Porcine circovirus type 2-based combined protein nanoparticles, and preparation and application thereof

Abstract

The invention discloses combined protein nanoparticles based on porcine circovirus type 2 as well as preparation and application of the combined protein nanoparticles. The combined protein nano-particle is formed by self-assembly of an RBD (Radial Basis Description)-Spatier fusion protein and a PCV2 (Poly Carbonaceous V2)-Spytag nano-particle. The combined protein nano-particle based on the porcine circovirus type 2 can generate a high-level RBD specific antibody earlier, and compared with an RBD monomer, the combined protein nano-particle based on the porcine circovirus type 2 can induce generation of a higher-level RBD specific IgG2a antibody and a same-level and same-RBD specific IgG1 antibody. Meanwhile, the level of the combined protein nanoparticles based on the porcine circovirus type 2 for generating neutralizing antibodies is highly consistent with that of wild PCV2 nanoparticles, and the immunogenicity of the PCV2 is not influenced. The combined protein nanoparticles based on the porcine circovirus type 2 can prevent infection of PCV2 and PEDV viruses at the same time, and time and cost of secondary vaccination are avoided.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to combined protein nanoparticles based on porcine circovirus type 2, and preparation and application thereof. Background Art

[0002] There are many pathogens in pig farms, such as ASFV, PEDV, PCV2, PRRSV, CSFV, PRV, etc., which have an impact on the healthy growth of pigs, causing serious economic losses and restricting the development of the breeding industry. For example, porcine epidemic diarrhea virus (PEDV) belongs to the Alphacoronavirus family of the Coronaviridae family and the Nidovirales order. It can cause serious health problems in newborn piglets, including acute diarrhea, vomiting, dehydration and high mortality. PEDV is transmitted through food, water quality and contact infection. During the infection period, the virus can survive in sewage, environmental surfaces and pig feces for up to several weeks. At present, PEDV vaccines have been widely used in some countries, including China, South Korea, Vietnam and Japan. However, in 2010, a highly virulent variant of PEDV broke out in China, making the existing vaccine ineffective against the disease. PEDV broke out again in the United States and neighboring countries in 2013, posing a major threat to the global pig industry. At present, there are limited vaccines for PEDV and their effectiveness has not yet been determined. Therefore, the development of more effective PEDV vaccines is crucial to prevent its spread and minimize its impact on the global swine industry.

[0003] Porcine circovirus type 2 (PCV2) belongs to the genus Circovirus of the family Circoviridae. It is a non-enveloped, single-stranded circular DNA virus with a genome size of about 1.7kb, encoding two major proteins (Cap and Rep / Rep'). The average size of the virus particles is 12-23nm, making it the smallest virus found in mammals so far. It causes dermatitis nephrotic syndrome (PDNS), which slows the growth of pigs, reduces feed-to-meat ratio, inhibits the body's immune response, and is prone to co-infection with other pathogens, and enhances the adverse symptoms caused by other pathogens. The commercialized VLPs vaccine of PCV2 is widely used in clinical practice and has a significant effect on the growth performance of pigs.

[0004] However, there is currently no commercial vaccine that can prevent both PEDV and PCV2. The development and application of multi-component and multivalent vaccines can avoid the time and cost of secondary vaccinations, and simultaneously prevent infection with PCV2 and PEDV viruses, providing an important guarantee for the healthy development of the pig industry, helping farmers to effectively control diseases, reduce economic losses, and improve farming efficiency and disease prevention levels. Summary of the invention

[0005] The main purpose of the present invention is to provide a combination protein nanoparticle based on porcine circovirus type 2, and its preparation and application, so as to solve the technical problems of poor effectiveness and biosafety of the above-mentioned multi-link multivalent vaccine.

[0006] To achieve the above objectives, the present application provides a combination protein nanoparticle based on porcine circovirus type 2, wherein the combination protein nanoparticle is formed by self-assembly of RBD-Spycatcher fusion protein and PCV2-Spytag nanoparticles.

[0007] The amino acid sequence of the RBD-Spycatcher fusion protein is shown in SEQ ID NO.1, and the amino acid sequence of the PCV2-Spytag nanoparticle is shown in SEQ ID NO.2.

[0008] The present application also provides a method for preparing combined protein nanoparticles based on porcine circovirus type 2, comprising the steps of:

[0009] S1, obtain RBD-Spycatcher fusion protein and PCV2-Spytag nanoparticles;

[0010] Wherein, the amino acid sequence of the RBD-Spycatcher fusion protein is shown in SEQ ID NO.1, and the amino acid sequence of the PCV2-Spytag nanoparticle is shown in SEQ ID NO.2;

[0011] S2, incubating the RBD-Spycatcher fusion protein and the PCV2-Spytag nanoparticles in a buffer solution to obtain the combined protein nanoparticles.

[0012] According to an embodiment of the present application, the nucleotide sequence encoding the RBD-Spycatcher fusion protein is shown as SEQ ID NO.3, and the nucleotide sequence encoding the PCV2-Spytag nanoparticle is shown as SEQ ID NO.4.

[0013] According to an embodiment of the present application, the molar concentration ratio of the RBD-Spycatcher fusion protein and the PCV2-Spytag nanoparticles in the buffer solution is 5:3-1:3.

[0014] According to an embodiment of the present application, the step S2 further comprises: after incubating the RBD-Spycatcher fusion protein and the PCV2-Spytag nanoparticles in a buffer solution, purifying the generated combined protein nanoparticles.

[0015] According to an embodiment of the present application, the incubation temperature is 4-37° C., and the incubation time is 1-72 h.

[0016] The present application also provides a combination protein nanoparticle based on porcine circovirus type 2, which is prepared using the above-mentioned preparation method.

[0017] The present application also provides the above-mentioned combined protein nanoparticles or the use of the above-mentioned combined protein nanoparticles in the preparation of a vaccine for simultaneously preventing porcine epidemic diarrhea virus and porcine circovirus type 2.

[0018] The present application also provides a multi-valent vaccine, which includes the combined protein nanoparticles as described above or the combined protein nanoparticles prepared by the above preparation method.

[0019] The present application also provides a use of the above-mentioned combined protein nanoparticles or the combined protein nanoparticles prepared by the above-mentioned preparation method in the preparation of a vaccine for simultaneously preventing porcine epidemic diarrhea virus and porcine circovirus type 2.

[0020] The beneficial effects of the present invention include at least:

[0021] The above-mentioned combined protein nanoparticles based on porcine circovirus type 2 can produce high levels of RBD-specific antibodies at an earlier stage. Compared with RBD monomers, they can induce the production of higher levels of RBD-specific IgG2a antibodies, as well as the same level of the same RBD-specific IgG1 antibodies. At the same time, the level of neutralizing antibodies produced by the combined protein nanoparticles based on porcine circovirus type 2 is highly consistent with that of wild PCV2 nanoparticles, and does not affect the immunogenicity of PCV2 itself. The above-mentioned combined protein nanoparticles based on porcine circovirus type 2 can prevent infection by PCV2 and PEDV viruses at the same time, avoiding the time and cost of secondary vaccination. In addition, the present invention also solves the problem of vaccine safety. Since the protein does not involve live viruses, it does not belong to inactivated vaccines and attenuated vaccines. It only has protein components and is safe and harmless, thereby ensuring the biosafety of the vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] Figure 1 This is an SDS-PAGE identification diagram of the RBD-Spycatcher fusion protein in Example 1 of the present invention;

[0024] Figure 2 This is an SDS-PAGE identification diagram of the PCV2-Spytag nanoparticles in Example 1 of the present invention;

[0025] Figure 3 This is an SDS-PAGE identification diagram after the RBD-Spycatcher fusion protein and PCV2-Spytag nanoparticles are connected in Example 1 of the present invention;

[0026] Figure 4 This is a transmission electron microscopy identification image of the PCV2-Spytag nanoparticles in Example 1 of the present invention;

[0027] Figure 5 This is a monitoring chart of the mouse RBD-specific IgG antibody titer at different time points in Example 1 of the present invention;

[0028] Figure 6 This is a titer distribution diagram of RBD-specific IgG1 and IgG2a antibody subtypes in the serum of mice in the tenth week in Example 1 of the present invention;

[0029] Figure 7 This is a graph evaluating the neutralizing antibody level in mouse serum at the tenth week in Example 1 of the present invention.

[0030] 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

[0031] 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.

[0032] 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.

[0033] The formation of PCV2 VLPs is mainly formed by the assembly of Cap protein, which is the only structural protein of PCV2. It is encoded by the ORF2 gene, has a molecular weight of about 27kDa, and is immunogenic. In order to produce PCV2 virus-like particles (VLPs), the ORF2 gene is cloned into a baculovirus vector and expressed in insect cells. The expression product can self-assemble to form highly pure PCV2 VLPs. However, the process of this method is complicated and costly, which limits its large-scale application. Therefore, many researchers have tried to use Escherichia coli to express PCV2 Cap protein. However, most attempts have ended in failure. In recent years, researchers have fused the Sumo tag to the 5' end of the PCV2 capsid gene, which has increased the expression level and solubility of the Cap protein in Escherichia coli. The purified fusion protein can be enzymatically cleaved to remove the Sumo tag, and then successfully assembled into PCV2 VLPs in vitro.

[0034] To this end, the present invention provides a combination protein nanoparticle based on porcine circovirus type 2, wherein the combination protein nanoparticle is formed by self-assembly of RBD-Spycatcher fusion protein and PCV2-Spytag nanoparticles;

[0035] The amino acid sequence of the RBD-Spycatcher fusion protein is shown in SEQ ID NO.1, and the amino acid sequence of the PC V2-Spytag nanoparticle is shown in SEQ ID NO.2.

[0036] The sequence of SEQ ID NO.1 is:

[0037] MSFVTLPSFNDHSFVNITVSAAFGGHSGANLIASDTTINGFSSFCVDTRQFTISLFYNVTNSYGYVSKSQDSNCPFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITG TPKPLEGVTDVSMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIHHHHHH.

[0038] The sequence of SEQ ID NO.2 is:

[0039] MTYPRRRYRRRRHRPRSHLGQILRRRPWLVHPRHRYRWRRKNGIFNTRLSRTIGYTVKATTVRTPSWNVDMMRFNINDFLPPGGGSNPLTVPFEYYRIRKVKVEFWPCSPITQGDRGVGSTAVILDD NFVTKANALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDRTIDYFQPNNKRNQLWLRLQTTGNVDHVGLGTAFENSIYDQDYNIRITMYVQFREFNLKDPPLNPKAHIVMVDAYKPTKHHHHHH.

[0040] In some embodiments, when the combined protein nanoparticles are subjected to electrophoresis, the molecular weight of the band corresponding to the PCV2-Spytag protein nanoparticles is significantly higher than that of the RBD-Spycatcher fusion protein, and in transmission electron microscopy observation, obvious irregular protrusions appear on the surface of the PCV2-Spytag nanoparticles.

[0041] In order to obtain the combined protein nanoparticles, the present invention also provides a method for preparing the combined protein nanoparticles based on porcine circovirus type 2, comprising the steps of:

[0042] S1, Obtain RBD-Spycatcher fusion protein and PCV2-Spytag nanoparticles.

[0043] Among them, the amino acid sequence of the RBD-Spycatcher fusion protein is shown in SEQ ID NO.1, and the amino acid sequence of the PCV2-Spytag nanoparticle is shown in SEQ ID NO.2.

[0044] Illustratively, the RBD-Spycatcher fusion protein and the PCV2-Spytag nanoparticles are obtained by plasmid construction, plasmid extraction and protein expression purification, respectively.

[0045] The nucleotide sequence encoding the RBD-Spycatcher fusion protein may be as shown in SEQ ID NO.3, and the nucleotide sequence encoding the PCV2-Spytag nanoparticle may be as shown in SEQ ID NO.4.

[0046] The sequence of SEQ ID NO.3 is: atgagcttcgtgaccctgcccagcttcaacgaccacagcttcgtgaacat caccgtgagcgccgccttcggcggccacagcggcgccaacctgatcgccagcgacaccaccatcaacggcttcagcagcttctgcgtggacaccaggcagttcaccatcagcctgttctacaacgtgaccaacagctacggctacgtgagcaagagccaggacagcaactgccccttcaccctgcagagcgtgaacgactacctgagcttcagcaagttctgcgtgagcaccagcctgctggccagcgcctgcaccatcgacctgttcggctaccccgagttcggcagcggcgtgaagttcaccagcctgtacttccagttcaccaagggcgagctgatcaccggcacccccaagcccctggagggcgtgaccgacgtgagcatggtggacaccctgagcggcctgagcagcgagcagggccagagcggcgacatgaccatcgaggaggacagcgccacccacatcaagttcagcaagagggacgaggacggcaaggagctggccggcgccaccatggagctgagggacagcagcggcaagaccatcagcacctggatcagcgacggccaggtgaaggacttctacctgtaccccggcaagtacaccttcgtggagaccgccgcccccgacggctacgaggtggccaccgccatcaccttcaccgtgaacgagcagggccaggtgaccgtgaacggcaaggccaccaagggcgacgcccacatccaccaccatcaccaccattga。

[0047] The sequence of SEQ ID NO.4 is: atgacctaccccaggaggaggtacaggaggaggaggcacaggccca.

[0048] S2, incubating the RBD-Spycatcher fusion protein and the PCV2-Spytag nanoparticles in a buffer solution to obtain the combined protein nanoparticles.

[0049] Exemplarily, the RBD-Spycatcher fusion protein and the PCV2-Spytag nanoparticles can be fully mixed in a buffer solution before incubation, the incubation temperature can be 4-37° C., and the incubation time can be 1-72 h.

[0050] In order to improve the purity of the combined protein nanoparticles, the step S2 may further include: after incubating the RBD-Spycatcher fusion protein and the PCV2-Spytag nanoparticles in a buffer solution, purifying the generated combined protein nanoparticles; the purification method may specifically be gel chromatography.

[0051] In some embodiments, the molar concentration ratio of the RBD-Spycatcher fusion protein and the PCV2-Spytag nanoparticles in the buffer solution is 5:3-1:3. Preferably, the molar concentration ratio of the two in the buffer solution is 1:2.

[0052] The present invention also provides a combined protein nanoparticle based on porcine circovirus type 2, which is prepared by any of the preparation methods described above.

[0053] The present invention also provides a use of any of the combined protein nanoparticles described above or the combined protein nanoparticles prepared by the above method in preparing a vaccine for simultaneously preventing porcine epidemic diarrhea virus and porcine circovirus type 2.

[0054] The present invention also provides a multi-valent vaccine, which comprises the above-mentioned combined protein nanoparticles or the combined protein nanoparticles prepared by the above-mentioned method.

[0055] The present invention also provides a use of the above-mentioned combined protein nanoparticles or the combined protein nanoparticles prepared by the above-mentioned method in preparing a vaccine for simultaneously preventing porcine epidemic diarrhea virus and porcine circovirus type 2.

[0056] The present invention is now specifically described with reference to examples:

[0057] Example 1

[0058] 1. Plasmid construction

[0059] According to the sequence of PEDV spike (GenBank: AFQ37598) in the NCBI database, the RBD region of PEDV was screened and fused with the Spycatcher sequence to obtain RBD-Spycatcher, which was synthesized by the company and subcloned into the pCDNA3 series eukaryotic expression vector (this plasmid is used to obtain the RBD-Spycatcher fusion protein).

[0060] In addition, the sequence of Spytag was fused with the gene sequence of PCV2 Cap protein to obtain PCV2-Spytag, which was synthesized by the company and then subcloned into the prokaryotic expression vector pET-28a (this plasmid was used to obtain PCV2-Spytag nanoparticles).

[0061] 2. Plasmid extraction

[0062] Take out the required plasmid (such as pCDNA3 series eukaryotic expression vector, prokaryotic expression vector pET-28a) from the refrigerator stored at -20℃, mix 1μL of the plasmid with 30μL of competent Escherichia coli DH5α, and let it stand on ice for 30 minutes, then place it in a 42℃ water bath for a short heat shock of 60 seconds, and then quickly put the mixture back on ice for 5 minutes to improve the transformation efficiency.

[0063] Next, 400 μL of LB medium without antibiotics was added and the mixture was cultured on a shaking platform at 37° C. and 200 rpm for 40 minutes to allow DH5α to fully absorb the plasmid.

[0064] Then, 20 μL of the mixture was spread on an LB plate containing antibiotics, inverted and placed in a constant temperature incubator at 37°C to allow colony formation and screening of transformed strains. After 12 hours of culture, LB solution containing antibiotics was added to the test tube, and a single colony was selected and transferred to the test tube, and cultured at 37°C and 200 rpm for 14 hours to expand the culture scale.

[0065] The culture volume can be further expanded as needed to facilitate subsequent plasmid extraction and purification. Finally, a commercial extraction kit is used for plasmid extraction to obtain high-quality plasmid DNA.

[0066] 3. Expression and purification of RBD-Spycatcher fusion protein

[0067] In order to express the RBD-Spycatcher fusion protein and obtain the target protein, 293F cells with good growth status were selected and the cell density was adjusted to 2×10 using fresh SMM 293-TII medium. 6 cells / mL.

[0068] Next, the plasmid of the RBD-Spycatcher fusion protein was transfected into 293F cells and cultured in a constant temperature shaker (140 rpm, 37°C, 5% CO2) for 4-5 days. During the culture process, the cells will express the target protein and secrete it into the culture medium.

[0069] Next, the supernatant was collected by centrifugation to obtain the total protein in the supernatant expressing the recombinant protein. In order to purify the target protein, Ni-NTA filler was used, and the total protein in the supernatant was mixed with the filler and incubated for 1 hour.

[0070] Purify the total protein in the supernatant by nickel column as follows:

[0071] The flow-through F was collected.

[0072] 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;

[0073] 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;

[0074] 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 high-concentration imidazole washing solution W3;

[0075] The collected liquid was subjected to SDS-PAGE electrophoresis detection and identification, purified and concentrated, and then stored at low temperature.

[0076] Experimental results:

[0077] The RBD-Spycatcher solution collected before and after the nickel column purification was identified by SDS-PAGE. Figure 1 As shown, after electrophoresis identification, obvious bands appeared around 35 to 40 kDa, indicating that the protein was successfully purified and the molecular weight was within the expected range.

[0078] The following is Figure 1 The specific meaning of each symbol in:

[0079] M: protein standard 26616; S: ​​total protein in supernatant; F: flow-through; W1-W3: washing solutions with different concentrations of imidazole; E1-E4: eluents.

[0080] 4. PCV2-Spytag Nanoparticle Expression and Purification

[0081] Mix 1 μL PCV2-Spytag plasmid with 100 μL competent cells BL21, place on ice for 30 min, place in a 42°C water bath for 60 s, and then place on ice for 5 min. Add 500 μL LB medium without antibiotics, resuscitate in a 37°C shaker for 45-60 min, apply to LB medium containing Kanamycin, and culture at 37°C for 12 h.

[0082] A single colony was selected from the culture dish and placed in LB containing Kanamycin, cultured at 37°C 200rpm for 12h, transferred to a large-volume shake flask, and the inducer IPTG was added, and cultured at 28°C 180rpm for 12h. After centrifugation for 10min, the supernatant was discarded, the colony precipitate was collected, resuspended and broken in an ultrasonic disruptor, centrifuged at 4°C 20,000g for 30min, and the total protein in the supernatant after centrifugation was collected.

[0083] The total protein in the supernatant was purified by nickel column, and the following steps were performed in sequence:

[0084] Collect the flow-through liquid F;

[0085] 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 W30;

[0086] 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 and obtain a high-concentration imidazole washing solution W50;

[0087] 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.

[0088] The collected fluid was subjected to SDS-PAGE electrophoresis.

[0089] The total protein in the supernatant was mixed with Ni-NTA filler and incubated for 1 hour, and the impurities were removed with Wash buffer (50mM NaH2PO4, 300mMNaCl, 30mM imidazole, pH 8.0) (flow-through W1, W2), and then the target protein was eluted with Elution buffer (50mMNaH2PO4, 300mM NaCl, 300mM imidazole, pH 8.0) (elution E1-E5). PCV2-Spytag nanoparticles were self-assembled, identified by SDS-PAGE electrophoresis, and stored at low temperature after concentration.

[0090] Experimental results:

[0091] The PCV2-Spytag nanoparticles collected before and after nickel column purification were identified by SDS-PAGE. Figure 2As shown in Figure 2, after electrophoresis, a clear band appeared near 28 kDa ( Figure 2 The band indicated by the arrow in the middle is consistent with the expected molecular weight.

[0092] The following is Figure 2 The specific meaning of each symbol in:

[0093] M: protein standard 26616; S: ​​supernatant; F: flow-through; W30, W50: washing solutions with different concentrations of imidazole; E1-E5: eluents.

[0094] 5. Ligation reaction of RBD-Spycatcher and PCV2-Spytag nanoparticles

[0095] In a 20 μL reaction system, take 5 μL of RBD-Spycatcher fusion protein with a concentration of 0.5 mg / mL, gradually increase the volume of PCV2-Spytag nanoparticles with a concentration of 0.28 mg / mL, and react with RBD-Spycatcher fusion protein at 0 μL, 3 μL, 5 μL, 7.5 μL, 10 μL and 15 μL, respectively. After fully mixing, centrifuge the residual liquid on the tube wall to the bottom of the tube, and then incubate. Finally, use SDS-PAGE electrophoresis for identification.

[0096] Experimental results:

[0097] The reactivity of the RBD-Spycatcher fusion protein with PCV2-Spytag was tested, see Figure 3 The results showed that when the volume of PCV2-Spytag nanoparticles was 10 μL, the band of RBD-Spycatcher fusion protein was significantly weakened or disappeared compared with Line 4 and Line 5, and the band of RBD-Spycatcher fusion protein in Line 6 was significantly higher than that in Line 2, indicating that a connection reaction occurred between RBD-Spycatcher fusion protein and PCV2-Spytag nanoparticles. As the volume of PCV2-Spytag nanoparticles increased, the band of RBD-Spycatcher fusion protein gradually disappeared, indicating that the connection reaction had reached saturation. These results further show that RBD-Spycatcher fusion protein can effectively react with PCV2-Spytag nanoparticles.

[0098] The following is Figure 3 The specific meaning of each symbol in:

[0099] M: protein standard 26616;

[0100] 1: 5 μL PCV2-Spytag nanoparticles and 0 μL RBD-Spycatcher fusion protein;

[0101] 2: 0 μL PCV2-Spytag nanoparticles and 5 μL RBD-Spycatcher fusion protein;

[0102] 3: 3 μL PCV2-Spytag nanoparticles and 5 μL RBD-Spycatcher fusion protein;

[0103] 4: 5 μL PCV2-Spytag nanoparticles and 5 μL RBD-Spycatcher fusion protein;

[0104] 5: 7.5 μL PCV2-Spytag nanoparticles and 5 μL RBD-Spycatcher fusion protein;

[0105] 6: 10 μL PCV2-Spytag nanoparticles and 5 μL RBD-Spycatcher fusion protein;

[0106] 7: 15 μL PCV2-Spytag nanoparticles and 5 μL RBD-Spycatcher fusion protein.

[0107] 6. Nanoparticle morphology identification

[0108] In order to observe the binding effect of RBD and PCV2 nanoparticles, the components whose main components are RBD-PCV2 nanoparticles were first selected, and PCV2-Spytag nanoparticles were set as the control group. 10μL of sample was added to the electron microscope copper grid and left to stand for 1min, and the residual solution was absorbed with filter paper, and then 10μL of uranyl acetate solution was added. After 30s, the residual solution was absorbed to complete the negative staining step. Finally, the morphology of PCV2 nanoparticles was observed using a 200KV transmission electron microscope, and the morphological differences produced after RBD was displayed on its surface were observed.

[0109] Experimental results:

[0110] like Figure 4 As shown, different samples were observed by transmission electron microscopy, such as Figure 4 The PCV2-spytag nanoparticle sample in a was observed to be spherical nanoparticles with a diameter of about 20 nm under an electron microscope. The morphology was regular and the uniformity between the particles was high. Figure 4 Compared with the RBD-PCV2 nanoparticles in b, additional electron density appeared on the surface, which was completely inconsistent with the smooth surface of PCV2-spytag nanoparticles, indicating that the RBD-Spycatcher fusion protein was successfully displayed on the surface of PCV2-spytag nanoparticles.

[0111] 7. Animal Immunization

[0112] In this experiment, mice were immunized. The mice were randomly divided into 4 groups, 6 in each group, and injected with different immunogens, including RBD-Spycatcher fusion protein, RBD-PCV2 nanoparticles, wild PCV2 nanoparticles, and the control group was injected with an equal volume of PBS solution. Each group of mice was subcutaneously injected with immunogens twice, each immunization dose was 5μg / mouse, and the interval between the first immunization and the second booster immunization was 4 weeks. During the immunization process, serum samples of mice were collected every two weeks and stored at -20℃ for subsequent testing. The antibody titer, subtype and immune response of each group of mice were evaluated by antibody detection of serum samples.

[0113] Experimental results:

[0114] exist Figure 5 In this study, sera from mice in different experimental groups after immunization at different time points were collected, and RBD-specific IgG was measured using ELISA. Figure 5 The results showed that 2-4 weeks after the first immunization, the level of RBD-specific antibodies in the serum of mice in the RBD-PCV2 nanoparticle group was significantly higher than that in the RBD-Spycatcher fusion protein group, with a difference of about 30-40 times.

[0115] This indicates that nanoparticles as carriers can more effectively improve the immunogenicity of the vaccine. In the fourth week after the second immunization, the titers of RBD-specific antibodies in all groups with RBD-PCV2 nanoparticles as the immunogen were above 10 5 The RBD-Spycatcher fusion protein group has a RBD-specific antibody titer of 10. 5 In addition, the levels of RBD antibodies produced by each group reached a plateau from week 6 to week 10 and remained stable. This suggests that the two different immunogens have comparable effects in improving the RBD-specific immunity of mice. It is worth noting that the RBD-PCV2 nanoparticle group was able to produce high levels of antibodies earlier, which suggests that nanoparticle carriers have greater potential in vaccine applications.

[0116] Specifically, in Figure 5 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.

[0117] 8. Immunogenicity assessment

[0118] The evaluation system for changes in antibody levels in mouse serum at different time points is as follows:

[0119] (1) RBD and PCV2 nanoparticles were diluted with PBS, coated separately, and incubated at 4°C overnight.

[0120] (2) Discard the liquid in the wells and wash with PBST five times, 300 μL each time.

[0121] (3) Add BSA and block at 24°C for 3 h. Wash five times with 300 μL PBST, discard the liquid in the wells, air-dry and seal, and store at 2-8°C in the dark.

[0122] (4) The mouse serum was diluted with PBS in a 10-fold gradient to 105. After each dilution, the serum was thoroughly vortexed on a vortexer to mix well. The liquid remaining on the tube wall was collected by centrifugation before the next gradient dilution was performed.

[0123] (5) According to the preliminary experiments, 100 μL of serum samples diluted at appropriate dilution factors were added to the ELISA plate and incubated at 37°C for 1 h.

[0124] (6) After repeating step 2, dilute the goat anti-mouse IgG (H+L) HRP-labeled secondary antibody at 1:7,000, take 100 μL and add it to the ELISA plate and incubate at 37°C for 30 min.

[0125] (7) Repeat step 2 and add 50 μL of TMB colorimetric solution to each well and incubate at 37°C for 10 min.

[0126] (8) Then, 50 μL of stop solution was added to each well to terminate the color development reaction and the value at OD450 nm was read using a microplate reader.

[0127] Using the endpoint method, according to the dilution multiple of the serum sample, when the serum sample test result is still positive at the highest dilution multiple, the corresponding dilution multiple value at this time is converted into Log 10 As the ordinate, different collection time points were plotted as the abscissa to analyze the changes in total IgG antibody levels between different experimental groups.

[0128] 9. Antibody subtype analysis

[0129] The mouse sera collected from each group at the 10th week were selected to detect the IgG antibody subtypes IgG1 and IgG2a at the same time. The sera of different groups of mice were gradiently diluted, and the gradient diluted serum samples were selected and added to the ELISA plate for incubation and washing. Then, the goat anti-mouse IgG1 HRP-labeled secondary antibody and the goat anti-mouse IgG2a HRP-labeled secondary antibody were diluted 1:5000, respectively, and added to different wells of the same serum sample for incubation for 30 minutes. The absorbance values ​​of all sample wells in the microplate reader were measured, and the endpoint method was used to convert the dilution multiple values ​​of IgG1 and IgG2a in the same sample at the maximum dilution multiple, and the test results were still positive into Log. 10 , and graphical analysis was performed to compare the differences in antibody subtype production between different experimental groups.

[0130] Experimental results:

[0131] In this experiment, the effects of the RBD-Spycatcher fusion protein group and the RBD-PCV2 nanoparticle group on the IgG antibody subtypes produced by mice after immunization were compared. Although the total IgG titers of the two groups were similar after week 6, it is still necessary to understand whether the titers of different IgG subtypes in each group are the same.

[0132] Therefore, at week 10, the titers of RBD-specific IgG subtypes (IgG1 and IgG2a) were measured in the RBD-Spycatcher fusion protein group and the RBD-PCV2 nanoparticle group. The results showed that although the titers of RBD-specific IgG1 in the two groups were the same, both reaching 10 5 However, the titer of RBD-specific IgG2a in the RBD-PCV2 nanoparticle group was significantly higher than that in the RBD-Spycatcher fusion protein group, reaching 10 2.6 , while the titer of RBD-specific IgG2a in the RBD-Spycatcher fusion protein group was lower and significantly lower than that in the RBD-PCV2 nanoparticle group. These results indicate that the RBD-PCV2 nanoparticle group can induce higher levels of RBD-specific IgG2a antibodies compared to RBD monomers, while the levels of RBD-specific IgG1 in the two groups are the same. These results emphasize the importance of nanoparticles in inducing specific immune responses, which will help to better understand the potential of nanoparticles in vaccine research.

[0133] Specifically, in Figure 6 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 converted into Log values ​​for statistical analysis. P values ​​were calculated by two-tailed test. ns represents P>0.05

[0134] 10. Neutralization potency assessment

[0135] A small amount of mouse serum collected in the wild PCV2 nanoparticle group, RBD-PCV2 nanoparticle group and PBS group in the 10th week was dispensed into centrifuge tubes, and then placed in a 56°C water bath for 30 minutes. The serum samples after the water bath were diluted 100 times with 2% FBS complete DMEM medium, filtered with a 0.22μM disposable needle filter, and then diluted in a 2-fold gradient. According to the original TCID50 of PCV2 stored in the laboratory, PCV2 was diluted with 2% FBS complete DMEM medium, and the serum with different gradient dilutions was mixed with an equal volume of PCV2 solution to form a mixture of serum and virus, which was placed in a 37°C 5% CO2 incubator for 1 hour. At the same time, the PK15 cells pre-plated in 96 wells were taken out of the incubator, the culture medium was discarded, 100 μL of serum and virus mixture was added to each well, and the cells were returned to the incubator and cultured at 37°C 5% CO2 for 72 hours. The IFA test was then performed to evaluate the titer of mouse serum against PCV2. The main operating steps are as follows:

[0136] (1) Take the 96-well plate out of the incubator, discard the solution in the wells, add 200 μL PBS to rinse three times, and add 100 μL 4% paraformaldehyde to fix at room temperature for 10 min.

[0137] (2) Add 100 μL of 0.1% Triton X-100 and incubate at room temperature for 10 min.

[0138] (3) Add 200 μL PBS to rinse three times, discard the liquid in the wells, and add 3% BSA solution to block for more than 30 minutes.

[0139] (4) After discarding the liquid in the wells, dilute PCV2 rabbit polyclonal antibody with 1% BSA at a ratio of 1:1000 and add 100 μL to each well and incubate at room temperature for 1 h.

[0140] (5) Add 200 μL PBST and wash five times, 5 min each time.

[0141] (6) Then, discard the liquid in the well and add 100 μL of donkey anti-rabbit FITC fluorescent secondary antibody prepared with 1% BSA solution at a dilution of 1:2000. Incubate at room temperature in the dark for 1 h.

[0142] (7) After repeating step 5, discard the liquid in the wells, add PBS to keep the wells moist, and then place the 96-well plate under a fluorescence microscope for observation, take pictures, and analyze the experimental results.

[0143] (8) Based on the number of fluorescent spots, the serum dilution factor corresponding to the ability to neutralize more than 50% of the virus was converted into Log10 as the ordinate, and the different experimental groups were plotted as the abscissa to evaluate the effect of using PCV2 nanoparticles as a carrier on the virus neutralization titer.

[0144] Experimental results:

[0145] Based on the obvious differences in the surface of RBD-PCV2 nanoparticles compared to wild PCV2 nanoparticles, the level of neutralizing antibodies produced was analyzed to evaluate whether the immunogenicity had changed, and the effect of neutralizing PCV2 in the sera of different groups of mice was evaluated.

[0146] The results are as follows Figure 7 As shown, in the RBD-PCV2 and PCV2-Spytag nanoparticle groups, when the mouse serum was diluted about 1600 times, it was still able to neutralize 50% of the PCV2 virus. At this time, the corresponding neutralization titer reached 103.2, and the neutralization titer between the two groups was 103.2, with no difference. This shows that after the PCV2 nanoparticles were connected to the RBD surface and then immunized with mice, the level of neutralizing antibodies produced was highly consistent with that of wild PCV2 nanoparticles, and did not affect the immunogenicity of PCV2 itself. In summary, the developed PCV2-Spytag nanoparticles can be used as a carrier to display other pathogen antigens, providing a new approach for the design and development of multi-linked and multivalent nanoparticle vaccines.

[0147] Specifically in Figure 7 In the experiment, the mouse sera collected in week 10 were diluted 2-fold to neutralize PCV2 virus. The serum dilution factor corresponding to 50% virus neutralization was determined by IFA method, and the value was converted into Log value for statistical analysis. P value was calculated by two-tailed test. ns represents P>0.05.

[0148] 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 combination protein nanoparticle based on porcine circovirus type 2, characterized in that: The combined protein nanoparticles are formed by self-assembly of RBD-Spycatcher fusion protein and PCV2-Spytag nanoparticles; The amino acid sequence of the RBD-Spycatcher fusion protein is shown in SEQ ID NO.1, and the amino acid sequence of the PC V2-Spytag nanoparticle is shown in SEQ ID NO.

2.

2. A method for preparing combined protein nanoparticles based on porcine circovirus type 2, characterized in that: Including steps: S1, obtain RBD-Spycatcher fusion protein and PCV2-Spytag nanoparticles; Wherein, the amino acid sequence of the RBD-Spycatcher fusion protein is shown in SEQ ID NO.1, and the amino acid sequence of the PCV2-Spytag nanoparticle is shown in SEQ ID NO.2; S2, incubating the RBD-Spycatcher fusion protein and the PCV2-Spytag nanoparticles in a buffer solution to obtain the combined protein nanoparticles.

3. The preparation method according to claim 2, characterized in that: The nucleotide sequence encoding the RBD-Spycatcher fusion protein is shown in SEQ ID NO.3, and the nucleotide sequence encoding the PCV2-Spytag nanoparticle is shown in SEQ ID NO.

4.

4. The preparation method according to claim 2, characterized in that: The molar concentration ratio of the RBD-Spycatcher fusion protein and the PCV2-Spytag nanoparticles in the buffer solution is 5:3-1:

3.

5. The preparation method according to claim 2, characterized in that: The step S2 further comprises: after incubating the RBD-Spycatcher fusion protein and the PCV2-Spytag nanoparticles in a buffer solution, purifying the generated combined protein nanoparticles.

6. The preparation method according to claim 2, characterized in that: The incubation temperature is 4-37° C., and the incubation time is 1-72 h.

7. A combined protein nanoparticle based on porcine circovirus type 2, characterized in that: The preparation is carried out by the preparation method according to any one of claims 3 to 6.

8. Use of the combined protein nanoparticles as claimed in claim 1 or the combined protein nanoparticles as claimed in claim 7 in the preparation of a vaccine for simultaneously preventing porcine epidemic diarrhea virus and porcine circovirus type 2.

9. A multi-component multivalent vaccine, characterized in that: The multi-combination multivalent vaccine comprises the combined protein nanoparticles according to claim 1 or the combined protein nanoparticles according to claim 7.

10. Use of the combined protein nanoparticles according to claim 1 or the combined protein nanoparticles according to claim 7 in preparing a vaccine for simultaneously preventing porcine epidemic diarrhea virus and porcine circovirus type 2.

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