Protein nanoparticle based on swine transmissible gastroenteritis virus, recombinant protein, preparation and application

Abstract

The invention provides a protein nanoparticle based on a swine transmissible gastroenteritis virus, a recombinant protein, and preparation and application of the protein nanoparticle and the recombinant protein. The TGEV protein nano particles are formed by self-assembly of a TGEV-CTD (Triglyceride-Cellulose Diphosphate)-SpyCatcher recombinant protein and a PCV2-Cap-SpyTag protein nano particle, and the TGEV-CTD-SpyCatcher recombinant protein and the PCV2-Cap The amino acid sequence of the TGEV-CTD-SpyCatc her recombinant protein is as shown in SEQ ID NO. 1, and the amino acid sequence of the PCV2-Cap-SpyTag protein nano-particles is as shown in SEQ ID NO. 2. The TGEV-CTD-Spy Catcher recombinant protein and the TGEV protein nanoparticles, which are constructed by the invention, can be used for preventing swine transmissible gastroenteritis virus infection, and the TGEV-CTD-Spy Catcher recombinant protein and the TGEV protein nanoparticles have relatively high immunogenicity and safety.

Description

Technical Field

[0001] The present invention relates to the technical field of research and development and application of porcine transmissible gastroenteritis virus vaccines, and in particular to protein nanoparticles and recombinant proteins based on porcine transmissible gastroenteritis virus, as well as preparation and application thereof. Background Art

[0002] Transmissible Gastroenteritis of Swine virus (TGEV) is one of the important pathogens affecting the current pig farming industry. It mainly causes intestinal diseases in pigs. Its clinical manifestations are mainly diarrhea, vomiting and dehydration. TGEV is susceptible to pigs of all ages, especially newborn piglets. The morbidity and mortality rates after infection of newborn piglets are extremely high, posing a huge threat and economic losses to the global pig farming industry.

[0003] Effective biosafety prevention and control is an effective means to block the spread of TGEV. In addition, vaccination is one of the most effective means of preventing and controlling TGEV. However, the current commercial TGEV vaccines are all traditional inactivated vaccines or attenuated live vaccines, and their clinical prevention effects are not very ideal, and their immunogenicity and safety need to be further improved.

[0004] In view of this, it is necessary to provide a protein nanoparticle, a recombinant protein, and a preparation and application based on porcine transmissible gastroenteritis virus to solve or at least alleviate the technical defect of the poor immunogenicity of the above-mentioned porcine transmissible gastroenteritis vaccine. Summary of the invention

[0005] The main purpose of the present invention is to provide a protein nanoparticle, a recombinant protein based on porcine transmissible gastroenteritis virus, and its preparation and application, so as to solve the technical problem of poor immunogenicity of the above-mentioned porcine transmissible gastroenteritis vaccine.

[0006] To achieve the above objectives, the present invention provides a TGEV-CT D-SpyCatcher recombinant protein based on porcine transmissible gastroenteritis virus, and the amino acid sequence of the TGEV-CTD-SpyCatcher recombinant protein is shown in SEQ ID NO.1.

[0007] The present invention also provides a TGEV protein nanoparticle based on porcine transmissible gastroenteritis virus, wherein the TGEV protein nanoparticle is formed by self-assembly of TGEV-CTD-SpyCatcher recombinant protein and PCV2-Cap-SpyTag protein nanoparticles;

[0008] The amino acid sequence of the TGEV-CTD-SpyCatcher recombinant protein is shown in SEQ ID NO.1, and the amino acid sequence of the PCV2-Cap-SpyTag protein nanoparticle is shown in SEQ ID NO.2.

[0009] The present invention also provides a method for preparing TGEV protein nanoparticles based on porcine transmissible gastroenteritis virus, comprising the steps of:

[0010] S1, obtain TGEV-CTD-SpyCatcher recombinant protein and PCV2-Cap-SpyTag protein nanoparticles;

[0011] Wherein, the amino acid sequence of the TGEV-CTD-SpyCatcher recombinant protein is shown in SEQ ID NO.1, and the amino acid sequence of the PCV2-Cap-SpyTag protein nanoparticle is shown in SEQ ID NO.2;

[0012] S2, connecting the TGEV-CTD-SpyCatcher recombinant protein and the PCV2-Cap-SpyTag protein nanoparticles in a buffer solution to obtain the TGEV protein nanoparticles.

[0013] Furthermore, the nucleotide sequence encoding the TGEV-CTD-SpyCatcher recombinant protein is shown in SEQ ID NO.3, and the nucleotide sequence encoding the PCV2-Cap-SpyTag protein nanoparticles is shown in SEQ ID NO.4.

[0014] Furthermore, the molar concentration ratio of the TGEV-CTD-SpyCatcher recombinant protein and the PCV2-Cap-SpyTag protein nanoparticles is 6:1-1:1.

[0015] The present invention also provides a use of any of the above-described TGEV-CTD-SpyCatcher recombinant proteins in the preparation of a porcine transmissible gastroenteritis vaccine.

[0016] The present invention also provides an application of any of the TGEV protein nanoparticles described above in the preparation of a porcine transmissible gastroenteritis vaccine.

[0017] The present invention also provides a porcine transmissible gastroenteritis vaccine, which includes the TGEV-CTD-SpyCatcher recombinant protein as described above, or the porcine transmissible gastroenteritis vaccine includes the TGEV protein nanoparticles as described above.

[0018] The present invention also provides a use of any of the above-described TGEV-CTD-SpyCatcher recombinant proteins in the preparation of TGEV-specific antibodies.

[0019] The present invention also provides a use of any of the TGEV protein nanoparticles described above in the preparation of TGEV-specific antibodies.

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

[0021] The TGEV-CTD-SpyCatcher recombinant protein and TGEV protein nanoparticles constructed by the present invention can be used to prevent porcine transmissible gastroenteritis virus infection, and have high immunogenicity and safety. Specifically, the present invention completes the expression and purification of TGEV-CTD-SpyCatcher recombinant protein and PCV2-Cap-SpyTag protein nanoparticles, and connects TGEV-CTD-SpyCatcher recombinant protein and PCV2-Cap-SpyTag protein nanoparticles to form TGEV protein nanoparticles. In the present invention, the antibody level of mice is in a stable state on the 35th to 70th day after immunization; among them, when the TGEV-CTD-SpyCatcher recombinant protein is used as an immunogen, the antibody titer can reach 7.4×10 4 -9×10 4 When TGEV protein nanoparticles were used as immunogens, the antibody titer could reach 6.2×10 5 -6.4×10 5 . 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 the recombinant plasmid map of pFastBac 1-TGEV-CTD-SpyCatcher in Example 1 of the present invention;

[0024] Figure 2 The results of the indirect immunofluorescence assay (IFA) in Example 1 of the present invention are shown at a magnification of 100 times; wherein (a) is a normal Sf9 cell (scale bar 100 μm), and (b) is a Sf9 cell infected with a P2 generation virus (scale bar 100 μm);

[0025] Figure 3The identification diagram of the TGEV-CTD-SpyCatcher recombinant protein in Example 1 of the present invention; wherein (a) is the SDS-PAGE test result; (b) is the Western blot test result;

[0026] Figure 4 This is an SDS-PAGE identification diagram of PCV2-Cap-SpyTag protein nanoparticles in Example 1 of the present invention;

[0027] Figure 5 The SDS-PAGE identification diagram of the connection samples at different reaction ratios in Example 1 of the present invention;

[0028] Figure 6 This is an electron micrograph of TGEV-CTD-NPs in Example 1 of the present invention;

[0029] Figure 7 This is a graph showing the result of mouse TGEV-CTD protein-specific IgG antibody titers at different time points 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] It should be known that the TGEV surface spike protein (S) is a type I transmembrane protein, which is a homotrimer in structure and embedded in the surface of virus particles. It is responsible for the binding of the virus to specific receptors on the cell surface and mediates the virus's cell invasion. The S protein has very good immunogenicity and can induce the body to produce effective neutralizing antibodies. The specific antibodies produced against the S protein can effectively block the invasion of the virus and is one of the ideal antigen targets for the current development of new TGEV subunit vaccines.

[0034] Protein nanoparticles are prepared through genetic engineering technology. They do not contain nucleic acid components of pathogens and have very good biosafety. They have stronger immunogenicity than recombinant protein subunit vaccines. Compared with recombinant soluble proteins, protein nanoparticles are more easily captured by antigen-presenting cells, thereby more effectively activating the body's humoral and cellular immune responses. They are the mainstream direction of current vaccine research and development.

[0035] However, obtaining a highly pure and immunogenic TGEV CTD recombinant protein still faces technical challenges; therefore, how to obtain a highly immunogenic recombinant protein and TGEV protein nanoparticles based on TGEV is a technical difficulty that needs to be overcome in the present invention.

[0036] To this end, the present invention provides a TGEV-CTD-SpyCatcher recombinant protein based on porcine transmissible gastroenteritis virus, and the amino acid sequence of the TGEV-CTD-SpyCatcher recombinant protein is shown in SEQ ID NO.1.

[0037] The TGEV-CTD-SpyCatcher recombinant protein is immunogenic and can induce the production of specific antibodies in the body. However, in order to obtain a higher performance porcine transmissible gastroenteritis vaccine, the immunogenicity of the TGEV-CTD-SpyCatcher recombinant protein needs to be further improved.

[0038] The present invention also provides TGEV protein nanoparticles (TGEV-CTD-NPs) based on porcine transmissible gastroenteritis virus, wherein the TGEV protein nanoparticles are self-assembled by TGEV-CTD-SpyCatcher recombinant protein and PCV2-Cap-SpyTag protein nanoparticles, that is, formed by spontaneous connection.

[0039] The amino acid sequence of the TGEV-CTD-SpyCatcher recombinant protein is shown in SEQ ID NO.1, and the amino acid sequence of the PCV2-Cap-SpyTag protein nanoparticle is shown in SEQ ID NO.2.

[0040] In the present invention, SEQ ID The amino acid sequence shown in NO.1 is: MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAGSLPSFYTHTIVNITIGLGMKRSGYGQPIASTLSNITLPMQDHNTDVYCIRSDQFSVYVHSTCKSALWDNIFKRNCTDVLDATAVIKTGTCPFSFDKLNNYLTFNKFCLSLSPVGANCKF DVAARTRTNEQVVRSLYVIYEEGDNIVGVPSDNSGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGSGDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIGSHHHHHHHH.

[0041] SEQ ID The amino acid sequence shown in NO.2 is: MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDHPFTNGIFNTRLSRTFGYTIKRTTVKTPSWAVDMMRFNINDFLPPGGGSNPRSVPFEYYRIRKVKVEFWPCSPITQGDRGVGSS AVILDDNFVTKATALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDSTIDYFQPNNKRNQLWLRLQTAGNVDHVGLGTAFENSIYDQEYNIRVTMYVQFREFNLKDPPLNPGGSGAHIVMVDAYKPTK.

[0042] The present invention also provides a method for preparing TGEV protein nanoparticles based on porcine transmissible gastroenteritis virus, comprising the steps of:

[0043] S1, obtain TGEV-CTD-SpyCatcher recombinant protein and PCV2-Cap-SpyTag protein nanoparticles.

[0044] Among them, the amino acid sequence of the TGEV-CTD-SpyCatcher recombinant protein is shown in SEQ ID NO.1, and the amino acid sequence of the PCV2-Cap-SpyTag protein nanoparticle is shown in SEQ ID NO.2.

[0045] In the present invention, the process of obtaining the TGEV-CTD-SpyCatcher recombinant protein may include: obtaining a secretory recombinant protein using a recombinant baculovirus-insect cell expression system; obtaining a recombinant Bacmid DNA by transposition, and then transfecting it into Sf9 cells to obtain a recombinant baculovirus that can stably express the recombinant protein, and then amplifying the obtained recombinant baculovirus to infect Sf9 for secretory expression of the recombinant protein, harvesting the culture medium supernatant, and purifying the recombinant protein by nickel column affinity chromatography.

[0046] The process of obtaining the PCV2-Cap-SpyTag protein nanoparticles may include: preparing by using an E. coli expression system, purifying the recombinant protein by nickel column affinity chromatography, and assembling the protein nanoparticles by dialyzing the purified protein in an assembly buffer.

[0047] S2, connecting the TGEV-CTD-SpyCatcher recombinant protein and the PCV2-Cap-SpyTag protein nanoparticles in a buffer solution (such as mixed connection) to obtain the TGEV protein nanoparticles.

[0048] In the present invention, the nucleotide sequence encoding the TGEV-CTD-SpyCatcher recombinant protein is shown in SEQ ID NO.3, and the nucleotide sequence encoding the PCV2-Cap-SpyTag protein nanoparticles is shown in SEQ ID NO.4.

[0049] In the present invention, the nucleotide sequence of SEQ ID NO.3 is: ATGCTACTGGTGAACCAGAGTCATCAAGGATTCAACAAGGAGCATACGTCAAAAATGGTATCAGCGATAGTCTTATACGTGCTCCTCGCTGCCGCTGCACACTCGGCCTTCGCTGGATCTTTGCCATCATTCTACACACACACCATCGTTAACATTACAATTGGGCTGGGTATGAAGAGGTCCGGTTATGGACAACCGATCGCATCGACCTTGTCGAACATTACGCTTCCTATGCAGGATCATAACACTGACGTGTACTGTATCCGGAGCGATCAGTTTAGCGTCTATGTACACTCGACGTGTAAGAGCGCGTTGTGGGACAATATTTTTAAAAGAAACTGCACTGATGTTTTAGATGCCACCGCCGTAATAAAAACGGGAACTTGCCCTTTTTCTTTTGATAAATTAAATAATTACTTAACTTTCAATAAATTTTGCCTGTCCCTTAGCCCCGTCGGTGCAAACTGTAAGTTCGACGTAGCGGCACGTACAAGAACCAATGAACAGGTTGTTCGATCATTATATGTCATTTACGAAGAGGGCGACAATATTGTGGGTGTTCCTTCCGATAATTCTGGCGGGGGGGGCTCGGGTTATATCCCTGAGGCACCCCGCGATGGACAAGCGTATGTTCGGAAAGATGGCGAGTGGGTGCTGCTTTCCACATTTCTCGGGTCCGGGGATTCAGCGACTCATATAAAATTCAGTAAGCGTGACGAGGACGGTAAAGAACTAGCCGGTGCTACGATGGAATTGCGAGATAGTAGTGGCAAAACCATAAGTACCTGGATCTCTGATGGCCAAGTCAAGGACTTTTACCTATATCCGGGGAAATATACGTTCGTCGAAACGGCCGCACCAGACGGTTACGAAGTAGCAACAGCGATAACTTTCACAGTTAATGAGCAAGGACAGGTGACAGTTAACGGGAAGGCTACTAAGGGCGACGCTCACATCGGAAGCCATCACCACCATCACCATCATCACTAA。

[0050] The nucleotide sequence of SEQ ID NO.4 is: ATGCGGGGTTCTCATCATCATCATCAT.

[0051] In order to effectively generate the TGEV protein nanoparticles, the molar concentration ratio of the TGEV-CTD-SpyCatcher recombinant protein and the PCV2-Cap-SpyTag protein nanoparticles in the buffer solution is 6:1-1:1, preferably 2:1-1.5:1.

[0052] In the present invention, by introducing the gp67 signal peptide sequence at the amino terminus, the technical difficulty in obtaining secretory expression of recombinant proteins in the art is overcome; by introducing T4 foldon trimerization at the carboxyl terminus, the technical difficulty in obtaining stable recombinant trimeric proteins in the art is overcome; by optimizing the codon gene sequence in insect cells, the technical difficulty in efficiently expressing recombinant TGEV CTD protein in the art is overcome; thereby obtaining the TGEV protein nanoparticles with superior immunogenicity.

[0053] The present invention also provides a use of any of the above-described TGEV-CTD-SpyCatcher recombinant proteins in the preparation of a porcine transmissible gastroenteritis vaccine.

[0054] The present invention also provides an application of any of the TGEV protein nanoparticles described above in the preparation of a porcine transmissible gastroenteritis vaccine.

[0055] The present invention also provides a porcine transmissible gastroenteritis vaccine, which includes the TGEV-CTD-SpyCatcher recombinant protein as described above, or the porcine transmissible gastroenteritis vaccine includes the TGEV protein nanoparticles as described above.

[0056] The present invention also provides a use of any of the above-described TGEV-CTD-SpyCatcher recombinant proteins in the preparation of TGEV-specific antibodies.

[0057] The present invention also provides a use of any of the TGEV protein nanoparticles described above in the preparation of TGEV-specific antibodies.

[0058] The following are specific examples of the present invention:

[0059] Example 1

[0060] 1. Expression and purification of TGEV-CTD-SpyCatcher recombinant protein

[0061] 1.1 Construction of pFastBac 1-TGEV-CTD-SpyCatcher recombinant plasmid

[0062] The TGEV-SHXB strain (GenBank number: KP202848) was selected as the reference strain, and the region (Carboxyl-Terminal Domain, CTD) located at the carboxyl terminus of the S1 subunit of the Spike protein (S) on the surface of the virus was screened. The gp67 signal peptide sequence and the T4 foldon trimerization motif were introduced at the amino terminus and the carboxyl terminus, and the SpyCatcher sequence was fused to the carboxyl terminus to obtain the recombinant TGEV-CTD-SpyCatcher fusion protein sequence. The sequence was codon-optimized and then synthesized to obtain the TGEV-CTD-SpyCatcher recombinant protein. The target gene was cloned into the pFastBac 1 vector using the EcoRⅠ and XbaⅠ restriction sites, named pFastBac 1-TGEV-CTD-SpyCatcher, and sequencing comparison analysis was performed.

[0063] In the present invention, the amino acid sequence of the TGEV-CTD-SpyCatcher recombinant protein is shown in SEQ ID NO.1, and the nucleotide sequence encoding the TGEV-CTD-SpyCatcher recombinant protein is shown in SEQ ID NO.3.

[0064] In the present invention, the recombinant plasmid map of pFastBac 1-TGEV-CTD-SpyCatcher is as follows Figure 1 As shown, the plasmid sequencing analysis was consistent with the target sequence.

[0065] 1.2 Construction and identification of recombinant baculovirus

[0066] (1) Extraction and identification of recombinant bacmid

[0067] Take 1 μL of recombinant plasmid (10 ng) and add it to DH10 Bac competent cells. After ice bath for 30 minutes, heat shock in 42℃ water bath for 90 seconds, immediately put it on ice for 2 minutes, add 900 μL of antibiotic-free LB liquid culture medium, place it in a 37℃ constant temperature shaker at 225r / min for 4-5 hours, and then spread it on LB agar plates containing Kan (50 μg / mL), Gen (7 μg / mL) and Tet (10 μg / mL) as well as IPTG (40 μg / mL) and X-gal (100 μg / mL). After culturing at 37℃ for 16-24 hours, blue-white screening was carried out, and the recombinant Bacmid DNA was extracted by alkaline lysis method, and PCR verification was performed with M13 primers. The positive Bacmid DNA was used for the next experiment.

[0068] (2) Transfection of recombinant Bacmid

[0069] 9×10 5 Sf9 cells were placed in a 27°C incubator for 1 hour to allow the cells to adhere to the wall. The positive Bacmid DNA verified by PCR was transfected into the culture medium using Cellfectin TM II transfection reagent was used to transfect Sf9 cells. Grace's Insect Medium, unsupplemented, was used during the transfection process. After transfection, the cells were cultured at 27°C for 4-5 days. When the cells became larger and showed obvious pathological changes, the P1 generation recombinant baculovirus was harvested.

[0070] (3) Recombinant baculovirus amplification

[0071] The P2 virus was amplified at an MOI (multiplicity of infection, MOI) of 0.05, and 0.5 mL of P1 was inoculated into 50 mL at a density of 1.5×10 6 Sf9 cells (insect cells) with a concentration of 1 cell / mL were amplified in a 27°C constant temperature shaker at 130 r / min for 2 days. The cell status was observed at all times. When the cells became significantly larger, they were centrifuged at 1,000 g for 10 min to harvest the P2 generation virus, and 2% FBS was added to the P2 generation virus for aliquoting and storage.

[0072] (4) Identification of recombinant baculovirus by IFA

[0073] 2×10 per well of a 24-well plate 5 Sf9 cells were plated, 20 μL of P2 virus was added to each well, and the cells were cultured in a 27°C constant temperature incubator for 72 h before IFA identification (normal Sf9 cells were set as a control); the primary antibody was mouse anti-His tag monoclonal antibody (1:2000), and the secondary antibody was FITC-labeled donkey anti-mouse IgG (1:2000). Finally, the results were observed under a fluorescence microscope.

[0074] In the present invention, the results of indirect immunofluorescence assay (IFA) are referred to as Figure 2 (Converted from color picture) It can be understood that Sf9 cells infected with P2 generation virus have green fluorescence signals, while normal Sf9 cells have no fluorescence signals, indicating that TGEV-CTD-SpyCatcher recombinant protein is successfully expressed in Sf9 cells.

[0075] 1.3 Expression and purification of recombinant proteins

[0076] The amplified P2 virus was inoculated at an MOI of 1 and a density of 2.0×10 6The Sf9 cells were expressed at 27°C constant temperature shaker at 130 r / min for 72 hours. The culture product was centrifuged at 1,000g for 10 minutes to collect the culture supernatant, which was then filtered with a 0.45μm filter and replaced with a loading buffer (10mM imidazole, pH=8) using a 10K membrane. The protein was purified by nickel column affinity chromatography. The supernatant was combined with Ni-NTA filler at room temperature for 20 minutes, and the impurities were eluted with a wash buffer (50mM Tris-HCl, 200mM NaCl, 10mM Imidazole, pH 8.0) with an imidazole concentration of 10mM. Finally, the target protein was eluted with an elution buffer (50mM Tris-HCl, 200mM NaCl, 200mM Imidazole, pH6.0) with an imidazole concentration of 200mM to obtain eluents E1 and E2. The purified protein was then identified by SDS-PAGE and Western blot.

[0077] In the present invention, reference is made to Figure 3 As shown, the results of SDS-PAGE and Western blot analysis showed that the target protein was around 35KDa, and its molecular weight was consistent with the expected protein molecular weight; indicating that the target protein was successfully expressed and could be secreted into the culture medium, and the present invention successfully purified and obtained TGEV-CTD-Spy Catcher protein (TGEV-CTD-SpyCatcher recombinant protein).

[0078] Figure 3 The markup in is as follows:

[0079] M: indicates protein marker;

[0080] E1, E2: represent the target protein after purification (derived from the eluent).

[0081] 2. Preparation of PCV2-Cap-SpyTag Protein Nanoparticles

[0082] 2.1 Construction of pET100-PCV2-Cap-SpyTag recombinant plasmid

[0083] The SpyTag sequence was fused to the carboxyl terminus of the Cap protein, and the codons were optimized and synthesized according to the codon bias of Escherichia coli to obtain PCV2-Cap-SpyTag protein nanoparticles. The target gene was cloned into the prokaryotic expression vector pET100_D / TOPO vector using the NdeⅠ and BamHⅠ restriction sites, named pET100-PCV2-Cap-SpyTag, and sequenced and compared.

[0084] In the present invention, the amino acid sequence of PCV2-Cap-SpyTag protein nanoparticles is shown as SEQ ID NO.2, and the nucleotide sequence encoding PCV2-Cap-SpyTag protein nanoparticles is shown as SEQ ID NO.4.

[0085] In the present invention, after the PCV2-Cap-SpyTag gene is cloned into the pET100 vector through the NdeⅠ and BamHI restriction sites, the plasmid sequencing comparison analysis is consistent with the target sequence.

[0086] 2.2 Expression and purification of recombinant proteins

[0087] (1) Expression of recombinant proteins

[0088] Take 1 μL of the recombinant plasmid and transform it into BL21 (DE3) Escherichia coli competent cells, spread it on an LB medium plate containing 100 μg / mL of ampicillin, and invert it at 37°C for 12-14 hours. Randomly pick a monoclonal colony to 10 mL of LB medium containing 100 μg / mL of ampicillin, and shake it at 37°C in a constant temperature shaker at 220 r / min for 12 hours. At a ratio of 1:100, add 10 mL of the bacterial solution to 1 L of autoclaved fermentation medium containing 100 μg / mL of ampicillin, and shake it at 37°C in a constant temperature shaker at 180 r / min for 3 hours until the OD 600 When it reached 0.8-1.0, the temperature was lowered to 25°C, and IPTG with a final concentration of 1 mM was added to induce protein expression. After shaking culture at 180 r / min for 16 h, the bacteria were collected by centrifugation at 6,000 g for 10 min and stored at -80°C for protein purification.

[0089] (2) Purification of recombinant protein

[0090] The E. coli pellet was resuspended in lysis buffer, and the cells were broken by ultrasonic cell disruptor. The broken samples were centrifuged at 14,000g for 30 minutes at 4°C to collect soluble supernatant proteins for purification. The protein was purified by nickel column affinity chromatography, and the supernatant collected by centrifugation was placed at room temperature for 1 hour for oscillation and binding with Ni-NTA filler.

[0091] Transfer the filler combined with the supernatant to the column and let the supernatant flow out of the column naturally to collect the flow-through;

[0092] Then, the impurities were washed with a wash buffer (50 mM NaH2PO4, 300 mM NaCl, 50 mM Imidazole, pH 8.0) containing 50 mM imidazole to obtain a washing solution;

[0093] Finally, the target protein was eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 300 mM Imidazole, pH 6.0) with an imidazole concentration of 300 mM to obtain eluents E1, E2, and E3; the purified protein was identified by SDS-PAGE.

[0094] In the present invention, see Figure 4 As shown, the SDS-PAGE results showed that there was an obvious target protein band at 28KDa, and the molecular weight was consistent with the expected protein molecular weight, indicating that the present invention successfully purified the PCV2-Cap-SpyTag protein (PCV2-Cap-SpyTag protein nanoparticles).

[0095] Figure 4 The markup in is as follows:

[0096] M: indicates protein marker;

[0097] S: indicates soluble supernatant protein;

[0098] FT: indicates proteins derived from the flow-through;

[0099] W: indicates impurity proteins originating from washing solution;

[0100] E1, E2, E3: represent the purified target protein (derived from the eluent).

[0101] 2.3 Assembly of PCV2-Cap-SpyTag protein nanoparticles

[0102] After the purified protein was identified by SDS-PAGE, the protein was placed in a 7K dialysis bag and dialyzed in assembly buffer for 48 hours. The dialyzed samples were collected and stored at 4°C for later use.

[0103] 3. Preparation and identification of TGEV protein nanoparticles (TGEV-CTD-NPs)

[0104] The obtained PCV2-Cap-SpyTag protein nanoparticles and TGEV-CTD-SpyCatcher recombinant proteins were connected in PBS solution at molar concentration ratios of 1:1, 1:1.5, 1:2, 1:4, and 1:6, respectively. After mixing, they were placed at 4°C for 16 hours, and the connected samples were analyzed by SDS-PAGE.

[0105] The present invention prepares TGEV-CTD-NPs by connecting PCV2-Cap-SpyTag protein nanoparticles and TGEV-CTD-SpyCatcher recombinant proteins.

[0106] And, refer to Figure 5It was understood that the SDS-PAGE analysis results showed that when the molar concentration ratio of PCV2-Cap-SpyTag protein nanoparticles and TGEV-CTD-SpyCatcher recombinant proteins was above 1:2, the added TGEV-CTD-SpyCatcher recombinant protein could not be completely connected to the PCV2-Cap-SpyTag protein nanoparticles, indicating that saturation had been reached; therefore, the connection samples with a molar concentration ratio of 1:1.5 were subsequently selected for immunogenicity evaluation.

[0107] Figure 5 The markup in is as follows:

[0108] M: indicates protein marker;

[0109] 1: indicates PCV2-Cap-SpyTag protein nanoparticles;

[0110] 2: indicates TGEV-CTD-Spycatcher recombinant protein;

[0111] 3: indicates the ligation product with a molar concentration ratio of TGEV-CTD-Spycatcher recombinant protein: PCV2-Cap-SpyTag protein nanoparticles of 1:1;

[0112] 4: indicates the ligation product with a molar concentration ratio of TGEV-CTD-Spycatcher recombinant protein: PCV2-Cap-SpyTag protein nanoparticles of 1.5:1;

[0113] 5: indicates the ligation product with a molar concentration ratio of TGEV-CTD-Spycatcher recombinant protein: PCV2-Cap-SpyTag protein nanoparticles of 2:1;

[0114] 6: represents the ligation product with a molar concentration ratio of TGEV-CTD-Spycatcher recombinant protein: PCV2-Cap-SpyTag protein nanoparticles of 4:1;

[0115] 7: represents the connection product with a molar concentration ratio of 6:1 between TGEV-CTD-Spycatcher recombinant protein and PCV2-Cap-SpyTag protein nanoparticles.

[0116] In the present invention, the connection sample (TGEV-CTD-NPs) obtained by connecting PCV2-Cap-SpyTag protein nanoparticles and TGEV-CTD-SpyCatcher recombinant protein at a ratio of 1:1.5 was negatively stained with 1% phosphotungstic acid staining solution for 5 minutes, and the morphology of TGEV-CTD-NPs was observed under an electron microscope.

[0117] Reference Figure 6 To understand, the morphology and size of TGEV-CTD-NPs were observed by transmission electron microscopy. The electron microscopy results showed that particles with regular and uniform morphology and a diameter of 30-40 nm were observed, further indicating that TGEV-CTD-NPs were successfully prepared in the present invention.

[0118] 4. Evaluation of immunogenicity of TGEV-CTD-NPs

[0119] 4.1 Immunization of mice

[0120] Fifteen 6-week-old female Balb / c mice were selected and randomly divided into three groups (5 mice / group), namely, the TGEV-CTD-NPs group, the TGEV-CTD-SpyCatcher recombinant protein group and the control group.

[0121] TGEV-CTD-NPs group:

[0122] The prepared TGEV-CTD-NPs were mixed in the form of a protein solution (containing a buffer) with Seppic Gel02ST adjuvant at a volume ratio of 9:1 to obtain an injection solution;

[0123] Each mouse was injected intramuscularly with 5 μg of TGEV-CTD-NPs (obtained by connecting PCV2-Cap-SpyTag protein nanoparticles and TGEV-CTD-SpyCatcher recombinant proteins in a ratio of 1:1.5) during each immunization; 5 μg of TGEV-CTD-NPs was present in 200 μL of injection solution; each mouse was immunized twice, with a booster immunization 21 days after the first immunization.

[0124] TGEV-CTD-SpyCatcher recombinant protein group:

[0125] The prepared TGEV-CTD-SpyCatcher recombinant protein was mixed in the form of a protein solution (containing a buffer) with Seppic Gel 02 ST adjuvant at a volume ratio of 9:1 to obtain an injection solution;

[0126] Each mouse was injected intramuscularly with 5 μg of TGEV-CTD-SpyCatcher recombinant protein during each immunization; 5 μg of TGEV-CTD-SpyCatcher recombinant protein was present in 200 μL of injection solution; each mouse was immunized twice, with a booster immunization 21 days after the first immunization.

[0127] Control group: At each immunization, an equal amount of PBS was injected.

[0128] In all groups, blood was collected on days 35, 49, and 70 after the first immunization, and serum was separated for antibody level testing.

[0129] 4.2 Indirect ELISA antibody titer determination

[0130] The indirect ELISA method was used to detect the titer of IgG antibody specific to mouse serum TGEV-CTD protein. The purified TGEV-CTD-Spycatcher recombinant protein was coated on the ELISA plate and placed at 4°C for 12 hours. After coating, it was washed with PBST for 5 times and blocked with 3% BSA for 3 hours. The mouse serum was diluted 10 times in a gradient, and 100 μL of diluted serum was added to each well and incubated at 37°C for 1 hour. The secondary antibody was incubated with HRP-labeled goat anti-mouse IgG (1:7000) for 30 minutes. Finally, 50 μL of TMB color development solution was added to each well and placed at 37°C for 10 minutes. After that, 50 μL of 2M concentrated H2SO4 was added to each well to terminate the reaction. 450 The OD value was read at nm wavelength.

[0131] In the present invention, the results of ELISA antibody titer determination of mouse sera on days 35, 49 and 70 after the first immunization are as follows: Figure 7 As shown, the results showed that the antibody levels were stable from 35 to 70 days after the first immunization.

[0132] Among them, the antibody titer of the TGEV-CTD-SpyCatcher recombinant protein group was basically 7.4×10 4 -9×10 4 The antibody titer of the TGEV-CTD-NPs group was basically 6.2×10 5 -6.4×10 5 The antibody titer of TGEV-CTD-NPs immunization was 7-8 times that of TGEV-CTD-SpyCatcher recombinant protein, and there was a significant difference (p < 0.05), indicating that TGEV-CTD-NPs can induce higher antibody levels.

[0133] Figure 7 The markup in is as follows:

[0134] TGEV-CTD indicates TGEV-CTD-SpyCatcher recombinant protein group;

[0135] TGEV-CTD-NPs indicates the TGEV-CTD-NPs group.

[0136] Since no specific antibodies were detected in the control group at 35-70 days, no Figure 7 Shown in.

[0137] 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 TGEV-CTD-SpyCatcher recombinant protein based on porcine transmissible gastroenteritis virus, characterized in that: The amino acid sequence of the TGEV-CTD-SpyCatcher recombinant protein is shown in SEQ ID NO.

1.

2. A TGEV protein nanoparticle based on porcine transmissible gastroenteritis virus, characterized in that: The TGEV protein nanoparticles are formed by self-assembly of TGEV-CTD-SpyCatcher recombinant protein and PCV2-Cap-SpyTag protein nanoparticles; The amino acid sequence of the TGEV-CTD-SpyCatcher recombinant protein is shown in SEQ ID NO.1, and the amino acid sequence of the PCV2-Cap-SpyTag protein nanoparticle is shown in SEQ ID NO.

2.

3. A method for preparing TGEV protein nanoparticles based on porcine transmissible gastroenteritis virus, characterized in that: Includes steps: S1, obtain TGEV-CTD-SpyCatcher recombinant protein and PCV2-Cap-SpyTag protein nanoparticles; Wherein, the amino acid sequence of the TGEV-CTD-SpyCatcher recombinant protein is shown in SEQ ID NO.1, and the amino acid sequence of the PCV2-Cap-SpyTag protein nanoparticle is shown in SEQ ID NO.2; S2, connecting the TGEV-CTD-SpyCatcher recombinant protein and the PCV2-Cap-SpyTag protein nanoparticles in a buffer solution to obtain the TGEV protein nanoparticles.

4. The preparation method according to claim 3, characterized in that: The nucleotide sequence encoding the TGEV-CTD-Sp yCatcher recombinant protein is shown in SEQ ID NO.3, and the nucleotide sequence encoding the PCV2-Cap-Spy Tag protein nanoparticles is shown in SEQ ID NO.

4.

5. The preparation method according to claim 3, characterized in that: The molar concentration ratio of the TGEV-CTD-SpyCatc her recombinant protein and the PCV2-Cap-SpyTag protein nanoparticles is 6:1-1:

1.

6. Use of the TGEV-CTD-SpyCatcher recombinant protein as claimed in claim 1 in the preparation of a porcine transmissible gastroenteritis vaccine.

7. An application of the TGEV protein nanoparticles as described in claim 2 in the preparation of porcine transmissible gastroenteritis vaccine.

8. A porcine transmissible gastroenteritis vaccine, characterized in that: The porcine transmissible gastroenteritis vaccine includes the TGEV-CTD-SpyCatcher recombinant protein as described in claim 1, or the porcine transmissible gastroenteritis vaccine includes the TGEV protein nanoparticles as described in claim 2.

9. Use of the TGEV-CTD-SpyCatcher recombinant protein as claimed in claim 1 in the preparation of TGEV-specific antibodies.

10. Use of the TGEV protein nanoparticles as described in claim 2 in the preparation of TGEV-specific antibodies.

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