a in each group of mice, serum samples were collected at the 10th week and tested using ELISA technology.

[0149] The serum samples of different groups of mice were serially diluted and added to the ELISA plate for incubation and washing. Next, goat anti-mouse IgG1 HRP-labeled secondary antibody and goat anti-mouse IgG2a HRP-labeled secondary antibody were added to different wells of the same sample at a dilution ratio of 1:5,000 and incubated for 30 minutes.

[0150] The absorbance of all samples was measured by ELISA, and the dilution multiple values ​​of IgG1 and IgG2a in the same sample at the highest dilution multiple at which the test results were still positive were converted into Log using the endpoint method. 10 , and graphical analysis was performed to compare the differences in antibody subtype production between different experimental groups.

[0151] Experimental results:

[0152] Although there was no significant difference in IgG antibody levels between the C type lectin-Spycatcher fusion protein group and the C type lectin-Ferritin protein nanoparticle group starting from week 6, it is unknown whether the titers of antibody subtypes are consistent. At week 10, the titers of C type lectin-specific IgG subtypes (IgG 1 and IgG2a) in the serum of each group were further studied.

[0153] The results are as follows Figure 8 As shown in Figure 2, the titers of C type lectin-specific IgG1 in both the C type lectin-Ferritin protein nanoparticle group and the C type lectin-Spycatcher fusion protein group reached 10 4.5 However, in the C typelectin-Spycatcher fusion protein group, the titer of C type lectin-specific IgG2a was only 10 3.3 , more than 100 times lower than that of the C typelectin-Ferritin protein nanoparticle group.

[0154] The above findings indicate that the C type lectin-Ferritin protein nanoparticle group was able to induce the same level of C type lectin-specific IgG1 and a higher level of C type lectin-specific IgG2a compared with C type lectin monomer.

[0155] Figure 8 In the experiment, the titers of IgG1 and IgG2a antibody subtypes were detected by endpoint method in the mouse sera collected at week 10, and then converted into Log values ​​for statistical analysis. P values ​​were calculated by two-tailed test. ns represents P>0.05, and ** represents p<0.01.

[0156] 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 C type lectin-Spycatcher fusion protein based on African swine fever virus, characterized in that: The amino acid sequence of the Ctype lectin-Spycatcher fusion protein is shown in SEQ ID NO.

1.

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

2.

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

4. The preparation method according to claim 3, characterized in that: The nucleotide sequence encoding the C type lectin-Spycatcher fusion protein is shown in SEQ ID NO.3, and the nucleotide sequence encoding the Ferritin-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 C type lectin-Spy catcher fusion protein and the Ferritin-Spytag nanoparticles in the buffer solution is 2:1-1:

3.

6. Use of the C type lectin-Spycatcher fusion protein as claimed in claim 1 in the preparation of African swine fever vaccine.

7. Use of the C type lectin 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 includes the Ctype lectin-Spycatcher fusion protein as described in claim 1, or the African swine fever vaccine includes the Ctype lectin protein nanoparticles as described in claim 2.

9. Use of the C type lectin-Spycatcher fusion protein according to claim 1 in preparing C type lectin specific antibodies.

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

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