CN113416236B - Porcine circovirus type 3 virus-like particle and preparation method and application thereof - Google Patents

Abstract

The invention provides porcine circovirus type 3 virus-like particles and a preparation method and application thereof. The present invention utilizes an insect-baculovirus expression system to express a variant of PCV3Cap protein to produce virus-like particles. The invention tries to start the expression of the Cap protein by using double promoters of baculovirus, deletes the amino acids from the 1 st to the 22 th positions and inserts a sequence which codes the basic amino acid rich at the P6.9N end of the baculovirus basic protein on the basis of keeping the basic amino acid rich at the N end of the Cap protein, so that the Cap protein is expressed in a fusion mode. The result shows that the expressed fusion protein can not only form virus-like particles, but also express the protein at a higher level, thereby providing a foundation for further developing PCV3 vaccine and realizing the industrial production of PCV3 virus-like particles.

Description

Porcine circovirus type 3 virus-like particle and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to porcine circovirus type 3 virus-like particles, and a preparation method and application thereof.

Background

Porcine Circovirus (PCV) belongs to the genus Circovirus (Circoviridae) and contains single-stranded circular DNA, is one of the smallest animal DNA viruses, and was first discovered in Porcine kidney cell line (PK-15 cell line) in 1974. Currently, 4 genotypes of PCV have been found, PCV1, PCV2, PCV3, PCV4, respectively. PCV1 is nonpathogenic, PCV4 is a genotype newly discovered in 2019, and the pathogenicity of PCV4 is to be further studied. PCV2 can infect monocyte, macrophage and the like to cause immune cell injury of pigs, and cause severe immunosuppression of infected pigs, thereby causing symptoms such as postweaning multisystemic asthenia syndrome (PMWS), dermatitis and nephrotic syndrome (PDNS), proliferative necrotizing interstitial pneumonia (PNP) and reproductive disorders of pigs. PCV3 was discovered in the United states in 2015, and tissues of the diseased pig were subjected to Immunohistochemistry (IHC) and quantitative PCR (IHC) detection, so that the diseased pig was determined to be free from viruses such as PCV2 and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) which may cause PDNS, and PCV3 is similar to PCV2 and can also cause the infected pig to have diseases such as PDNS and reproductive disturbance. Later studies showed that nasal vaccination of 4 week old piglets without Specific Pathogen (SPF) with PCV3 obtained from infectious clones resulted in PDNS disease in piglets. PCV3 is currently prevalent in several countries of the world and is detected in several provinces of china. Since PCV2 has low homology with PCV3 and the possibility of cross protection is very low, PCV3 may bring huge loss to pig breeding industry, and therefore, the development of detection kits and vaccines aiming at PCV3 has important significance for the detection and control of PCV 3.

PCV virus particles have stronger resistance to the outside, are difficult to eradicate by adopting conventional physical and chemical methods, and are an important means for effectively controlling PCV infection by carrying out immunization and prevention on pig vaccination. The PCV3 is not commercialized at present, and the main reasons are that PCV3 is extremely difficult to separate and culture in vitro, and is difficult to establish an animal infection model and obtain live viruses for preparing inactivated vaccines. Virus-like particles are structurally similar to parental virions, consisting of only viral structural proteins, and contain no viral nucleic acids, and can be expressed by gene synthesis using prokaryotic or eukaryotic expression systems without the need to obtain active virus. Therefore, the virus-like particles have immunogenicity similar to that of parental viruses, but have higher safety, and the production and preparation of the virus-like particles are lower in conditions such as environment, equipment and the like, thereby being beneficial to reducing the production cost and realizing large-scale production. PCV3 virions contain the only structural protein or protective antigen, i.e. the capsid protein Cap, which can be formed into virus-like particles by expression of Cap alone. Therefore, the expression and preparation of the virus-like particle consisting of Cap protein against PCV3 are important directions for vaccine development.

For preparing PCV3 virus-like particles, many research units or enterprises often adopt recombinant Escherichia coli expression systems. Although soluble expression of Cap protein can be realized at present, the expression quantity is low, and a prokaryotic expression system cannot modify expressed protein in eukaryotic cells such as phosphorylation and glycosylation, and also has the problem of endotoxin, so that endotoxin removal treatment is required in industrial production; before protein purification, it is necessary to lyse the bacterial cells and sometimes to cleave the fusion sequence and purify the protein. The factors make the process for preparing PCV3 virus-like particles by using a prokaryotic expression system complicated and the immunogenicity of the obtained vaccine possibly poor. In addition, secretory expression is also performed using a yeast expression system, and a technical strategy is to add a nucleotide sequence encoding a secretory signal peptide to a nucleotide sequence encoding a Cap to secretly express the Cap protein, but the secretory expression level is low. Baculovirus expression systems have many advantages of accommodating larger foreign insertion genes, high protein expression level, simple operation, post-translational processing modification and the like, and are widely used. At present, although the full-length and N-terminal 32-amino-acid-truncated Cap protein is expressed in baculovirus and can form VLP (virus-like particle), the 1 st to 32 nd amino acids at the N terminal of the Cap protein are rich in basic amino acids and can mediate nuclear transport of the Cap protein, the expression of the complete Cap protein may influence the expression quantity of the Cap protein in an expression system, and the deletion of the whole segment may influence the surface characteristics of the virus-like particles and the stability of the virus-like particles, thereby influencing the immunogenicity of the Cap protein. Initial studies find that the expression level of the complete PCV3Cap protein and the Cap protein lacking the N-terminal 32 amino acids is extremely low in insect cells, and the industrialization can not be satisfied. In addition, in the above scheme using the baculovirus expression system, only the codon non-optimized Cap gene was inserted into the expression vector, and no other element was added or the vector was modified to further increase the expression amount of the protein.

In conclusion, the development of PCV3Cap virus-like particles is of great significance, but on the expression of Cap protein at present, the process of a prokaryotic expression system is more complex, the protein is not subjected to post-translational modification, the immunogenicity of the obtained virus-like particles is poorer than that of eukaryotic expression, the secretory expression quantity of a yeast expression system is lower, and an insect-baculovirus expression system still needs to be further optimized and improved. Therefore, the development of a method for preparing highly efficient PCV3 virus-like particles is urgently needed.

Disclosure of Invention

The invention aims to provide a novel porcine circovirus type 3 virus-like particle, and a preparation method and application thereof.

To achieve the object of the present invention, in a first aspect, the present invention provides a variant Cap protein of porcine circovirus type 3, said variant Cap protein comprising or consisting of the amino acid sequence as follows:

i) an amino acid sequence (consisting of 23-214 amino acids of porcine circovirus type 3Cap protein) shown as SEQ ID NO. 5; or

ii) an amino acid sequence obtained by connecting a label at the N end and/or the C end of the i); or

iii) protein with same function obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequence of i) or ii).

In a second aspect, the invention provides a nucleic acid molecule encoding the protein variant.

In a third aspect, the present invention provides biological materials containing the above-described nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

In a fourth aspect, the present invention provides a porcine circovirus type 3 virus-like particle, wherein the virus-like particle is obtained by expressing the protein variant using an insect-baculovirus expression system.

Preferably, the insect-baculovirus expression system uses a dual promoter to promote the expression of the porcine circovirus type 3Cap protein variant, namely, a PH promoter is used and the promoter of the baculovirus P6.9 gene and a 5' part coding sequence (namely, a nucleic acid sequence for coding the N-terminal part amino acid of the P6.9 protein are fused at the same time, wherein the N-terminal part amino acid of the baculovirus P6.9 protein at least comprises the 2 nd to 9 th amino acids at the N-terminal of the baculovirus P6.9 protein, such as VYRRRRS) are inserted into the downstream of the promoter in a single copy or multi-copy mode.

In a fifth aspect, the present invention provides a method for preparing porcine circovirus type 3 virus-like particles, the method comprising: firstly, constructing recombinant baculovirus for expressing the protein variant, then infecting insect cells with the recombinant baculovirus, and separating virus-like particles from cell culture solution after the cells are diseased;

in the method, a nucleic acid sequence encoding the variant Cap protein is operably linked to a PH-P6.9 dual promoter, and a nucleic acid sequence encoding the N-terminal amino acid of the baculovirus P6.9 protein is inserted before the initiation codon of the nucleic acid sequence encoding the variant Cap protein; wherein the N-terminal amino acid of the baculovirus P6.9 protein at least comprises 2 nd to 9 th amino acids (VYRRRRS) at the N-terminal of the baculovirus P6.9 protein, preferably comprises 11 th amino acids (MVYRRRRRSST) at the N-terminal of the baculovirus P6.9 protein, and the PH-P6.9 double promoter is formed by connecting a baculovirus PH promoter and a P6.9 promoter in series.

Preferably, the vector for constructing the recombinant baculovirus may be selected from a series of vectors for insect-baculovirus expression systems such as pFastBac1, pFastBac HTA, pFastBac HTB, pFastBac HTC, pFastBacDual, pIEx-Bac, pOET or pQB, and vectors modified on the basis of these vectors; more preferably pfastpac 1.

Preferably, the insect cell may be selected from Sf9, Sf21, High Five (Hi5), and the like, and insect cells modified on the basis of these cells by gene knockout, insertion, modification, or the like.

In the invention, the sequence of the p6.9 promoter is shown as SEQ ID NO. 6.

The PH-p6.9 double promoter sequence is shown in SEQ ID NO. 7.

A fusion protein of PCV3Cap protein variant with the N end connected with the N-end part amino acid of P6.9 protein (the 1 st to 11 th amino acids at the N end of the P6.9 protein + the 23 rd to 214 th amino acids of PCV3Cap protein) is named as P6-Cap, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 4.

In a fifth aspect, the invention provides a porcine circovirus type 3 genetically engineered subunit vaccine comprising said protein variant, or said vaccine comprising said virus-like particle, or said vaccine comprising a virus-like particle prepared according to the above method.

In a sixth aspect, the present invention provides any one of the following uses of the protein variant, or the virus-like particle, or a virus-like particle prepared according to the above method:

1) is used for preparing porcine circovirus type 3 polyclonal antibody or monoclonal antibody;

2) is used for preparing a porcine circovirus type 3 antibody detection reagent;

3) the kit is used for detecting porcine circovirus type 3 virus infection;

4) used for preparing porcine circovirus type 3 genetic engineering subunit vaccine;

5) can be used for preventing porcine circovirus type 3 infection and related diseases caused by the infection.

Further, the invention also relates to the optimization of the nucleotide sequence for coding the Cap protein of porcine circovirus type 3, the construction of recombinant baculovirus for efficiently expressing the Cap protein and the preparation of PCV3 virus-like particles (VLP).

By means of the technical scheme, the invention at least has the following advantages and beneficial effects:

the present invention utilizes an insect-baculovirus expression system to express a variant of PCV3Cap protein to produce virus-like particles. The invention firstly tries to delete partial sequences at the N end of the Cap protein so as to keep the characteristic of the Cap protein rich in basic amino acids, and proves that the deletion of the 1 st to 22 th amino acids at the N end does not influence the assembly and formation of the Cap protein virus-like particles; meanwhile, the invention tries to start the expression of PCV3Cap protein by using the dual promoter of baculovirus for the first time, and fuses the N-terminal basic amino acid-rich sequence of baculovirus basic protein P6.9, thereby further retaining the characteristic of the N-terminal basic amino acid-rich of Cap protein. The results show that the expressed fusion protein can not only form a virus-like particle structure, but also express the protein at a higher level, thereby providing a basis for further developing virus-like particle vaccines aiming at PCV3 and industrial production of PCV3 virus-like particles.

Drawings

FIG. 1 shows the result of PCR amplification of the protein encoding PCV3Cap according to the preferred embodiment of the present invention. Wherein, Lane 2 and Lane 3 are the result of amplification of the sequence encoding the full-length PCV3Cap protein (Cap-FL); lanes 4 and 5 show the amplification of the sequence encoding the P6-Cap fusion protein.

FIG. 2 shows the identification of clones by PCR in a preferred embodiment of the present invention. Wherein M is a DNA molecular weight standard, and 1-4 respectively represent pFB1-PCV3-Cap-FL positive clone, pFB1-PP6-PCV3-P6-Cap positive clone, a control taking pFastBac1 empty plasmid vector as a template and a blank control without the template.

FIG. 3 is a diagram illustrating indirect immunofluorescence detection of PCV3Cap protein expressed by recombinant viruses in a preferred embodiment of the present invention. Wherein, Mock is a healthy Sf9 cell, Bac-PCV2-Cap is a virus expressing the Cap protein of the porcine circovirus type 2.

FIG. 4 shows the expression and identification of PCV3Cap protein in a preferred embodiment of the present invention. Wherein, 1) is the SDS-PAGE detection of PCV3Cap protein, M: protein molecular weight standards; 1: a Bac-PCV3-Cap-FL virus infected whole cell sample; 2: Bac-PP6-PCV3-PP6-Cap virus infected whole cell sample. 2) For Western blot detection of PCV3Cap protein, M: protein molecular weight standards; 1: a Bac-PCV3-Cap-FL virus infected whole cell sample; 2: Bac-PP6-PCV3-PP6-Cap virus infected whole cell sample; control Bac-PCV2-Cap virus infected whole cell sample. Wherein the first antibody is a polyclonal antibody against PCV3Cap protein.

FIG. 5 shows the results of the observation of PCV3Cap forming virus-like particles expressed by recombinant baculovirus under the electron microscope 1) and the Western blot identification 2) in the preferred embodiment of the invention.

FIG. 6 shows the expression of P6-Cap protein under different conditions of inoculation in the preferred embodiment of the present invention.

FIG. 7 is a SDS-PAGE pattern of P6-Cap protein purification in accordance with a preferred embodiment of the present invention.

FIG. 8 shows the result of ELISA detection of the PCV3Cap antibody level of the immunized piglet according to the preferred embodiment of the present invention.

Detailed Description

One of the objectives of the present invention is to provide a novel nucleotide and amino acid sequence of PCV3Cap capable of self-assembling to form virus-like particles, so as to retain the characteristic of rich basic amino acids at the N-terminal of Cap protein, and reduce the difference between the formed virus-like particles and the natural virus structure, thereby reducing the difference in immunogenicity between the two.

The invention also aims to provide a construction method for improving the expression quantity of PCV3Cap protein in a baculovirus expression system so as to reduce the production cost.

The invention also provides PCV3 virus-like particles obtained based on the purpose, and provides immunogen or antigen for vaccine preparation and serological detection of PCV 3.

In order to achieve the purpose, the invention clones a coding nucleic acid sequence to the downstream of a PH promoter of a pFastBac1 vector by deleting 1 st to 22 th amino acids at the N terminal of the Cap protein of PCV3, fuses the promoter of a baculovirus P6.9 gene and a 5' part coding sequence (namely, the nucleic acid sequence coding the 1 st to 11 th amino acids at the N terminal of the baculovirus P6.9 protein) at the same time, then constructs a recombinant baculovirus to express the Cap protein, and detects the formation of virus-like particles by an electron microscope. The obtained recombinant virus antigen has high expression level, and the fused Cap protein can well form virus-like particles and has good immunogenicity. The virus-like particles prepared by the invention have high expression level, the vaccine prepared by the virus-like particles has high safety, and the body animal can generate high-level specific antibody after immunizing piglets.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

The vector used in the examples below, pFastBac1, was purchased from Invitrogen, and all endonucleases and T4 DNA ligase were purchased from Thermo Scientific.

EXAMPLE 1 construction of recombinant viruses

The specific technical route for obtaining the recombinant baculovirus is as follows:

1. obtaining Cap protein coding sequence

S1, designing forward and reverse amplification primers which are respectively 5' -TAAGGATCCAATCATGCGTCACCGTGCTATCTTCCGTCGCCGTTCCCGCCCTAGGAGACGTCGACGCCACAGAAGGCGCTACGTGCGCCGCAAGCTGTTCATCCGCCGCCCCACAGCTGGCACATACTACACCAA-3 '(underlined represents a BamHI restriction enzyme site, italicized represents a nucleotide sequence inserted between the cleavage site and the ATG initiation codon) and 5' -GACAAGCTTTTACAGCACGCTCTT GTACCTGATCCA-3' (underlined HindIII restriction sites), sequence synthesis was performed by Wuhan Strongylocentron.

S2, using pFastdeal-PCV 3 ORF2 recombinant transfer vector disclosed in CN111558037A as a template, and forward and reverse primers in step S1, Polymerase chain amplification (PCR) was performed using PrimerSTAR Max DNA Polymerase (Takara Bio Inc.) with reference to the instruction manual, to obtain a full-length nucleotide sequence encoding PCV3Cap, which is shown in SEQ ID NO:1 and encoded amino acid sequence is shown in SEQ ID NO: 2.

The PCR conditions in this step were set as follows:

pre-denaturation at 98 ℃ for 3min → (98 ℃, 15S denaturation → 55 ℃, 15S annealing → 72 ℃, 10S elongation), cycle 34 times → 72 ℃, 5 min.

S3, the amplified fragment was subjected to 1% agarose Gel electrophoresis (FIG. 1, Cap-FL), cut, and recovered by Gel Extraction Kit (Gel Extraction Kit, Omega) according to the instructions, and named Cap-FL.

S4, primers designed to amplify Cap coding sequences containing baculovirus p6.9 gene fragment fusions (Table 1):

TABLE 1 relevant primer Synthesis for amplification of Cap coding sequences fused to baculovirus p6.9 Gene fragment

S5, using P6-for-BamHI and P6-Rev as a pair of primers and DH10Bac bacterial liquid (from Bac-to-Bac system) as a template, and using PrimerSTAR Max DNA Polymerase to perform PCR amplification, thereby obtaining a DNA fragment (33bp) containing a P6.9 promoter sequence (133bp, SEQ ID NO:6) and encoding amino acids 1 to 11 at the N end of the P6.9 protein.

S6, using P6-Cap-for and Cap-Rev-XbaI as a pair of primers and pFastdeal-PCV 3 ORF2 as a template, and using PrimerSTAR Max DNA Polymerase to perform PCR amplification, a DNA fragment (579bp) containing amino acid residues 23 to 214 of the encoded Cap protein was obtained.

S7, performing overlapped PCR amplification by using P6-for-BamHI and Cap-Rev-XbaI as a pair of primers and using the DNA fragments obtained by amplification in the steps S2 and S3 as a template (equal amount) to obtain a fused DNA fragment (shown in figure 1, P6-Cap), running agarose gel, and recovering to obtain a Cap coding sequence containing baculovirus P6.9 gene fragment fusion, wherein the sequence is shown as SEQ ID NO:3, and the sequence of the coded fusion protein is shown as SEQ ID NO: 4.

2. Plasmid construction

S1, carrying out double digestion on a fragment coding P6-Cap and pFastBacI by using BamHI and XbaI, then carrying out ligation by using T4 DNA ligase, and standing overnight at 4 ℃;

s2, transforming the ligation product in the step S1 into Top10 chemical competent cells (Invitrogen), shaking the cells in a shaker at 37 ℃ for 1h, then coating all the cells on a solid plate containing gentamicin, and culturing at 37 ℃ overnight (16 h);

s3, picking single colony, and detecting positive clone by PCR.

In this step, primers P6-Cap-for and Cap-Rev-HindIII were used for PCR identification using rTaq DNA polymerase under the following conditions:

95 ℃, 3min pre-denaturation → (95 ℃, 20S denaturation → 55 ℃, 30S annealing → 72 ℃, 30S elongation), cycling 19 times → 72 ℃, 5 min.

S4, taking a clone identified as positive (figure 2, lane 2), shaking in a shaker at 37 ℃ for 16h, extracting plasmids by using a plasmid extraction kit, sequencing, and storing the bacteria, wherein the sequencing result is correct. The plasmid was named pFB1-PP6-PCV 3-P6-Cap.

Thereafter, pFB1-PCV3-Cap-FL was constructed in the same manner as the construction of pFB1-PP6-PCV3-P6-Cap plasmid except that the fragment encoding Cap-FL was double-digested with BamHI and HIndIII in the S1 step, unlike pFastBacI.

3. Construction and acquisition of recombinant viruses

S1, adopting a Bac-to-Bac system (Invitrogen) and according to the instructions, transforming the donor plasmids pFB1-PP6-PCV3-P6-Cap and pFB1-PCV3-Cap-FL obtained in the step 2 into DH10Bac, then carrying out two rounds of blue-white spot screening and PCR detection, extracting and identifying correct recombinant baculovirus genomes which are named as Bac-PP6-PCV3-P6-Cap and Bac-PCV3-Cap-FL respectively.

S2, transfecting Sf9 cells with Bac-PP6-PCV3-P6-Cap and Bac-PCV3-Cap-FL respectively by using Cellfectin transfection reagents, observing the transfection condition, collecting a culture medium on the 6 th day after transfection, storing at 4 ℃, and marking as P0 virus.

S3, according to 2.0X 10⁶Inoculating P0 seed viruses at the density of cell/mL for blind transmission, and sequentially marking the obtained seed viruses as P1 and P2 seed viruses; among them, P2 virus was used as the subsequently expressed virus.

Example 2 identification of recombinant viruses and expression of Cap

To identify recombinant viruses, indirect immunization and western immunoblotting were used:

indirect connectionThe main process of immunization is as follows: in 24-well plates, 1X 10 inoculate per well⁵cell, then Bac-PCV3-Cap-FL, Bac-PP6-PCV3-P6-Cap or Bac-PCV2-Cap (baculovirus expressing PCV2 Cap protein was constructed and stored by Probiotics of Wuhan family, Ltd.), while healthy Sf9 cells (Mock) without virus infection were blanked. At 72h of virus infection, cells were fixed with 4% paraformaldehyde solution for 10min, permeabilized with 0.2% Triton X-100 solution for 10min, blocked with 5% BSA at room temperature for 3h, blocked with polyclonal antibody against PCV3Cap protein with cells at room temperature for 3h, gently washed with PBS for 3 times, further blocked with fluorescently labeled goat anti-mouse IgG secondary antibody (Invitrogen) at room temperature for 30min, gently washed with PBS for 3 times, and then observed with a fluorescence microscope. The fluorescence results of the indirect immunization are shown in FIG. 3.

Wherein, the preparation process of the polyclonal antibody against PCV3Cap protein mainly comprises the following steps:

s1: design of Forward primer Cap-NcoI-For (5' -ATA)CCATGGATACCTACCAGAACAACTGGCGTTCTGGCGGCTCTCCCACAGCTGGCACATACTACACC-3 '), followed by the reverse primer (5' -GAC) of example 1AAGCTTTTACAGCACGCTCTTGTACCTGATCCA-3') as a pair of primers, and using the Cap-FL nucleic acid fragment as a template to carry out PCR amplification to obtain a nucleic acid sequence (SEQ ID NO:8) which codes the amino acids from the 32 nd to the 214 th positions of the Cap protein, wherein the amino acid sequence is shown in SEQ ID NO: 9.

S2: the nucleic acid fragment obtained in S1 is subjected to double enzyme digestion of NcoI and HindIII with pET-28a (Novagen) vector, recovered after running 1% agarose gel, then subjected to enzyme ligation by using T4 DNA ligase, transformed, plated and identified by PCR to obtain a positive clone, and then extracted for plasmid sequencing.

S3: the plasmid with correct sequencing is taken and transformed into BL21(DE3) competent cells (Beijing holotype gold) to express protein under the induction condition of 37 ℃, the final concentration of IPTG is 1mM/L, and the induction time is 6h, and then the cells are collected for protein purification.

S4: the protein is expressed by inclusion body, the purification is carried out by the inclusion body purification method, the main process is to collect the sediment after cracking the bacteria, resuspend the sediment by PBS buffer solution containing 1 percent Triton X-100, carry out ultrasonic treatment, then centrifuge and take the sediment, and repeat for 3 times.

S5: resuspending the precipitate with 1M urea-containing PBS buffer, standing at room temperature for 10min, centrifuging again to obtain precipitate, and precipitating with 8M urea-containing phosphate buffer (0.1M NaH)₂P0₄(ii) a 0.01M Tris-Cl, pH8.0), at 4 ℃ overnight, centrifuged and the supernatant taken for full gradient dialysis.

S6: balb/c mice (purchased from Wuhan's Biochemical research institute) were immunized with 100. mu.g/mouse of total amount plus Freund's complete adjuvant (Sigma) using the dialyzed protein solution as antigen, and a second immunization was performed 14 days later with the same dose but with Freund's incomplete adjuvant (Sigma), and a third immunization with the same vaccine and dose as the second immunization was performed 14 days later.

S7: and (3) collecting blood from an eyeball 7 days after the third immunization, standing at normal temperature, and centrifuging after blood coagulation to obtain serum, namely the polyclonal antibody against PCV3Cap protein.

The main process of western blotting is as follows: Bac-PCV3-Cap-FL, Bac-PP6-PCV3-PP6-Cap or Bac-PCV2-Cap recombinant virus is used for infecting cells with the density of 2.0 multiplied by 10⁶cell/mL Sf9 suspension cells (0.5moi), 5 days after infection, taking 40 μ L of whole cell culture solution, adding SDS-PAGE Loading Buffer, boiling for 10min, then carrying out electrophoresis by SDS-PAGE, transferring protein to PVDF membrane by wet transfer, sealing by skimmed milk, incubating by anti-PCV 3Cap antibody, washing membrane, incubating by horseradish peroxidase-coupled goat anti-mouse IgG (Proteintetech), washing membrane, and developing color by chemiluminescence color developing instrument. The results of protein immunization are shown in fig. 4, 2).

EXAMPLE 3 Electron microscopy

200mL of a solution having a density of 2X 10⁶cell/mL Sf9 cells were infected with Bac-PCV3-Cap-FL and Bac-PP6-PCV3-P6-Cap, respectively, wherein the inoculation dose was 0.5 moi. On day 4 of infection, expression of Cap was detected by SDS-PAGE, see 1 in FIG. 4). After infected cells are broken (8 d after infection), collecting cell culture solution, centrifuging for 0.5h at 10000g to remove cell debris, taking supernatant, centrifuging for 2h at 200,000 g, and adding double distilled water (ddH)₂O) resuspending the pellet, followed by a 25% -45% sucrose density gradientCentrifuging at 180,000g for 2h, taking sediment at the bottom of a centrifuge tube, and using ddH₂Resuspend O, then burden stain with phosphotungstate, and observe with electron microscope, see 1) in fig. 5.

The electron microscopy results show that about 20-25nm structures are formed in both expression strategies, and in order to determine that the obtained structures are VLPs assembled from Cap proteins, samples for preparing electron microscopy are subjected to SDS-PAGE separation and then subjected to Western blot detection, as shown in 2 in FIG. 5). Western blot results show that the sample prepared by the electron microscope is indeed a suspension containing Cap protein. Electron microscopy results show that both Cap proteins expressed by the two expression strategies can well form VLPs. This indicates that deletion of the N-terminal portion of the Cap protein and fusion of the N-terminal amino acids 1-11 of P6.9 did not affect the formation of virus-like particles.

Example 4 protein expression conditions

To further investigate suitable expression conditions for proteins to increase the expression amount of proteins, Bac-PP6-PCV3-P6-Cap of example 1 was used to infect proteins with a density of 2X 10 at different moi⁶cell/mL High Five cells, at 6 days post infection, protein expression was detected by SDS-PAGE and Western blot, and the results are shown in FIG. 6. The results showed that 2X 10 were infected with virus amounts of 0.5moi and 1moi⁶cell/mL High Five cell, P6-Cap protein can get higher level expression.

Example 5 vaccine preparation and animal experiments

1. Vaccine preparation

Infected Sf9 cells are crushed under high pressure, after cell fragments are removed by centrifugation, the cells are centrifuged by 25% -45% sucrose density gradient twice, sediment at the bottom of a centrifuge tube is taken and resuspended by 0.02M PBS, and the sediment is taken as an antigen after aseptic filtration. The content of P6-Cap protein was then assessed by running SDS-PAGE protein gel and using 50. mu.g/mL BSA as reference (FIG. 7) and diluted to 50. mu.g/mL for formulation of vaccines:

1) 12mL of P6-Cap antigen solution (50. mu.g/mL);

2) aluminum hydroxide adjuvant (2mg/mL)3mL was purchased from CHEMTRADE, USA.

Fully mixing the antigen liquid of 1) and the adjuvant of 2) to obtain the porcine circovirus type 3 virus-like particle vaccine with the antigen content of 40 mu g/mL.

2. Animal immunization and antibody detection

(1) Porcine circovirus type 3 virus-like particle vaccine safety evaluation

The experimental piglets (the long and white pig species with the average weight of 5.8 +/-0.6 kg) of 21-27 days old are selected from PCV2, PCV3 antigen and antibody negative and PRRSV antigen negative pigs. 10 pigs were randomly divided into 2 groups, a blank control group and a vaccine group. Wherein the blank control group has 5 heads, and sterile normal saline is injected into neck muscles at multiple points by 1 mL; vaccine group 5 head, neck muscle multiple injection vaccine 1 mL. And (3) observing the mental state, food intake, activity, injection part lesion and other conditions of the immune pig, continuously observing for 14 days, and simultaneously measuring the rectal temperature of the pig every 3 days. The results show that the vaccine group has normal mental state, food intake and activity of the immunized piglets, no red swelling, ulceration and the like at the injection part, and the body temperature of the two groups of piglets is within the normal range (38-39.5 ℃) like the blank control group, which indicates that the circovirus type 3 virus-like particle vaccine prepared by using the baculovirus expression system is safe for the body animals.

(2) Immunogenicity evaluation of porcine circovirus type 3 virus-like particle vaccine

The experimental piglets (the long and white pig species with the average weight of 6.1 +/-0.5 kg) of 21-27 days old are selected from PCV2, PCV3 antigen and antibody negative and PRRSV antigen negative pigs. 10 pigs were randomly divided into 2 groups, a blank control group and a vaccine group. Wherein the blank control group has 5 heads, and the neck muscle is injected with 1mL of sterilized normal saline at multiple points; vaccine group 5 head, neck muscle multiple injection vaccine 1 mL. After 14 days of injection immunization, a second immunization is carried out with the same vaccine and dosage, and 14 days of the second immunization, a pig serum sample is taken, the vaccine group serum is diluted by 1:1000, and then the antibodies of two groups of experimental piglets to the Cap protein are detected by an ELISA method (an antigen coated plate is prepared by coating PCV3Cap protein (SEQ ID NO:9) which is expressed by pronucleus onto a 96-well enzyme-labeled plate according to the concentration of 100 mu g/well by using carbonate coated buffer), and the result is shown in figure 8. The result shows that the vaccine prepared by the insect-baculovirus expression system can stimulate the immune piglet to generate the anti-PCV 3Cap protein antibody with higher titer.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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P,Shang P，et al.A Novel Porcine Circovirus Distantly Related to Known Circoviruses Is Associated with Porcine Dermatitis and Nephropathy Syndrome and Reproductive Failure.J Virol.2016；91(1):e01879-16.doi:10.1128/JVI.01879-16.

(4)Jiang H，Wang D，Wang J，et al.Induction of Porcine Dermatitis and Nephropathy Syndrome in Piglets by Infection with Porcine Circovirus Type 3.J Virol.2019；93(4):e02045-18.doi:10.1128/JVI.02045-18.

(5) detection of porcine circovirus type 3 and prediction analysis of its Cap structural sequence and antigenicity [ J ]. Proc. veterinary Commission on livestock, 2017, 48(6):1076-1084.

(6) Liu Ying 26163, Zhou Zhi, Jie, etc. the epidemic and detection method of porcine circovirus type 3 infection has been studied and developed [ J ]. zootechnics and veterinarians, 2019, 51(01): 114-.

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(8)Zhang S，Wang D，Jiang Y,et al.Development and application of a baculovirus-expressed capsid protein-based indirect ELISA for detection of porcine circovirus 3IgG antibodies.BMC Vet Res.2019；15(1):79.doi:10.1186/s12917-019-1810-3.

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Sequence listing

<110> Wuhan Keshi Probiotics GmbH

<120> porcine circovirus type 3 virus-like particle, and preparation method and application thereof

<130> KHP211111116.5

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 645

<212> DNA

<213> Porcine circovirus (Portone circovirus)

<400> 1

atgcgtcacc gtgctatctt ccgtcgccgt tcccgcccta ggagacgtcg acgccacaga 60

aggcgctacg tgcgccgcaa gctgttcatc cgccgcccca cagctggcac atactacacc 120

aagaagtact ccaccatgaa cgtgatcagc gtgggcaccc ctcagaacaa caagccttgg 180

cacgctaacc acttcatcac ccgcctgaac gagtgggaga ccgccatcag cttcgagtac 240

tacaagatcc tgaagatgaa ggtgaccctg agccctgtga tcagccctgc tcagcagacc 300

aagaccatgt tcggtcacac cgccatcgac ctggacggtg cctggaccac caacacctgg 360

ctgcaggacg acccttacgc tgaaagcagc acccgtaagg ttatgacctc caagaagaag 420

cactcccgct acttcacccc taagcctatc ctggctggta ctaccagcgc tcaccctggc 480

cagtccctgt tcttcttctc ccgtcctacc ccttggctga acacctacga ccctaccgtg 540

cagtggggcg ctctgctgtg gagcatctac gtgcctgaaa agaccggtat gaccgacttc 600

tacggtacta aggaagtgtg gatcaggtac aagagcgtgc tgtaa 645

<210> 2

<211> 214

<212> PRT

<213> Porcine circovirus (Portone circovirus)

<400> 2

Met Arg His Arg Ala Ile Phe Arg Arg Arg Ser Arg Pro Arg Arg Arg

1 5 10 15

Arg Arg His Arg Arg Arg Tyr Val Arg Arg Lys Leu Phe Ile Arg Arg

20 25 30

Pro Thr Ala Gly Thr Tyr Tyr Thr Lys Lys Tyr Ser Thr Met Asn Val

35 40 45

Ile Ser Val Gly Thr Pro Gln Asn Asn Lys Pro Trp His Ala Asn His

50 55 60

Phe Ile Thr Arg Leu Asn Glu Trp Glu Thr Ala Ile Ser Phe Glu Tyr

65 70 75 80

Tyr Lys Ile Leu Lys Met Lys Val Thr Leu Ser Pro Val Ile Ser Pro

85 90 95

Ala Gln Gln Thr Lys Thr Met Phe Gly His Thr Ala Ile Asp Leu Asp

100 105 110

Gly Ala Trp Thr Thr Asn Thr Trp Leu Gln Asp Asp Pro Tyr Ala Glu

115 120 125

Ser Ser Thr Arg Lys Val Met Thr Ser Lys Lys Lys His Ser Arg Tyr

130 135 140

Phe Thr Pro Lys Pro Ile Leu Ala Gly Thr Thr Ser Ala His Pro Gly

145 150 155 160

Gln Ser Leu Phe Phe Phe Ser Arg Pro Thr Pro Trp Leu Asn Thr Tyr

165 170 175

Asp Pro Thr Val Gln Trp Gly Ala Leu Leu Trp Ser Ile Tyr Val Pro

180 185 190

Glu Lys Thr Gly Met Thr Asp Phe Tyr Gly Thr Lys Glu Val Trp Ile

195 200 205

Arg Tyr Lys Ser Val Leu

210

<210> 3

<211> 745

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ggaaacacag caacaaaaat tcaaacgacc aagacgagtt aaacatatat ttgggagttc 60

agtcgtcgaa tgcaaagcgt aaaaaatatt aataaggtaa aaattacagc tacataaatt 120

acacaattta aacatggttt atcgtcgccg tcgccgttct tcaacctacg tgcgccgcaa 180

gctgttcatc cgccgcccca cagctggcac atactacacc aagaagtact ccaccatgaa 240

cgtgatcagc gtgggcaccc ctcagaacaa caagccttgg cacgctaacc acttcatcac 300

ccgcctgaac gagtgggaga ccgccatcag cttcgagtac tacaagatcc tgaagatgaa 360

ggtgaccctg agccctgtga tcagccctgc tcagcagacc aagaccatgt tcggtcacac 420

cgccatcgac ctggacggtg cctggaccac caacacctgg ctgcaggacg acccttacgc 480

tgaaagcagc acccgtaagg ttatgacctc caagaagaag cactcccgct acttcacccc 540

taagcctatc ctggctggta ctaccagcgc tcaccctggc cagtccctgt tcttcttctc 600

ccgtcctacc ccttggctga acacctacga ccctaccgtg cagtggggcg ctctgctgtg 660

gagcatctac gtgcctgaaa agaccggtat gaccgacttc tacggtacta aggaagtgtg 720

gatcaggtac aagagcgtgc tgtaa 745

<210> 4

<211> 203

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Val Tyr Arg Arg Arg Arg Arg Ser Ser Thr Tyr Val Arg Arg Lys

1 5 10 15

Leu Phe Ile Arg Arg Pro Thr Ala Gly Thr Tyr Tyr Thr Lys Lys Tyr

20 25 30

Ser Thr Met Asn Val Ile Ser Val Gly Thr Pro Gln Asn Asn Lys Pro

35 40 45

Trp His Ala Asn His Phe Ile Thr Arg Leu Asn Glu Trp Glu Thr Ala

50 55 60

Ile Ser Phe Glu Tyr Tyr Lys Ile Leu Lys Met Lys Val Thr Leu Ser

65 70 75 80

Pro Val Ile Ser Pro Ala Gln Gln Thr Lys Thr Met Phe Gly His Thr

85 90 95

Ala Ile Asp Leu Asp Gly Ala Trp Thr Thr Asn Thr Trp Leu Gln Asp

100 105 110

Asp Pro Tyr Ala Glu Ser Ser Thr Arg Lys Val Met Thr Ser Lys Lys

115 120 125

Lys His Ser Arg Tyr Phe Thr Pro Lys Pro Ile Leu Ala Gly Thr Thr

130 135 140

Ser Ala His Pro Gly Gln Ser Leu Phe Phe Phe Ser Arg Pro Thr Pro

145 150 155 160

Trp Leu Asn Thr Tyr Asp Pro Thr Val Gln Trp Gly Ala Leu Leu Trp

165 170 175

Ser Ile Tyr Val Pro Glu Lys Thr Gly Met Thr Asp Phe Tyr Gly Thr

180 185 190

Lys Glu Val Trp Ile Arg Tyr Lys Ser Val Leu

195 200

<210> 5

<211> 192

<212> PRT

<213> Porcine circovirus (Portone circovirus)

<400> 5

Tyr Val Arg Arg Lys Leu Phe Ile Arg Arg Pro Thr Ala Gly Thr Tyr

1 5 10 15

Tyr Thr Lys Lys Tyr Ser Thr Met Asn Val Ile Ser Val Gly Thr Pro

20 25 30

Gln Asn Asn Lys Pro Trp His Ala Asn His Phe Ile Thr Arg Leu Asn

35 40 45

Glu Trp Glu Thr Ala Ile Ser Phe Glu Tyr Tyr Lys Ile Leu Lys Met

50 55 60

Lys Val Thr Leu Ser Pro Val Ile Ser Pro Ala Gln Gln Thr Lys Thr

65 70 75 80

Met Phe Gly His Thr Ala Ile Asp Leu Asp Gly Ala Trp Thr Thr Asn

85 90 95

Thr Trp Leu Gln Asp Asp Pro Tyr Ala Glu Ser Ser Thr Arg Lys Val

100 105 110

Met Thr Ser Lys Lys Lys His Ser Arg Tyr Phe Thr Pro Lys Pro Ile

115 120 125

Leu Ala Gly Thr Thr Ser Ala His Pro Gly Gln Ser Leu Phe Phe Phe

130 135 140

Ser Arg Pro Thr Pro Trp Leu Asn Thr Tyr Asp Pro Thr Val Gln Trp

145 150 155 160

Gly Ala Leu Leu Trp Ser Ile Tyr Val Pro Glu Lys Thr Gly Met Thr

165 170 175

Asp Phe Tyr Gly Thr Lys Glu Val Trp Ile Arg Tyr Lys Ser Val Leu

180 185 190

<210> 6

<211> 133

<212> DNA

<213> Autographa californica nuclear polyhedrosis virus strain E2

<400> 6

ggaaacacag caacaaaaat tcaaacgacc aagacgagtt aaacatatat ttgggagttc 60

agtcgtcgaa tgcaaagcgt aaaaaatatt aataaggtaa aaattacagc tacataaatt 120

acacaattta aac 133

<210> 7

<211> 267

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atcatggaga taattaaaat gataaccatc tcgcaaataa ataagtattt tactgttttc 60

gtaacagttt tgtaataaaa aaacctataa atattccgga ttattcatac cgtcccacca 120

tcgggcgcgg atccggaaac acagcaacaa aaattcaaac gaccaagacg agttaaacat 180

atatttggga gttcagtcgt cgaatgcaaa gcgtaaaaaa tattaataag gtaaaaatta 240

cagctacata aattacacaa tttaaac 267

<210> 8

<211> 588

<212> DNA

<213> Porcine circovirus (Portone circovirus)

<400> 8

atggatacct accagaacaa ctggcgttct ggcggctctc ccacagctgg cacatactac 60

accaagaagt actccaccat gaacgtgatc agcgtgggca cccctcagaa caacaagcct 120

tggcacgcta accacttcat cacccgcctg aacgagtggg agaccgccat cagcttcgag 180

tactacaaga tcctgaagat gaaggtgacc ctgagccctg tgatcagccc tgctcagcag 240

accaagacca tgttcggtca caccgccatc gacctggacg gtgcctggac caccaacacc 300

tggctgcagg acgaccctta cgctgaaagc agcacccgta aggttatgac ctccaagaag 360

aagcactccc gctacttcac ccctaagcct atcctggctg gtactaccag cgctcaccct 420

ggccagtccc tgttcttctt ctcccgtcct accccttggc tgaacaccta cgaccctacc 480

gtgcagtggg gcgctctgct gtggagcatc tacgtgcctg aaaagaccgg tatgaccgac 540

ttctacggta ctaaggaagt gtggatcagg tacaagagcg tgctgtaa 588

<210> 9

<211> 195

<212> PRT

<213> Porcine circovirus (Portone circovirus)

<400> 9

Met Asp Thr Tyr Gln Asn Asn Trp Arg Ser Gly Gly Ser Pro Thr Ala

1 5 10 15

Gly Thr Tyr Tyr Thr Lys Lys Tyr Ser Thr Met Asn Val Ile Ser Val

20 25 30

Gly Thr Pro Gln Asn Asn Lys Pro Trp His Ala Asn His Phe Ile Thr

35 40 45

Arg Leu Asn Glu Trp Glu Thr Ala Ile Ser Phe Glu Tyr Tyr Lys Ile

50 55 60

Leu Lys Met Lys Val Thr Leu Ser Pro Val Ile Ser Pro Ala Gln Gln

65 70 75 80

Thr Lys Thr Met Phe Gly His Thr Ala Ile Asp Leu Asp Gly Ala Trp

85 90 95

Thr Thr Asn Thr Trp Leu Gln Asp Asp Pro Tyr Ala Glu Ser Ser Thr

100 105 110

Arg Lys Val Met Thr Ser Lys Lys Lys His Ser Arg Tyr Phe Thr Pro

115 120 125

Lys Pro Ile Leu Ala Gly Thr Thr Ser Ala His Pro Gly Gln Ser Leu

130 135 140

Phe Phe Phe Ser Arg Pro Thr Pro Trp Leu Asn Thr Tyr Asp Pro Thr

145 150 155 160

Val Gln Trp Gly Ala Leu Leu Trp Ser Ile Tyr Val Pro Glu Lys Thr

165 170 175

Gly Met Thr Asp Phe Tyr Gly Thr Lys Glu Val Trp Ile Arg Tyr Lys

180 185 190

Ser Val Leu

195

Claims (4)

1. A method for preparing porcine circovirus type 3 virus-like particles, the method comprising: firstly, constructing recombinant baculovirus for expressing porcine circovirus type 3Cap protein variants, then infecting insect cells with the recombinant baculovirus, and separating virus-like particles from cell culture solution after cytopathic effect; wherein the amino acid sequence of the porcine circovirus type 3Cap protein variant is shown as SEQ ID NO. 5;

in the method, a nucleic acid sequence encoding the variant Cap protein is operably linked to a PH-P6.9 dual promoter, and a nucleic acid sequence encoding the N-terminal amino acid of the baculovirus P6.9 protein is inserted before the initiation codon of the nucleic acid sequence encoding the variant Cap protein; the amino acid at the N-terminal part of the baculovirus P6.9 protein at least comprises amino acids MVYRRRRRSST at the 1 st to 11 th sites of the N-terminal of the baculovirus P6.9 protein, and the PH-P6.9 double promoter is formed by connecting a baculovirus PH promoter and a P6.9 promoter in series.

2. The method according to claim 1, wherein the vector for constructing the recombinant baculovirus is selected from the group consisting of pFastBac1, pFastBac HTA, pFastBac HTB, pFastBac HTC, pFastBac dual, pIEx-Bac, pOET and pQB, and vectors modified based on these vectors;

the insect cell is selected from Sf9, Sf21 or High Five, and the insect cell is modified by gene knockout, insertion or modification on the basis of the Sf9, Sf21 or High Five.

3. A porcine circovirus type 3 genetically engineered subunit vaccine, characterized in that the vaccine comprises virus-like particles prepared according to the method of claim 1 or 2.

4. Use of a virus-like particle prepared according to the method of claim 1 or 2, in any one of the following applications:

1) is used for preparing porcine circovirus type 3 polyclonal antibody or monoclonal antibody;

2) is used for preparing a porcine circovirus type 3 antibody detection reagent;

3) is used for preparing porcine circovirus type 3 genetic engineering subunit vaccine.

Images