Recombinant antigen protein, recombinant vector, recombinant host cell, virus-like particle, application of virus-like particle and vaccine

Abstract

The invention discloses a recombinant antigen protein, a recombinant vector, a recombinant host cell, a virus-like particle and application thereof and a vaccine, and relates to the technical field of antigen preparation, the recombinant antigen protein comprises an icosahedral porcine circovirus capsid protein and an A104R protein loaded on the surface of the icosahedral porcine circovirus capsid protein, the A104R protein comprises a protein coded by an African swine fever virus A104R gene. The recombinant antigen protein capable of simultaneously reacting with positive serum of the African swine fever virus and positive serum of the porcine circovirus is prepared, and the vaccine prepared by mixing the recombinant antigen protein with an adjuvant can effectively stimulate a pig body to generate specific antibodies of the African swine fever virus and the porcine circovirus; therefore, reference is provided for joint defense of the two viruses. The recombinant antigen protein provided by the invention has the characteristics of stable expression, high yield and high solubility.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of antigen preparation, in particular to a recombinant antigen protein, a recombinant carrier, a recombinant host cell, a virus-like particle and application and a vaccine thereof. BACKGROUND

[0002] African swine fever (ASF) is an acute and severe infectious disease caused by African swine fever virus (ASFV) and infecting pigs of various breeds and various ages, and the mortality rate is as high as 100%. The World Organization for Animal Health (OIE) lists it as a must-report animal epidemic, and China lists it as a class A animal infectious disease that needs to be prevented.

[0003] The African swine fever virus was first reported in 1921, and has been more than a hundred years since then. Although the development of vaccines has never stopped, there is still a lack of safe and effective commercial vaccines and no specific drugs. ASFV is extremely complex, and due to the limitations of its research, the protective antigen and antigen epitope spectrum have not been clearly understood, which has become a key factor restricting the research of genetic engineering vaccines.

[0004] In addition, there is currently a lack of measures to prevent both African swine fever virus and porcine circovirus infection. SUMMARY

[0005] The main purpose of the application is to provide a recombinant antigen protein, a recombinant carrier, a recombinant host cell, a virus-like particle and application and a vaccine, which aims to solve the problem of lack of effective African swine fever virus antigens in the prior art.

[0006] To achieve the above-mentioned purpose, the application provides a recombinant antigen protein, which comprises: an icosahedral porcine circovirus capsid protein and A104R protein loaded on the surface of the icosahedral porcine circovirus capsid protein, wherein the A104R protein comprises a protein encoded by an African swine fever virus A104R gene.

[0007] In an embodiment, the African swine fever virus comprises one or more of a type I strain, a type II strain, and a type I+II recombinant strain; or,

[0008] The amino acid sequence of the A104R protein is shown in SEQ ID No. 1 or SEQ ID No. 2.

[0009] In an embodiment, the genotype of the porcine circovirus comprises porcine circovirus type II, and the porcine circovirus type II comprises one or more of genotypes 2a, 2b, 2c, 2d, 2e, etc.; or,

[0010] The amino acid sequence of the icosahedral porcine circovirus capsid protein is shown as SEQ ID No. 4.

[0011] In an embodiment, the icosahedral porcine circovirus capsid protein and the A104R protein are connected by at least one flexible polypeptide; and / or,

[0012] The recombinant antigen protein further comprises a TorA signal peptide, and the amino acid sequence of the TorA signal peptide is shown as SEQ ID No. 6; and / or,

[0013] The amino acid sequence of the recombinant antigen protein is shown as SEQ ID No. 8.

[0014] The present application further provides a recombinant vector for encoding the aforementioned recombinant antigen protein, and the recombinant vector comprises a nucleotide sequence encoding an A104R protein and a nucleotide sequence encoding an icosahedral porcine circovirus capsid protein.

[0015] In an embodiment, the nucleotide sequence encoding the A104R protein is shown as SEQ ID No. 3; and / or,

[0016] The nucleotide sequence encoding the icosahedral porcine circovirus capsid protein is shown as SEQ ID No. 5; and / or,

[0017] The nucleotide sequence of the recombinant vector is shown as SEQ ID No. 9.

[0018] The present application further provides a recombinant host cell comprising the aforementioned recombinant vector.

[0019] The present application further provides a virus-like particle comprising the aforementioned recombinant antigen protein.

[0020] The present application further provides use of the aforementioned recombinant antigen protein, or the aforementioned recombinant vector, or the aforementioned recombinant host cell, or the aforementioned virus-like particle in the preparation of a preparation capable of causing an organism to simultaneously respond immunologically to porcine circovirus type II and African swine fever virus.

[0021] The present application further provides a vaccine comprising an oil adjuvant and the aforementioned recombinant antigen protein or the aforementioned virus-like particle.

[0022] The technical scheme of the present application is that the icosahedral porcine circovirus capsid protein is used as a carrier to load the A104R protein coded by the A104R gene of the African swine fever virus, so that a recombinant antigen protein capable of reacting with the African swine fever virus positive serum and the porcine circovirus positive serum is prepared, the vaccine prepared by mixing the recombinant antigen protein with an adjuvant can effectively stimulate the pig to produce the African swine fever virus and the porcine circovirus specific antibody, and therefore the reference for the joint defense of the two viruses is provided. The recombinant antigen protein provided by the present application has the characteristics of stable expression, high yield and high solubility. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained according to the structures shown in the drawings without creative labor for those skilled in the art.

[0024] Figure 1 The SDS-PAGE graph of the recombinant antigen protein expression and purification in the embodiment 2 of the present application (SDS-PAGE lane 1: Marker; 2: expression supernatant; 3: Ni column flow; 4: purification of impurities; 5: purified protein);

[0025] Figure 2 The reaction graph of the recombinant antigen protein and the African swine fever virus positive serum in the embodiment 2 of the present application (SDS-PAGE lane 1: Marker; 2: recombinant protein; 3: negative control);

[0026] Figure 3 The reaction graph of the recombinant antigen protein and the porcine circovirus type II positive serum in the embodiment 2 of the present application (SDS-PAGE lane 1: Marker; 2: recombinant protein; 3: negative control);

[0027] Figure 4 The transmission electron microscope graph of the recombinant antigen protein in the embodiment 2 of the present application;

[0028] Figure 5 The porcine circovirus type II antibody titer graph of the pig immunized by the recombinant antigen protein in the embodiment 3 of the present application;

[0029] Figure 6 The African swine fever virus A104R protein antibody titer graph of the pig immunized by the recombinant antigen protein in the embodiment 3 of the present application.

[0030] The implementation, functional characteristics and advantages of the present application will be further described with reference to the embodiments and the drawings. DETAILED DESCRIPTION

[0031] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. If specific conditions are not indicated in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. If the used reagents or instruments are not indicated by the manufacturers, they are all the conventional products that can be purchased in the market. In addition, the meaning of "and / or" appearing in the whole text includes three parallel solutions. Taking "A and / or B" as an example, it includes the solution of A, or the solution of B, or the solution of A and B. In addition, the technical solutions of various embodiments can be combined with each other, but it must be based on the fact that the technical solutions can be realized by the ordinary skilled in the art. When the combination of the technical solutions appears contradictory or unachievable, it should be considered that the combination of the technical solutions does not exist and is not within the protection scope of the present application. Based on the embodiments in the present application, all other embodiments obtained by the ordinary skilled in the art without creative labor are within the protection scope of the present application.

[0032] African swine fever (ASF) is an acute and severe infectious disease caused by African swine fever virus (ASFV) infection in pigs of various breeds and various ages, with a mortality rate of 100%. The World Organization for Animal Health (OIE) lists it as a must-report animal disease, and China lists it as a class A animal infectious disease that needs to be prevented.

[0033] African swine fever virus was first reported in 1921, and has been more than a hundred years since then. Although the development of vaccines has never stopped, there is still a lack of safe and effective commercial vaccines and specific drugs. ASFV is extremely complex, and due to the limitations of its research, the protective antigen and antigen epitope spectrum have not been clearly understood, which has become a key factor restricting the research of genetic engineering vaccines.

[0034] In addition, there is currently a lack of measures to prevent both African swine fever virus and porcine circovirus infection.

[0035] In view of this, the present application provides a recombinant antigen protein, which comprises: an icosahedral porcine circovirus capsid protein and an A104R protein loaded on the surface of the icosahedral porcine circovirus capsid protein, wherein the A104R protein comprises a protein encoded by an A104R gene of African swine fever virus.

[0036] The icosahedral porcine circovirus capsid protein is used as a carrier to load the A104R protein encoded by the A104R gene of the African swine fever virus, so as to prepare a recombinant antigen protein which can simultaneously react with the African swine fever virus positive serum and the porcine circovirus positive serum. The vaccine prepared by mixing the recombinant antigen protein with an adjuvant can effectively stimulate the pig to produce the African swine fever virus and the porcine circovirus specific antibodies, and thus provides a reference for the joint prevention of the two viruses. The recombinant antigen protein has the characteristics of stable expression, high yield and high solubility. Further, the recombinant antigen protein can be directly assembled into the finally obtained virus-like particles in the expression process.

[0037] In some embodiments, the African swine fever virus comprises one or more of a type I strain, a type II strain, and a type I+II recombinant strain, wherein the type I strain is, for example, Pig / SD / DY-I / 2021 strain, pig / HeN / ZZ-P1 / 2021 strain, the type II strain is, for example, pig / HLJ / 2018 strain, Georgia 2007 strain, and the type I+II recombinant strain is, for example, pig / JS / LG / 2021 strain, pig / HN / 2022 strain, Pig / Inner Mongolia / DQDM / 2022 strain. Preferably, the African swine fever virus is a type I+II recombinant strain. The amino acid sequences of A104R of the above different strains are highly consistent.

[0038] Preferably, in some embodiments, the amino acid sequence of the A104R protein is as shown in SEQ ID No. 1 or SEQ ID No. 2. More preferably, the amino acid sequence of the A104R protein is as shown in SEQ ID No. 2, that is, 16 amino acids at the N-terminus and 10 amino acids at the C-terminus are deleted based on SEQ ID No. 1, the AU-rich region in the nucleotide sequence is optimized, the RNase E cleavage site is reduced, the protease cleavage site is reduced, the aggregation tendency of the recombinant antigen protein is reduced, and the yield and solubility of the recombinant antigen protein are improved.

[0039] Porcine circovirus type II (PCVII) belongs to the porcine circovirus genus, the virus particle has a diameter of 14-17 nm and exhibits icosahedral symmetry, can be mixedly infected with various viruses and bacteria in pigs, aggravates the clinical manifestations, is widely prevalent in the world, and causes serious economic losses to the pig industry. In some embodiments, the genotype of the porcine circovirus comprises one or more of porcine circovirus type II, 2a type, 2b type, 2c type, 2d type, 2e type, etc. Preferably, the genotype of the porcine circovirus type II is 2d type, which is widely prevalent.

[0040] Preferably, in some embodiments, the amino acid sequence of the icosahedral porcine circovirus capsid protein (Cap protein) is shown as SEQ ID No. 4. The Cap protein of the amino acid sequence has less antibody-dependent enhancement related epitopes.

[0041] In some embodiments, the icosahedral porcine circovirus capsid protein and the A104R protein are connected by at least one flexible polypeptide. Preferably, the icosahedral porcine circovirus capsid protein and the A104R protein are connected by three flexible polypeptides, and the amino acid sequence of the flexible polypeptide is GGSGG. By the above connection mode, the influence of the A104R sequence on the self-assembly of the Cap protein can be reduced, and the self-assembly of the Cap protein into an icosahedron can be ensured.

[0042] In some embodiments, the recombinant antigen protein further comprises a TorA signal peptide, and the amino acid sequence of the TorA signal peptide is shown as SEQ ID No. 6. The TorA signal peptide sequence enhances the solubility and native conformation formation of the recombinant antigen protein, and improves the yield of the recombinant antigen protein in Escherichia coli and the immunoreactivity and effectiveness of the recombinant protein.

[0043] In some embodiments, the amino acid sequence of the recombinant antigen protein is shown as SEQ ID No. 8.

[0044] The present application also provides a recombinant vector for encoding the aforementioned recombinant antigen protein, and the recombinant vector comprises a nucleotide sequence encoding an A104R protein and a nucleotide sequence encoding an icosahedral porcine circovirus capsid protein.

[0045] In some embodiments, the nucleotide sequence encoding the A104R protein is shown as SEQ ID No. 3. In some embodiments, the nucleotide sequence encoding the icosahedral porcine circovirus capsid protein is shown as SEQ ID No. 5. In some embodiments, the nucleotide sequence of the recombinant vector is shown as SEQ ID No. 9. It should be noted that the designed protein genome can be optimized according to the codon preference of the expression system, and the expression system is preferably an Escherichia coli expression system.

[0046] The present application also provides a recombinant host cell comprising the aforementioned recombinant vector. Preferably, the cell source of the recombinant host cell is an Escherichia coli BL21 (DE3) competent cell. The recombinant host cell has all the beneficial effects of the aforementioned recombinant vector, which will not be described here.

[0047] The application also provides a virus-like particle comprising the aforementioned recombinant antigen protein. The virus-like particle (VLP) is identical or similar to a real virus particle in morphology, has good safety and can strongly stimulate the immune system of the body, and can produce a good immune protection response. The PCVII-Cap self-assembles into a icosahedral virus-like particle surface attached to the exogenous A104R protein antigen can cause a strong immune response, that is, the virus-like particle of the application can enhance the immune response of the A104R protein.

[0048] The application also provides use of the aforementioned recombinant antigen protein, or the aforementioned recombinant vector, or the aforementioned recombinant host cell, or the aforementioned virus-like particle in the preparation of a preparation capable of causing the organism to simultaneously respond to porcine circovirus type II and African swine fever virus. Therefore, all the beneficial effects of the aforementioned recombinant antigen protein, or the aforementioned recombinant vector, or the aforementioned recombinant host cell, or the aforementioned virus-like particle are possessed, which will not be repeated here. It can be understood that the preparation includes a vaccine (which can be a subunit vaccine), an immune enhancer, a diagnostic reagent or an immunotherapeutic drug.

[0049] The application also provides a vaccine comprising an oil adjuvant and the aforementioned recombinant antigen protein or the aforementioned virus-like particle. Therefore, all the beneficial effects of the aforementioned recombinant virus antigen or the aforementioned virus-like particle are possessed, which will not be repeated here.

[0050] The technical solutions of the application will be further described in detail below in combination with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the application and do not limit the application.

[0051] Example 1 Determination and optimization of the gene sequence of the recombinant antigen protein A104R-Cap

[0052] 1. Determination and optimization of the A104R gene sequence

[0053] The amino acid sequence of A104R of ASFV was obtained from GenBank in the National Center for Biotechnology Information (NCBI), including: A104R in I type Pig / SD / DY-I / 2021 strain (MZ945537.1) (UEN73202.1), A104R in I type pig / HeN / ZZ-P1 / 2021 strain (MZ945536.1) (UEN73044.1), A104R in II type pig / HLJ / 2018 strain (MK333180.1) (QBH90522.1), A104R in II type Georgia 2007 strain (FR682468.1) (CBW46700.1), A104R in I+II type pig / JS / LG / 2021 strain (OQ504956.1) (WFS78491.1), A104R in I+II type pig / HN / 2022 strain (OQ504954.1) (WFS78147.1), A104R in I+II type Pig / Inner Mongolia / DQDM / 2022 strain (OQ504955.1) (WFS78319.1), etc., sequence alignment was performed, and it was found that the amino acid sequences of A104R between the above different strains were highly consistent.

[0054] The final determination of the amino acid sequence of A104R is shown in SEQ ID No. 1:

[0055] MSTKKKPTITKQELYSLVAADTQLNKALIERIFTSQQKIIQNALKHNQEVIIPPGIKFTVVTVKAKPARQGHNPATGEPIQIKAKPEHKAVKIRALKPVHDMLN

[0056] Sequence optimization step:

[0057] (1) The following optimization was performed on SEQ ID No. 1: 16 amino acids at the N-terminus were deleted, and 10 amino acids at the C-terminus were deleted, and the truncated sequence after deletion is shown in SEQ ID No. 2:

[0058] LVAADTQLNKALIERIFTSQQKIIQNALKHNQEVIIPPGIKFTVVTVKAKPARQGHNPATGEPIQIKAKPEHKAVKIR

[0059] The optimization step has the following advantages: a) deletion of 16 amino acids at the N-terminus containing non-antigen epitope disordered regions reduces protease degradation sites; b) deletion of 10 amino acids at the C-terminus containing hydrophobic motifs reduces the aggregation tendency of the recombinant antigen protein. Overall, the optimization step improves the yield and solubility of the recombinant antigen protein.

[0060] (2) According to the amino acid sequence of SEQ ID No. 2 and the preference of the E. coli expression system, the coding nucleotide sequence is designed and optimized, and the AU-rich region in the nucleotide sequence is optimized to reduce the RNase E cleavage site, so as to facilitate expression, and obtain SEQ ID No. 3:

[0061] CTGGTGGCGGCGGATACCCAGCTGAACAAAGCGCTGATTGAACGCATCTTTACCAGCCAGCAGAAAATTATTCAGAACGCGCTGAAACATAACCAGGAAGTGATTATTCCGCCGGGCATCAAATTCACCGTGGTGACCGTGAAAGCGAAACCGGCGCGCCAGGGCCATAACCCGGCGACCGGCGAACCGATTCAGATTAAAGCGAAACCGGAACATAAAGCGGTGAAAATTCGC

[0062] The optimization step can improve the stability of the recombinant antigen protein.

[0063] 2. Confirmation and optimization of PCVII-Cap gene sequence

[0064] The amino acid sequence of the Cap protein of PCVII type 2d genotype was obtained from GenBank.

[0065] Sequence optimization step:

[0066] (1) The amino acid sequence of the Cap protein of PCVII type 2d genotype is optimized as follows: deletion of 10 amino acids at the N-terminus and 4 amino acids at the C-terminus, mutation of Pro32 at the N-terminus to Ala32, mutation of Lys41 to Glu41, and the optimized amino acid sequence is shown in SEQ ID No. 4:

[0067] RRHRPRSHLGQILRRRPWLVHARHRYRWRRENGIFNTRLSRTIGYTVKKTTVRTPSWNVDMMRFNINDFLPPGGGSNPLTVPFEYYRIRKVKVEFWPCSPITQGDRGVGSTAVILDDNFVTKANALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDRTIDYFQPNNKRNQLWLRLQTTGNVDHVGLGTAFENSIYDQDYNIRITMYVQFREFNLKDP

[0068] The optimization step has the following advantages: a) deleting 10 amino acids at the N-terminus and 4 amino acids at the C-terminus that are irrelevant to the formation of VLPs enhances the expression and secretion of the recombinant protein; b) the directional mutation of Pro32 at the N-terminus to Ala32 and the directional mutation of Lys41 to Glu41 destroy the antibody-dependent enhancement (ADE)-related epitope, reduce the ADE action site, and reduce the possibility of ADE.

[0069] (2) According to the amino acid sequence of SEQ ID No. 4 and the preference of the E. coli expression system, the coding nucleotide sequence is designed and optimized, and the AU-rich region in the nucleotide sequence is optimized and the RNase E cleavage site is reduced to facilitate expression, obtaining SEQ ID No. 5:

[0070] CGCCGCCATCGCCCGCGCAGCCATCTGGGCCAGATTCTGCGCCGCCGCCCGTGGCTGGTGCATGCGCGCCATCGCTATCGCTGGCGCCGCGAAAACGGCATCTTTAACACCCGCCTGAGCCGCACCATTGGCTATACCGTGAAAAAAACCACCGTGCGCACCCCGAGCTGGAACGTGGATATGATGCGCTTTAACATTAACGATTTTCTGCCGCCGGGCGGCGGCAGCAACCCGCTGACCGTGCCGTTTGAATATTATCGCATTCGCAAAGTGAAAGTGGAATTTTGGCCGTGCAGCCCGATTACCCAGGGCGATCGCGGCGTGGGCAGCACCGCGGTGATTCTGGATGATAACTTTGTGACCAAAGCGAACGCGCTGACCTATGATCCGTATGTGAACTATAGCAGCCGCCATACCATTACCCAGCCGTTTAGCTATCATAGCCGCTATTTCACCCCGAAACCGGTGCTGGATCGCACCATTGATTATTTTCAGCCGAACAACAAACGCAACCAGCTGTGGCTGCGCCTGCAGACCACCGGCAACGTGGATCATGTGGGCCTGGGCACCGCGTTTGAAAACAGCATCTATGATCAGGATTATAACATTCGCATTACCATGTATGTGCAGTTCAGAGAATTTAATCTTAAAGACCCC

[0071] 3. Determination and optimization of TorA signal peptide gene sequence

[0072] The introduction of TorA signal peptide is beneficial to the formation of protein structure and the transport characteristics. The introduction of TorA signal peptide sequence at the front end of the sequence of recombinant antigen protein can enhance the solubility and natural conformation formation of recombinant antigen protein, improve the yield of recombinant antigen protein in E. coli and the immunoreactivity and effectiveness of recombinant antigen protein.

[0073] The amino acid sequence of the introduced TorA signal peptide is shown in SEQ ID No. 6:

[0074] MNNNDLFQASRRRFLAQLGGLTVAGMLAQA

[0075] According to the amino acid sequence of SEQ ID No. 6 and the preference of the E. coli expression system, the coding nucleotide sequence is designed and optimized, and the optimized nucleotide sequence is shown as SEQ ID No. 7 for facilitating the expression thereof:

[0076] ATGAACAACAACGATCTGTTTCAGGCGAGCCGCCGCCGCTTTCTGG CGCAGCTGGGCGGCCTGACCGTGGCGGGCATGCTGGCGCAGGCG

[0077] 4. Determination and optimization of the gene sequence of the recombinant antigen protein

[0078] A 6×His tag is introduced at the C-terminal of the recombinant antigen protein for facilitating the purification, and thus the target connection sequence of the recombinant antigen protein is in the order of: TorA signal peptide sequence-PCVII-Cap protein sequence-A104R truncated sequence-His tag sequence.

[0079] Sequence optimization steps:

[0080] (1) The above recombinant antigen protein is optimized as follows: the Cap skeleton and the TorA signal peptide, the A104R truncated body and the His are all connected in series through a flexible linker, and the Cap skeleton and the A104R truncated sequence are connected in series through 3 flexible linkers. The determined amino acid sequence of the recombinant antigen protein is shown as SEQ ID No. 8:

[0081] MGMNNNDLFQASRRRFLAQLGGLTVAGMLAQAGGSGGRRHRPRSHLGQILRRRPWLVHARHRYRWRRENGIFNTRLSRTIGYTVKKTTVRTPSWNVDMMRFNINDFLPPGGGSNPLTVPFEYYRIRKVKVEFWPCSPITQGDRGVGSTAVILDDNFVTKANALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDRTIDYFQPNNKRNQLWLRLQTTGNVDHVGLGTAFENSIYDQDYNIRITMYVQFREFNLKDPGGSGGGGSGGGGSGGLVAADTQLNKALIERIFTSQQKIIQNALKHNQEVIIPPGIKFTVVTVKAKPARQGHNPATGEPIQIKAKPEHKAVKIRGGSGGHHHHHH

[0082] This optimization step has the following advantages: reducing the influence of the A104R truncated body on the self-assembly of the Cap protein.

[0083] (2) According to the amino acid sequence of SEQ ID No. 8 and the preference of E. coli expression system, the coding nucleotide sequence is designed and optimized, in order to facilitate its expression, NcoI and XhoI enzyme digestion sites are introduced at the front and back of the sequence respectively, the AU-rich region of the whole nucleotide sequence is optimized, and a stop codon is introduced between the enzyme digestion sites at the C terminal. The final nucleotide sequence of the recombinant antigen protein A104R-Cap is shown in SEQ ID No. 9:

[0084]

[0085] Preparation of virus-like particles of recombinant antigen protein A104R-Cap

[0086] 1. Preparation of recombinant vector

[0087] According to the nucleotide sequence of the recombinant antigen protein obtained in Example 1, the nucleotide sequence of A104R-Cap (SEQ ID No. 9) was artificially synthesized, and the nucleotide sequence encoding the recombinant antigen protein A104R-Cap was cloned into the pET-28a(+) plasmid as a vector to construct a recombinant plasmid: pET-A104R-Cap.

[0088] 2. Transformation and induction of protein expression

[0089] The recombinant plasmid pET-A104R-Cap obtained in step 1 was transformed into E. coli BL21(DE3) competent cells. The main operations are as follows:

[0090] After thawing the E. coli BL21(DE3) competent cells on ice, 5 μL of 2 ng / mL recombinant plasmid obtained in step 1 was added, mixed well, and placed on ice for 30 min; heat shock at 42°C for 30 s, place on ice for 5 min, add 500 μL of culture medium, mix well, and incubate at 37°C, 200 rpm on a shaker for 1 h to obtain the cultured bacterial solution;

[0091] 100 μL of the bacterial solution was taken and evenly spread on an LB plate containing kanamycin 50 μg / mL, and incubated at 37°C overnight to obtain the cultured colonies;

[0092] A single colony was picked for colony PCR identification and sequencing identification to ensure correct transformation, and a correctly transformed strain was obtained;

[0093] The above correctly transformed strain was selected and inoculated into LB culture medium containing kanamycin 50 μg / mL, and cultured until the OD 600 = 0.6-0.8, then 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) was added, and the culture was continued at 20°C for 12 h to induce the expression of the recombinant antigen protein;

[0094] The bacterial solution before and after induction of the recombinant antigen protein expression was taken, and the sample (supernatant) was taken after ultrasonic bacterial disruption for SDS-PAGE detection of the expression of the recombinant antigen protein. The SDS-PAGE graph of the supernatant of the recombinant antigen protein corresponds to lane 2 in Figure 1 .

[0095] 3. Purification of recombinant antigen protein and detection of virus-like particles

[0096] After the bacterial culture in step 2 is induced and expressed, it is broken down and purified to obtain the recombinant antigen protein. The main steps are as follows:

[0097] Centrifuge the bacterial culture induced in step 2 at 12000 rpm for 15 min to collect the bacterial cells, resuspend the bacterial cells in superphosphate buffer (PBS), and lyse the bacterial cells by sonication on ice; then centrifuge at 4°C, 12000 rpm for 30 min to collect the supernatant.

[0098] The supernatant was passed through a nickel (Ni) column and washed with 10 column volumes of 50 mM imidazole followed by elution with 500 mM imidazole to obtain the purified recombinant antigen protein. The purified recombinant antigen protein was detected by Western blotting using positive sera for porcine circovirus type II and African swine fever virus, respectively. The results showed that the recombinant antigen protein reacted with both positive sera. Figure 2 and Figure 3 ).

[0099] The morphology of the assembled nanoparticle vaccine was observed by transmission electron microscopy (TEM) using a negative staining method. Figure 4 It can be seen that the recombinant antigen protein can self-assemble into virus-like particles, and the A104R protein has been successfully assembled onto the surface of the Cap protein.

[0100] Example 3: Preparation and application of immunogens based on recombinant antigen proteins

[0101] Vaccine preparation: Purified A104-Cap recombinant protein was combined with Montanide from Seppic, France. TM Mix ISA563VG oil adjuvant to achieve a final concentration of 50 μg / mL for the recombinant antigen protein, ensuring homogeneous emulsification. Perform sterility, viscosity, and stability tests according to the current Chinese Veterinary Pharmacopoeia appendix; store at 2-8℃.

[0102] Ten healthy 4-5 week old piglets, both negative for PCV2 and ASFV antigens and antibodies, were randomly divided into two groups of five each. The first group was the immunization group, where each piglet received an intramuscular injection of the prepared vaccine (2 mL / pig) into the neck. The second group was the control group, receiving no injection. Twenty-one days later, the immunized piglets underwent a second immunization in the same manner. Following the first immunization, blood samples were collected weekly from each group to separate serum. The antibody levels for porcine circovirus type 2 (PCV2 ELISA antibody detection kit) and African swine fever virus (ASFV indirect ELISA antibody detection kit) in the serum were detected using a commercially available kit produced by Sinopharm Animal Health Co., Ltd. The results are as follows: Figure 5 and Figure 6 As shown.

[0103] The antibody results show that the prepared recombinant antigen protein immunogen can rapidly produce antibodies after immunizing piglets. The antibody levels of porcine circovirus type 2 Cap protein and African swine fever virus A104R protein after immunization increase significantly. Among them, for the antibodies of porcine circovirus type 2: the antibodies of the immunized pigs increased at 7 days, the antibodies of 3 / 5 immunized pigs turned positive at 14 days, and the antibodies of 5 / 5 immunized pigs turned positive at 21 days; the antibodies of the African swine fever virus A104 protein increased at 7 days, the antibodies of 2 / 5 immunized pigs turned positive at 14 days, and the antibodies of 3 / 5 immunized pigs turned positive at 21 days; the antibodies of 5 / 5 immunized pigs turned positive at 28 days. The antibodies of the control group piglets to porcine circovirus II Cap protein and African swine fever virus A104R are negative. The results show that the recombinant protein vaccine of African swine fever virus A104R protein and porcine circovirus Cap protein prepared by the genetically engineered strain of the application can effectively stimulate pigs to produce African swine fever virus A104R and porcine circovirus II specific antibodies, and the antibody level produced after immunization has a significant difference compared with the control group.

[0104] The application is based on the A104R protein truncation, and the Cap protein of porcine circovirus 2d is used as a virus-like particle carrier to prepare a virus-like particle antigen containing A104R protein which can react with positive serum of African swine fever virus and porcine circovirus II. The antibody results of the antigen antibody double-negative piglets immunized after mixing with adjuvant show that the recombinant antigen protein vaccine can effectively stimulate pigs to produce African swine fever virus A104R and porcine circovirus II specific antibodies, and the antibody level produced after immunization has a significant difference compared with the control group, which lays a good technical foundation for the development of vaccines related to African swine fever virus and porcine circovirus, and provides reference and guidance for the joint prevention of the two diseases.

[0105] The above is only a preferred embodiment of the application, and does not limit the patent scope of the application. For those skilled in the art, the application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the application shall be included in the patent protection scope of the application.

Claims

1. A recombinant antigen protein, characterized in that, The recombinant antigen protein includes: icosahedral porcine circovirus capsid protein and A104R protein loaded on the surface of the icosahedral porcine circovirus capsid protein, wherein the A104R protein includes the protein encoded by the African swine fever virus A104R gene.

2. The recombinant antigen protein as described in claim 1, characterized in that, The African swine fever virus includes one or more of type I strains, type II strains, and type I+II recombinant strains; or, The amino acid sequence of the A104R protein is shown in SEQ ID No. 1 or SEQ ID No.

2.

3. The recombinant antigen protein as described in claim 1, characterized in that, The porcine circovirus genotype includes porcine circovirus type II, which includes one or more genotypes such as 2a, 2b, 2c, 2d, and 2e; or, The amino acid sequence of the icosahedral porcine circovirus capsid protein is shown in SEQ ID No.

4.

4. The recombinant antigen protein according to any one of claims 1 to 3, characterized in that, The icosahedral porcine circovirus capsid protein and the A104R protein are linked by at least one flexible polypeptide; and / or, The recombinant antigen protein further includes a TorA signal peptide, the amino acid sequence of which is shown in SEQ ID No. 6; and / or, The amino acid sequence of the recombinant antigen protein is shown in SEQ ID No.

8.

5. A recombinant vector, characterized in that, The recombinant vector is used to encode the recombinant antigen protein as described in any one of claims 1 to 4, wherein the recombinant vector comprises a nucleotide sequence encoding the A104R protein and a nucleotide sequence encoding the icosahedral porcine circovirus capsid protein.

6. The recombinant vector as described in claim 5, characterized in that, The nucleotide sequence encoding the A104R protein is shown in SEQ ID No. 3; and / or, The nucleotide sequence encoding the icosahedral porcine circovirus capsid protein is shown in SEQ ID No. 5; and / or, The nucleotide sequence of the recombinant vector is shown in SEQ ID No.

9.

7. A recombinant host cell, characterized in that, The recombinant host cell includes the recombinant vector as described in claim 5 or 6.

8. A virus-like particle, characterized in that, Includes the recombinant antigen protein as described in any one of claims 1 to 4.

9. The use of a recombinant antigen protein as described in any one of claims 1 to 4, or a recombinant vector as described in any one of claims 5 to 6, or a recombinant host cell as described in claim 7, or a virus-like particle as described in claim 8, in the preparation of an agent capable of inducing an immune response in an organism against both porcine circovirus type II and African swine fever virus.

10. A vaccine, characterized in that, The vaccine includes an oil adjuvant and a recombinant antigen protein as described in any one of claims 1 to 4 or a virus-like particle as described in claim 8.

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