CN114736310B

CN114736310B - Protein and biological material for producing circovirus type 2 virus-like particles and application

Info

Publication number: CN114736310B
Application number: CN202210649246.6A
Authority: CN
Inventors: 伏显华
Original assignee: Beijing Centre Biology Co ltd; Beijing Huaxia Xingyang Biological Science & Technology Co ltd
Current assignee: Beijing Centre Biology Co ltd; Beijing Huaxia Xingyang Biological Science & Technology Co ltd
Priority date: 2022-06-10
Filing date: 2022-06-10
Publication date: 2022-10-28
Anticipated expiration: 2042-06-10
Also published as: CN114736310A

Abstract

The invention discloses a protein and a biological material for generating a circovirus type 2 virus-like particle and application thereof, belonging to the field of peptides. The invention aims to solve the technical problem of how to improve the immune effect of the porcine circovirus type 2 Cap virus-like particles and/or how to solve the problem that the porcine circovirus type 2 Cap virus-like particles are easy to degrade in normal temperature storage. The name of the protein provided by the invention is recombinant protein Cap286W6, which is the protein with an amino acid sequence of SEQ ID No. 2. The invention also provides a nucleic acid molecule for coding the recombinant protein Cap286W6, an expression cassette, a recombinant vector, a recombinant microorganism and other biological materials. The invention also mineralizes the obtained Cap286W6 VLPs by using a phosphate buffer solution to obtain the virus-like particles mineralized by the circovirus Cap, thereby improving the immune effect on the circovirus.

Description

Protein and biological material for producing circovirus type 2 virus-like particles and application

Technical Field

The invention relates to a protein and a biological material for producing circovirus type 2 virus-like particles in the field of peptides and application thereof.

Background

Porcine Circovirus (PCV) belongs to the genus Cyclovirous of the family Cycloviroviridae, has a virion size of 14-25 nm, an average diameter of 17 nm, a symmetrical icosahedron shape, no cyst membrane, a single-stranded circular DNA genome, and a DNA suspension density of 1.37 cm in tissue ³ The sedimentation coefficient was 52S, which is the smallest animal virus found. The replication of PCV is a rolling circle replication mode, which firstly passes through a double-stranded Replicative Form (RF), and then transcribes and encodes protein by the double-stranded replicative form. PCV is classified into PCV1, PCV2, PCV3 and PCV4 according to differences in pathogenicity, antigenicity and nucleic acid sequence. PCV1 genome total length 1759bp, PCV2 total length 1768 bp or 1767 bp, and the sequence homology of the two is less than 80%, but among PCV-1 strainsThe nucleotide sequence homology is more than 99 percent, and the nucleotide sequence homology among PCV2 strains is more than 96 percent. PCV1 is nonpathogenic, was first discovered in 1974 from a plurality of serially passaged PK-15 cells, and was confirmed in 1982 to be derived from porcine kidney tissue from which PK-15 cells were originally prepared. Later, the virus was confirmed to be a conventional virus capable of infecting pigs, and no harm was caused to the infected pigs. PCV2 is pathogenic and causes pigs to exhibit a variety of clinical symptoms. PCV2 is the main cause of Postweaning Multisystemic Wasting Syndrome (PMWS), dermatitis and nephrotic Syndrome (PNDS).

PCV has strong resistance to the outside, and the virus is insensitive to chloroform and does not agglutinate red blood cells of various animals such as cattle, sheep, pigs, chickens and the like and human beings. PCV is capable of growing on PK-15 cells but is incapable of causing cytopathic effects, but contains many intracytoplasmic inclusions in PK-15 cells infected with PCV. A few infected cells contain nuclear inclusion bodies. PCV is unable to grow on primary fetal pig kidney cells, rhesus monkey kidney cells, BHK-21 cells.

PCV1 and PCV2 both have an open reading frame ORF1 encoding a viral replication protein. The interrelatedness of the ORF1 encoding protein sequences among strains is more than 86 percent, and the interrelatedness of the proteins encoded by the PCV reading frames is highest. It has been demonstrated that PCV has two major reading frames, ORF1 and ORF2, both of which are greater than 600 bp in length. ORF1 is the largest reading frame, encodes the viral replication protein (Rep), has a molecular weight of 37kDa, and is involved in the replication and transcription of the virus. ORF2 encodes a structural protein of the virus, which is reactive with antisera to PCV 2.

Porcine circovirus type 2 (PCV 2) ORF2 gene encodes the nucleocapsid protein (Cap) of the virus, which belongs to the virus protective antigen and has important immune function. The preparation of subunit or virus-like particle (VLPS) vaccines by using a genetic engineering method is mainly focused on the Cap antigen region, and the Cap antigen vaccine is also one of the important ways for controlling the disease at present. Although the supply of subunit vaccines of gene engineering for expressing porcine circular Cap such as Escherichia coli and baculovirus is available in the market at present, people still seek a new preparation method of long-acting antigen vaccines with low cost.

To dateHeretofore, there have been various ways and materials for preparing biodegradable nanoparticles, such as polyesters, polyanhydrides, polysaccharides, and the like. Nowadays, biomineralization peptides offer a new possibility for the preparation of nanoparticles, which can be prepared by chelating calcium phosphate (CaP), silicon dioxide (SiO) ₂ ) Or inorganic substances such as gold (Au), and further induces the formation of protein nanoparticles. Several studies have shown that CaP is a safe and effective protein adjuvant.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the immune effect of the porcine circovirus type 2 Cap virus-like particles and/or how to solve the problem that the porcine circovirus type 2 Cap virus-like particles are easy to degrade in normal temperature storage.

In order to solve the technical problems, the invention provides a protein, wherein the protein is formed by connecting a porcine circovirus type 2 nucleocapsid protein Cap and a recombinant mineralization peptide W6 and is named as a recombinant protein Cap286W6, and the recombinant protein Cap286W6 can be any one of the following A1) -A3):

a1 Protein with the amino acid sequence of 1-220 th site of SEQ ID No. 2;

a2 A protein having 80% or more identity to the protein represented by A1) obtained by substituting and/or deleting and/or adding at least one amino acid residue in the amino acid sequence represented by A1);

a3 A) and a tag is attached to the N-terminus and/or C-terminus of A1) or A2).

Wherein the mineralization peptide in the recombinant protein Cap286W6 can be combined with calcium and phosphorus and spontaneously form mineralization, thereby mineralizing the recombinant protein Cap286W6.

SEQ ID No.2 consists of amino acid residue 228.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then performing biological expression.

The protein, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is the substitution and/or deletion and/or addition of no more than 10 amino acid residues; or substitution and/or deletion and/or addition of not more than 9 amino acid residues; or a substitution and/or deletion and/or addition of not more than 8 amino acid residues; or a substitution and/or deletion and/or addition of not more than 7 amino acid residues; or substitution and/or deletion and/or addition of not more than 6 amino acid residues; or a substitution and/or deletion and/or addition of not more than 5 amino acid residues; or a substitution and/or deletion and/or addition of not more than 4 amino acid residues; or substitution and/or deletion and/or addition of not more than 3 amino acid residues; or a substitution and/or deletion and/or addition of not more than 2 amino acid residues; or substitution and/or deletion and/or addition of not more than 1 amino acid residue.

The protein-tag refers to a polypeptide or protein which is expressed by fusion with a target protein by using a DNA in vitro recombination technology so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag, and/or a SUMO protein tag, etc.

In the invention, the tag of the recombinant protein Cap286W6 can be His tag, and the amino acid sequence of the recombinant protein Cap286W6 with the His tag is shown as SEQ ID No. 2.

In order to solve the above technical problems, in a second aspect, the present invention provides a biomaterial related to the above protein, wherein the biomaterial may be any one of the following B1) to B4):

b1 Nucleic acid molecule which is used for coding the recombinant protein Cap286W6, wherein the nucleic acid molecule can be any one of g 1) to g 3),

g1 A DNA molecule shown as 5076-5735 th site of SEQ ID No.1 in the nucleotide sequence of the coding strand;

g2 The coding sequence of the coding chain is a DNA molecule shown in 5076-5762 of SEQ ID No. 1;

g3 A DNA molecule which has 80% or more than 80% of identity with the nucleotide sequence limited by g 1) or g 2) and codes the recombinant protein Cap286W 6;

b2 A nucleic acid molecule according to B1);

b3 A recombinant vector containing the nucleic acid molecule according to B1) or containing the expression cassette according to B2);

b4 B1) or a recombinant microorganism which comprises the nucleic acid molecule according to B1) or comprises the expression cassette according to B2) or comprises the recombinant vector according to B3).

The coding sequence of the Cap286W6 gene is shown in 5076-5762 of SEQ ID No.1, wherein, the 5076 th to 5204 th sites of the SEQ ID No.1 are N-terminal protein coding genes of the Cap, the 5205 th to 5216 th sites of the SEQ ID No.1 are GS connecting peptides, and the 5217 th to 5282 th sites of the SEQ ID No.1 are coding genes of mineralized peptide W6. The sites 5283-5735 of SEQ ID No.1 are coding genes of Cap C-terminal protein, the sites 5736-5762 of SEQ ID No.1 are XhoI enzyme cutting sites, his tag protein genes and stop codon genes, and the protein coded by the Cap286W6 gene is the recombinant protein Cap286W6 with His tag, and the amino acid sequence of the recombinant protein Cap286W6 is shown in SEQ ID No. 2.

Wherein the genes coding the porcine circovirus type 2 nucleocapsid protein Cap and the mineralization peptide W6 are required to be contained.

Further, the recombinant microorganism may be escherichia coli.

In order to solve the above technical problems, in a third aspect, the present invention provides a virus-like particle comprising the above protein.

In order to solve the above technical problems, in a fourth aspect, the present invention provides a vaccine for porcine circovirus type 2, comprising the above protein or the above virus-like particle.

In order to solve the above technical problems, according to a fifth aspect, the present invention provides a method for producing the above protein, comprising: expressing the coding gene of the recombinant protein Cap286W6 in a biological cell to obtain the recombinant protein Cap286W6.

Further, in the above preparation method, the organism may be a microbial cell, a plant or a non-human animal cell.

Further, in the above-mentioned production method, the organism may be a microorganism.

Further, in the above production method, the microorganism may be a prokaryotic microorganism.

Further, in the above preparation method, the prokaryotic microorganism may be escherichia coli.

In order to solve the above technical problems, according to a sixth aspect, the present invention provides a method for preparing the above virus-like particles, the method comprising the step of mineralizing the above proteins to obtain virus-like particles.

In the present invention, the virus-like particle may be a mineralized virus-like particle. The virus-like particles can be used for preventing porcine circovirus type 2 infection.

In the present invention, the step of mineralizing the protein to obtain the virus-like particle comprises:

adding the purified recombinant protein Cap286W6 into 10 XVLPs buffer solution, standing at 4 ℃ for 24 hours, assembling into recombinant protein Cap286W6 VLPs, and then supplementing calcium phosphate buffer solution for calcification for 48 hours to obtain mineralized Cap286W6 VLPs;

wherein, the 10 XVLPs buffer solution comprises the following components: 0.5% arginine, 0.5M NaCl,10% glycerol, 6% PEG3350, the remainder water;

the calcium phosphate buffer was a 10mM calcium phosphate solution pH 7.5: 3.1 g of Ca are taken ₃ (PO ₄ ) ₂ Dissolving 800mL of water, stirring, adding hydrochloric acid to promote dissolution, and adjusting the pH value to 7.5 by using NaOH to fix the volume to 1L for later use after complete dissolution.

In order to solve the above technical problem, in a seventh aspect, the present invention provides an application, where the application is any one of P1) to P3):

p1) and the application of the protein in the preparation of products for preventing porcine circovirus type 2 infection;

p2), the application of the biological material in preparing products for preventing porcine circovirus type 2 infection;

p3) and the application of the virus-like particles in preparing products for preventing porcine circovirus type 2 infection.

In the present invention, the product may be a reagent or a kit or a pharmaceutical product.

Further, in the present invention, the pharmaceutical product may be a vaccine.

In the present invention, identity means the identity of an amino acid sequence or a nucleotide sequence. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI home web site. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, per residual Gap cost, and Lambda ratio to 11,1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the present invention, the 80% or more identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

VLPs particle vaccines are currently better vaccines delivered in vivo, and can obtain higher immune antibodies. The mineralization peptide is a polypeptide segment, and is a polypeptide capable of combining calcium and phosphorus. This combines the assembly and chemical reactions of protein VLPs to improve protein stability and immune stimulatory responses. The invention utilizes a protein engineering method to recombine and coexpress a CapliopCD region and a mineralization peptide W6 to finally form the VLPs mineralization peptide.

The nucleic acid molecule encoding the recombinant protein Cap286W6 can be easily mutated in the nucleotide sequence of the nucleic acid molecule encoding the Cap and W6 polypeptide protein of the present invention by known methods, such as directed evolution and point mutation. Those artificially modified, having 75% or more identity to the nucleotide sequence of the nucleic acid molecule of the present invention isolated and encoding the Cap recombinant W6 polypeptide protein, and encoding the Cap or W6 polypeptide protein, are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention.

The term "coding sequence" means a sequence of nucleotides (positions including stop codons) corresponding to the sequence of amino acids in a protein; features include conceptual translation of amino acids.

The term "expression" includes any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

In the above biological material, the expression cassette refers to a DNA capable of expressing the recombinant protein Cap286W6 in the recombinant cell, and the DNA may include not only a promoter for initiating transcription of the biologically encoded gene of the recombinant protein Cap286W6, but also a terminator for terminating transcription of the biologically encoded gene of the recombinant protein Cap286W6. Further, the expression cassette may also include an enhancer sequence. The recombinant expression vector containing the recombinant protein Cap286W6 coding gene can be specifically a recombinant expression vector obtained by inserting the recombinant protein Cap286W6 coding gene into a multiple cloning site of a vector PET30a (+). The recombinant microorganism can be specifically bacteria such as escherichia coli, mammalian cells and insect cells, and can also be other expression systems such as yeasts, algae and plants. The mammalian cell can be 293, CHO, BHK, MDCK, etc., and the insect cell can be SF9 and Hifv5.

The term "recombinant vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide operably linked to regulatory sequences that provide for its expression. The recombinant vector comprises a polynucleotide of the invention linked to one or more control sequences, such as a promoter and transcriptional and translational stop signals, which direct the production of the polypeptide in an expression host. The various nucleotides and control sequences may be joined together to produce a recombinant vector, which may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting a nucleic acid construct or polynucleotide comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression. The recombinant vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the polynucleotide. The choice of the vector will generally depend on the compatibility of the vector with the recombinant cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid. The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for ensuring self-replication; alternatively, the vector may be one which, when introduced into a recombinant cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the entire DNA to be introduced into the genome of the recombinant cell may be used, or a transposon may be used.

The vector preferably contains one or more selectable markers that allow for easy selection of transformed, transfected, transduced, or the like cells. The vector preferably contains elements that allow the vector to integrate into the recombinant cell genome or the vector to replicate independently of the genome in the cell. For integration into the recombinant cell genome, the vector may rely on the sequence of the polynucleotide encoding the polypeptide or any other vector element for integration into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the host cell genome at a precise location in the chromosome. To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10000 base pairs, 400 to 10000 base pairs, and 800 to 10000 base pairs, which have a high degree of sequence identity with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. Alternatively, the vector may be integrated into the genome of the recombinant cell by non-homologous recombination. For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the recombinant cell. The origin of replication may be any plasmid replicon mediating autonomous replication, which functions in a cell. The term "origin of replication" or "plasmid replicon" means a polynucleotide capable of replicating a plasmid or vector in vivo.

More than one copy of a polynucleotide of the invention may be inserted into a recombinant cell to increase production of the polypeptide. The increase in the copy number of the polynucleotide can be obtained by: integrating at least one additional copy of the sequence into the host cell genome, or including an amplifiable selectable marker gene with the polynucleotide, wherein cells containing amplified copies of the selectable marker gene, and thus additional copies of the polynucleotide, can be selected for by culturing the cells in the presence of the appropriate selectable agent. Methods for ligating the above elements to construct the recombinant expression vectors of the invention are well known to those skilled in the art.

The recombinant protein can be expressed in Escherichia coli in a soluble way; expression is preferentially carried out in BL21 (DE 3) transfected with the recombinant vector PET30-Cap286W6. The application of the Escherichia coli cell strain is to prepare an anti-porcine circovirus antigen, and the antigen and an adjuvant can play a role in protecting animals after being mixed and immunized with the animal, and are safe for the animals.

The beneficial technical effects obtained by the invention are as follows: the immune effect of the mineralized recombinant protein Cap286W6 virus-like particles is better than that of unmineralized recombinant protein Cap286W6 virus-like particles and wild type Cap virus-like particles; the vaccine is relatively stable to store under the normal temperature (37 ℃), the low-temperature storage condition of the traditional vaccine is not needed, and the storage cost of the vaccine can be reduced.

Drawings

FIG. 1 is a map of the recombinant vector PET30-Cap286W6.

FIG. 2 is a map of the recombinant vector PET30-Cap2.

FIG. 3 shows the expression of the recombinant protein Cap286W6 and wild type Cap in the culture supernatants of the strain BL21/PET30-Cap286W6 and the strain BL21/PET30-Cap2.

FIG. 4 shows the result of microscopic examination of the assembly of the recombinant protein Cap286W6 in VLPs buffer.

FIG. 5 shows the microscopic examination of wild-type Cap assembly in VLPs buffer.

Figure 6 shows the grouping of animals and their immunizations.

Figure 7 is a three vaccine mice serological potency test.

FIG. 8 is a summary of the efficacy test results of three batches of vaccine PCV 2.

FIG. 9 shows the results of immunohistochemical assays for mineralized Cap286W6 and unmineralized Cap 286W.

FIG. 10 shows the results of the vaccine stability test.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise specified, were carried out in a conventional manner according to the techniques or conditions described in the literature in this field or according to the product instructions. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The recombinant expression vector PET30-Cap286W6 is provided by Oncorhiki Kagaku Bio Inc. The recombinant gene is synthesized according to the preference of escherichia coli codon, is synthesized by Nanjing Biotech of Onghamaceae, and has a nucleotide sequence shown as SEQ ID No. 1. Enzymes and other biochemical reagents: yeast powder peptone is purchased from Angel corporation, and the others are all made in China.

The invention has the following idea: can chelate Ca by a molecular cloning technology ²⁺ Or PO4 ^3- The biomineralization peptide W6 polypeptide sequence is inserted into and recombined and expressed with 86-87 amino acid sites of a circovirus type 2 nucleocapsid protein Cap coding amino acid LooP CD region, VLPs are formed by protein expression and then Ca is enriched ²⁺ Or PO4 ^3- In weak alkaline solution, the protein of Cap2 VLPs containing W6 polypeptide is chelated Ca respectively ²⁺ Or PO4 ^3- Binding calcium phosphate in a buffer to form particles mineralized by VLPs; the mineralization condition of the particles is detected by an electron microscope, and the immunoprotection safety of the antigen nanoparticles of the mineralized nanoparticles is evaluated by a target animal pig.

Example 1 expression of CaP286W6 recombinant protein

1.1 Synthesis of coding Gene and recombinant expression vector

In order to improve the high immunogenicity and stability of wild type Cap2, porcine circovirus type 2 nucleocapsid protein Cap and mineralized peptide W6 are recombined to obtain recombinant protein Cap286W6. The coding gene of the recombinant protein is synthesized according to the codon preference of escherichia coli and is synthesized by Scophthalmus sp, wherein the coding gene of the recombinant protein Cap286W6 is named Cap286W6 gene, and the Cap286W6 gene is constructed to PET30 to obtain a recombinant expression vector PET30-Cap286W6.

The coding sequence of the Cap286W6 gene is shown in the SEQ ID No.1 at 5076-5762, wherein the No. 5076-5204 of the SEQ ID No.1 is the N-terminal protein coding gene of the Cap, the No. 5205-5216 of the SEQ ID No.1 is the GS connecting peptide, and the No. 5217-5282 of the SEQ ID No.1 is the coding gene of the mineralized peptide W6. The sites 5283-5735 of SEQ ID No.1 are coding genes of Cap C-terminal protein, the sites 5736-5762 of SEQ ID No.1 are XhoI enzyme cutting sites, his tag protein genes and stop codon genes, and the protein coded by the Cap286W6 gene is named as recombinant protein Cap286W6, and the amino acid sequence of the protein is shown in SEQ ID No. 2.

The recombinant expression vector PET30-Cap286W6 is a recombinant vector obtained by replacing the sequence between NdeI and XhoI recognition sites of pET30a (+) vector with DNA molecule with the nucleotide sequence of 5076-5735 th site of SEQ ID No.1, and the nucleotide sequence of the recombinant expression vector PET30-Cap286W6 is shown in SEQ ID No. 1. The map of the recombinant expression vector PET30-Cap286W6 is shown in FIG. 1.

The nucleotide sequence of the wild type Cap coding gene without mineralized peptide is 5076-5657 th site of SEQ ID No.3, and the amino acid sequence of the wild type Cap protein is 1-194 th site of SEQ ID No. 4. The recombinant expression vector PET30-Cap2 of the control group is the recombinant expression vector PET30-Cap2 obtained by replacing the sequence between NdeI and XhoI of the PET30 vector with a DNA molecule (wild type Cap coding gene) with the nucleotide shown in the 5076-5657 th site of SEQ ID No.3, the nucleotide sequence of the recombinant expression vector PET30-Cap2 is SEQ ID No.3, and the map of the control recombinant expression vector PET30-Cap2 is shown in FIG. 2.

1.2 expression of Cap286W6 recombinant protein

1.2.1, transforming the synthesized recombinant expression vector PET30-Cap286W6 into host bacteriaE.coli In BL21 (DE 3), a recombinant strain BL21 was obtained/PET30-Cap286W6。

The method comprises the following specific steps: 0.5 mu L of recombinant expression vector PET30-Cap286W6 is added into 100ul of full-scale goldE.coli BL21 (DE 3) competence-EP tube, ice bath 30min,42 ℃ heat shock 90S ice bath for 2min, adding LB 500. Mu.L without antibiotics, culturing for 1 hour at 37 ℃, then coating on Kana (kanamycin) resistant LB plate culture medium, culturing overnight at 37 ℃, selecting single bacteria, inoculating in LB culture medium, culturing for 6 hours at 37 ℃ and 200rpm in a shaking way, and storing the bacteria liquid at-80 ℃ according to the proportion of the bacteria liquid to 50% glycerol 7.

1.2.2 expression of wild-type Cap the method described in 1.2.1 was followed, except that the recombinant expression vector PET30-Cap286W6 was replaced with the recombinant expression vector PET30-Cap2 to obtain the recombinant strain BL21/PET30-Cap2.

1.3 determination of product expression and expression Format

1.2 of the recombinant strain-preserved bacterium solution prepared using glycerol was inoculated at 1% into a 250 mL-capacity flask containing 100mL of kana (kanamycin) -resistant LB medium, 1mM TPTG was added to induce expression for 6 hours, and after the induction expression, the resulting solution was ultrasonically disrupted and centrifuged (centrifugation conditions: 12000rpm, centrifugation 10 min) to obtain a supernatant of the recombinant strain BL21/PET30-Cap286W6 and a supernatant of the recombinant strain BL21/PET30-Cap2, and the supernatants were subjected to SDS-PAGE, respectively.

The results are shown in FIG. 3, in which MK in FIG. 3 represents Marker and the band sizes are 180, 130, 100, 70, 55, 40, 35, 25 and 15kDa, respectively, from the top to the bottom; 3-1 and 3-2 in FIG. 3 are the supernatant running glue bands of the recombinant strain BL21/PET30-Cap286W6, 2-1 and 2-2 in FIG. 3 are the supernatant running glue bands of the recombinant strain BL21/PET30-Cap2, and the arrows in FIG. 3 are the target bands. The results show that: the recombinant strain BL21/PET30-Cap286W6 for expressing the recombinant protein Cap286W6 has obvious product expression at about 25-35 kDa.

The supernatant from the shake flask was added to 10 XVLPs buffer solution and treated overnight at 4 ℃ for electron microscopy as shown in FIGS. 4 and 5. Wherein, the components of the 10 XVLPs buffer solution are as follows: 0.5% arginine, 0.5M NaCl,10% glycerol, 6% PEG3350. FIG. 4 shows the result of electron microscopy of the recombinant protein Cap286W6 in VLPs buffer solution, and FIG. 5 shows the result of electron microscopy of wild type Cap in VLPs buffer solution, which indicates that the recombinant protein Cap286W6 with mineralized peptide and the wild type Cap protein without mineralized peptide can form virus-like particles (VLPs) without structural difference.

Example 2 high-Density fermentation of E.coli in a fermenter

2.1 seed liquid preparation

The recombinant strain BL21/PET30-Cap286W6 was inoculated in LB medium at 37 ℃ and 280rpm for 12 hours at an inoculum size of 1%. Wherein the LB culture medium comprises the following components in percentage by weight: 1% peptone, 0.5% NaCl, 0.5% yeast powder, 50. Mu.g/mL kanamycin, and the balance water.

2.2, second-stage seed preparation

The seed solution of the recombinant strain BL21/PET30-Cap286W6 prepared in 2.1 was inoculated in an improved LB medium at 37 ℃ in an inoculum size of 1% and cultured at 250rpm for 12 hours. Wherein the formula of the improved LB culture medium is as follows: 0.5% glycerol, 1% peptone, 0.5% NaCl, 1% yeast powder, kanamycin 25. Mu.g/mL, and water as the rest.

2.3 fermentation production

The fermentation culture is divided into three stages

(1) Stage of culturing thallus

After initial 3L fermentation medium in a 5L fermenter was sterilized (wherein the initial medium and the feed medium were formulated as follows:

the culture medium is a semi-synthetic culture medium, and the initial culture medium comprises the following components: 5g of glucose, 5g of peptone, 5g of yeast powder and KH ₂ PO ₄ 1g、K ₂ HPO ₄ 2g、Na ₂ HPO ₄ .12H ₂ O 3g、(NH ₄ ) ₂ SO ₄ 0.2g、NH ₄ Cl 0.2g、MgSo ₄ .7H ₂ o 0.3g, defoaming agent 0.2g, and adding distilled water to make the volume constant to 1L.

The composition of the feed medium is as follows: 60g of glycerol, 60g of glucose, 15g of peptone, 15g of yeast powder and Mgso ₄ .7H ₂ O2.5 g, adding distilled water to a constant volume of 1L, and sterilizing at 105 ℃ for 10min for use. Adding kana (kanamycin) 25 μ g/mL per liter of the initial culture medium, inoculating the seed solution prepared by 2.1.2 at an inoculum size of 10%, and culturing at 30 ℃ for about 7 hours under aeration and stirring, wherein the culture is carried out along with the cultureWhen the bacterial strain grows, sugar in the culture medium is gradually consumed, the bacterial strain does not grow after the carbon source is consumed, dissolved Oxygen (DO) is increased back to 10%, and the pH value is maintained to be 7.0 by ammonia water in the culture process.

(2) Source feeding stage

This step was started after the sugar consumption was confirmed (as indicated by a 20% rise in DO with a pH rise in the absence of acid). DO control with feed was between 10-20%. The pH is maintained at 7.0 with ammonia, and this step can be maintained for 3 hours. Expression induction was initiated later.

(3) Inducible expression phase

When the induction is started after feeding for 3 hours, the induction is carried out for 6 hours by adding IPTG with the final concentration of 0.1mM, the DO is controlled between 10 and 30 percent by feeding, the pH value is maintained at 7.0 by ammonia water in the whole induction process, and the fermentation liquor is collected after the induction for 6 hours.

2.4 fermentation Process of wild type Cap

The fermentation process of wild-type Cap without adding mineralized peptide refers to the fermentation process of recombinant protein Cap286W6, and the only difference is that the recombinant strain BL21/PET30-Cap286W6 is replaced by the recombinant strain BL21/PET30-Cap2.

Example 3 purification of the protein of interest

3.1 purification of soluble proteins

The fermentation broth collected in example 2 was centrifuged at 5000rpm for 5-10min to collect the cells, stored at-20 ℃, suspended in 10% amount in a buffer lysate (buffer lysate: 10mM Tris-HCl, 2mM NaCl,2mM EDTA,0.02% lysozyme pH 8.0) for disruption in ice bath, centrifuged at 10000 rpm for 20min to separate cell debris, and the supernatant was passed through a 0.45um membrane as a crude sample for purification. Loading crude sample into His column, before loading, balancing the column with buffer solution (balance buffer solution: 20mM Tris-HCl,20mM NaCl,20mM imidazole pH 8.0), then loading, washing with balance buffer solution, and finally eluting with target protein elution buffer solution (target protein elution buffer solution: 20mM Tris-HCl,0.2M NaCl,300mm imidazole, 3% glycerol pH 8.0) to obtain target protein, wherein the purified protein is recombinant protein Cap286W6.

Wild-type Cap protein preparation reference Cap286W6 process preparation.

The protein concentration detection method comprises the following steps:

the concentration of the purified protein is determined by adopting a Coomassie Brilliant Blue (Coomassie Brilliant Blue) method, and the mineralized Cap286W6 VLPs protein and wild type Cap group purified samples are detected, wherein the detection curve is as follows: y =9.106X; r ² =0.9995. In the formula: y is protein concentration in mg/mL; x is OD595nm.

The detection concentration result is as follows: cap286W6 VLPs were 1.35mg/mL, wild type Cap was 1.08mg/mL.

3.2 viral-like particle Assembly and mineralization treatment of recombinant protein Cap286W6

Recombinant proteins were divided into three treatment groups:

treatment group 1: adding the recombinant protein Cap286W6 prepared by 3.1 into 10 XVLPs buffer solution, standing at 4 ℃ for 24 hours, assembling into recombinant protein Cap286W6 VLPs, and then supplementing calcium phosphate buffer solution for calcification for 48 hours to obtain mineralized Cap286W6 VLPs.

Treatment group 2: the recombinant protein Cap286W6 prepared in 3.1 was added to 10 XVLPs buffer solution and left at 4 ℃ for 24 hours to assemble unmineralized Cap286W6 VLPs.

Treatment group 3: wild-type Cap proteins obtained from 3.1 preparation were added to 10 XVLPs buffer, respectively, and left at 4 ℃ for 24 hours to assemble wild-type Cap VLPs.

The composition of the 10 × VLPs buffer for each of the three treatment groups was: 0.5% arginine, 0.5M NaCl,10% glycerol, 6% PEG3350, the remainder water;

the calcium phosphate buffer was a 10mM calcium phosphate solution pH 7.5: 3.1 g of Ca are taken ₃ (PO ₄ ) ₂ Dissolving 800mL of water, stirring, adding hydrochloric acid to promote dissolution, adjusting the pH value to 7.5 by using NaOH after complete dissolution, and metering the volume to 1L for later use.

Example 4 vaccine formulation and emulsification

4.1.1 aqueous phase preparation

50 mu g of vaccine is prepared per head, and mineralized Cap286W6 VLPs, unmineralized Cap286W6 VLPs and wild type Cap VLPS antigens are respectively diluted to an antigen mother liquor with a final concentration of 100 mu g by PBS (0.04 mol/L, pH value of 7.6) for standby.

From Spiransys Saibox Co Monanide ^TM 1 part of ISA206 VG adjuvant, 1 part of qualified antigen, and the mass percentages of the antigens are as follows: adjuvant 50:50 (v/v) mixing in an emulsifying cylinder, stirring at low speed for emulsifying for 30 minutes to prepare the porcine circovirus Cap VLPS protein water-in-oil-in-water vaccine of each group.

The specific formula is as follows:

preparing a mineralized vaccine: mineralized Cap286W6 VLPs were diluted with PBS to a final concentration of 100. Mu.g/mL, 250mL was taken plus Monanide ^TM 250mL of ISA206 VG adjuvant is mixed in an emulsification cylinder, stirred and emulsified at low speed for 30 minutes, and the temperature is controlled to be 4 ℃, thus obtaining the porcine circovirus type 2 mineralized Cap286W6 VLPs water-in-oil formulation subunit vaccine.

Preparation of unmineralized vaccine: unmineralized Cap286W6 VLPs were diluted with PBS to a final concentration of 100. Mu.g/mL, 250mL plus Monanide ^TM 250mL of ISA206 VG adjuvant is mixed in an emulsifying cylinder, stirred at a low speed for emulsification for 30 minutes, and the temperature is controlled to be 4 ℃, so that the non-mineralized protein water-in-oil-in-water vaccine formulation of Cap286W6 VLPs of porcine circovirus type 2 is prepared.

Preparation of wild-type Cap vaccine: wild-type Cap VLPs were diluted to a final concentration of 100. Mu.g/mL with PBS, 250mL was taken and Monanide was added ^TM 250mL of ISA206 VG adjuvant is mixed in an emulsification cylinder, stirred and emulsified at low speed for 30 minutes, and the temperature is controlled to be 4 ℃, thus obtaining the porcine circovirus type 2 unmineralized wild type Cap protein water-in-oil-in-water vaccine.

Example 5 evaluation of vaccine efficacy

5.1 materials

5.1.1 test vaccine

The mineralized Cap286W6 VLPs protein water-in-oil-in-water dosage form vaccine, the unmineralized Cap286W6 VLPs protein water-in-oil-in-water dosage form vaccine, and the unmineralized wild-type Cap protein water-in-oil-in-water dosage form vaccine prepared in example 4.

5.1.2 antibody detection kit

Porcine circovirus type 2 antibody detection kit (Spanish Ingezim CIRCO IgG, product number (SKU) 11.PCV. K.1/5, lot number 180815).

5.1.3 test animals

40 male BALB/c mice, 18-22g, purchased from Wentotongliwa laboratory animal technologies, beijing. 40 healthy piglets of 14-21 days old, which are negative to the type 2 antigen-antibody of the porcine circovirus, grow white, wherein 20 piglets are purchased from a certain pig farm in the city of Baoding city, hebei province and a certain pig farm in Beijing Mount House; 20 were purchased from a certain SPF pig farm in Beijing, and all animal testing sites were performed in the animal house of Huaxia Xingyang.

5.2 serological effect test of mice

5.2.1 test methods

(1) Immunization

The vaccine is divided into three experimental groups and a blank control group according to the type of the vaccine, and the three experimental groups and the blank control group are specifically arranged as follows:

a first group: mice were immunized with the mineralized Cap286W6 VLPs protein water-in-oil-in-water formulation vaccine, this experimental group was named mineralized Cap286W6 group;

second group: mice were immunized with the non-mineralized Cap286W6 VLPs protein water-in-oil-in-water formulation vaccine, and this experimental group was named non-mineralized Cap286W6 group;

third group: immunizing mice with a wild-type Ccap protein water-in-oil-in-water vaccine, wherein the experimental group is named as a wild-type Cap group;

and a fourth group: mice were immunized with PBS instead of vaccine, which was a blank control group.

(2) Antibody assay

And (3) collecting blood and separating serum 28 days after the mice are immunized by the vaccine, and detecting the porcine circovirus type 2 antibody in the mouse serum by adopting a porcine circovirus type 2 antibody ELISA detection kit.

5.2.2, results

28 days after the vaccine immunization of the mice, blood is collected and serum is separated, and the porcine circovirus 2 antibody is detected by adopting an ELISA method. As shown in fig. 7, "/" in fig. 7 indicates an uncalculated value. The geometric mean values of the porcine circovirus type 2 antibodies are respectively 1:2640, higher than unmineralized 1 2320 and wild type 1:1840, the mouse serological potency test passed, and the blank mouse antibodies were all below 1.

5.3 immune challenge test

The vaccine is divided into groups and treated according to the way of piglets (long and white pigs) in figure 6, 10 heads of each test group are taken, the vaccine is inoculated by neck intramuscular injection, 1 mL/head, 14 days after the first immunization, the vaccine is immunized by 10 heads in the same method and dosage, and the non-immune vaccine is fed separately under the same condition to serve as a control. In FIG. 6 "/" indicates no vaccine immunization.

Porcine circovirus type 2 challenge: in the figure 6, four test pigs are extracted in groups, and each pig is injected with 3ml of PCV2 CAU0673 strain virus (purchased from China veterinary microbial strain preservation management center, CVCC) and 2ml of nasal drops (with the toxic content of 10%) through neck muscle ^6.0 TCID ₅₀ And/ml) as challenge groups, respectively named as a mineralized Cap286W6 group, an unmineralized Cap286W6 group, a wild type Cap group and a challenge control group. Wherein, the 5 pigs extracted from 10 pigs in the blank control group are used as the control group for counteracting the toxin according to the method; in addition, 5 animals were isolated and raised without toxic challenge, and named as blank control group. On days 4 and 7 after challenge, all pigs (except the placebo group) were co-inoculated with keyhole limpet hemocyanin (KLH/ICFA, 1 mg/mL) emulsified with Freund's incomplete adjuvant at 4 spots per pig in both axilla and both buttocks, and 1mL per spot.

Continuously observing for 28 days, weighing on 28 th day, collecting blood, separating serum, and detecting PCV2 virus nucleic acid in the serum according to PCV2 virus PCR detection method; all test pigs were killed, inguinal lymph nodes were taken, and PCV2 antigen was detected by PCV2 antigen immunohistochemical detection in lymph nodes. And simultaneously detecting the PCV2 nucleic acid load in the inguinal lymph node sample.

And the relative weight gain rate is that compared with the piglets of the blank control group, the relative weight gain rate of the piglets of the challenge group is lower than the average relative weight gain rate of the piglets of the blank control group, and if the relative weight gain rate is more than or equal to 5.0 percent, the piglets are judged to have poor growth and development and have clinical symptoms caused by the porcine circovirus type 2. The calculation formula is as follows:

relative gain (%) = (average daily gain of piglets in blank control group-average daily gain of piglets in challenge test)/average daily gain of piglets in blank control group is multiplied by 100%;

average daily gain of the piglets in the challenge group = (weight of the piglets in 28 days after challenge-weight of the piglets in the day of challenge)/28 days;

average daily gain of piglets in the blank control group = (sum of weights of piglets in blank control group at 28 days after challenge-sum of weights of piglets in blank control group at the same day of challenge)/(28 days × 5).

5.3.3 detection of viremia

Collecting blood from the anterior vena cava 28 days after virus challenge, separating serum, detecting viral nucleic acid of porcine circovirus type 2 (PCV 2) in the serum by using a PCR method, and judging the strip with PCV2 specificity as positive, otherwise, judging the strip as negative;

wherein, the nucleotide sequence of the PCR upstream primer is as follows: 5 'CACGGATATTGTAGTCCTGGT-3' (SEQ ID No. 5); nucleotide sequence of PCR downstream primer: 5 'CGCACCTTCGGATATACTGTC-3' (SEQ ID No. 6).

And analyzing the relative weight gain rate, the virus blood PCR detection and the immunohistochemical result, and judging the disease onset if the three meet the two criteria according to the disease onset judgment. The results show that the mineralized Cap286W6 group and the unmineralized Cap286W6 group are all negative, the wild type Cap group is positive for 1 piglet, the challenge control group is positive for 4 piglets, and the blank control is all negative, the detection results of the effectiveness of the vaccine are summarized as the figure 8, and the "/" in the figure 8 represents an undetected value.

5.3.4 immunohistochemical detection

According to the obvious brown-yellow colored signal in the cytoplasm of the lymph node cell of the diseased piglet, the signal is judged to be positive (A in figure 9), otherwise, the signal is judged to be negative (B in figure 9). The mineralized Cap286W6 group and the unmineralized Cap286W6 group were all negative, the unmineralized wild Cap group was positive for 1 piglet, the challenge control group was positive for 4 piglets, and the blank control was all negative (fig. 9, B). The partial immunohistochemistry results are shown in figure 9.

Example 6 vaccine stability experiment

6.1 test methods

Vaccine prepared in example 4: porcine circovirus type 2 mineralized Cap286W6 VLPs water-in-oil-in-water dosage form subunit vaccines (mineralized group), porcine circovirus type 2 unmineralized Cap286W6 VLPs protein water-in-oil-in-water dosage form vaccines (unmineralized group), and porcine circovirus type 2 wild type Cap protein water-in-oil-in-water dosage form vaccines (wild type Cap group).

And randomly selecting 20 bottles (with antigen specification of 10 mL/bottle) from each group, storing at 4 deg.C and 37 deg.C, sampling at 1, 2 and 3 months (the antigen is used up in 3 months), testing the antigen content, and comparing whether the antigen content in each group has change during the storage period.

The antigen concentration detection method comprises the following steps:

antigen concentration purified protein concentration was determined by Coomassie Brilliant Blue (Coomassie Brilliant Blue) method and vaccine samples of mineralized, unmineralized and wild type Cap groups were tested, where the standard curve is: y =9.106X; r ² =0.9995. In the formula: y is antigen concentration, and the unit is mg/mL; x is OD595nm.

6.2 results

Respectively storing 20 bottles of randomly extracted antigens in each group at 4 ℃ and 37 ℃ for 3 months, detecting the attenuation of the content of the porcine circovirus type 2 Cap, and ensuring that the porcine circovirus type 2 Cap286W6 VLPs water-in-oil dosage form subunit vaccine group (mineralization group) has the most stable 3 months at 4 ℃ and loses about 0.12-0.83%; the loss rate of the porcine circovirus type 2 unmineralized Cap286W6 VLPs protein water-in-oil-in-water type vaccine group (unmineralized group) is 0.13-2.81%; the vaccine loss rate of the porcine circovirus type 2 wild-type Cap protein water-in-oil-in-water agent vaccine group (wild-type Cap group) is the largest and is 2.11-4.65%. The relative loss rate of each group at 37 ℃ is slightly larger, wherein the loss rate of the mineralized group vaccine is about 0.4% -6.43% at 37 ℃ for 3 months, while the loss rate of the unmineralized group is larger than the loss rate of the mineralized vaccine group at 3.29-19.11%, the loss rate of the wild type Cap group vaccine is 7.89-29.32%, the determination result of the vaccine stability experiment is shown in figure 10, and the "/" in figure 10 represents the undetected value.

According to the analysis of the test result, the content of the vaccine prepared by the invention is attenuated by 0.83-6.43 percent after the antigen is stored for three months; to ensure efficacy of each batch of product over the shelf life, the final formula design was proposed to achieve efficacy standards with a maximum decay rate of 7% at 3 months.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.

Sequence listing

<110> Beijing Huaxia xing ocean Biotech Co., ltd

BEIJING CENTRE BIOLOGY Co.,Ltd.

<120> proteins and biomaterials for producing circovirus type 2 virus-like particles and application

<160> 6

<170> SIPOSequenceListing 1.0

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cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340

caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400

ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460

gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760

acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820

ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180

catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240

ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300

gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360

gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420

ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480

atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600

tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780

atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc 4980

gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa 5040

ataattttgt ttaactttaa gaaggagata tacatatgaa gaacggcatc ttcaacaccc 5100

gcctgagccg caccatcggc tacaccgtga agaagaccac cgtgcgcacc cccagctgga 5160

acgtggacat gatgcgcttc aacatcaacg acttcctgcc ccccggcggc ggcagccgct 5220

ggcgcctgga gggcaccgac gacaaggagg agcccgagag ccagcgccgc atcggccgct 5280

tcggcggcgg cagcaacccc ctgaccgtgc ccttcgagta ctaccgcatc cgcaaggtga 5340

aggtggagtt ctggccctgc agccccatca cccagggcga ccgcggcgtg ggcagcaccg 5400

ccgtgatcct ggacgacaac ttcgtgacca aggccaacgc cctgacctac gacccctacg 5460

tgaactacag cagccgccac accatcaccc agcccttcag ctaccacagc cgctacttca 5520

cccccaagcc cgtgctggac cgcaccatcg actacttcca gcccaacaac aagcgcaacc 5580

agctgtggct gcgcctgcag accaccggca acgtggacca cgtgggcctg ggcaccgcct 5640

tcgagaacag catctacgac caggactaca acatccgcat caccatgtac gtgcagttcc 5700

gcgagttcaa cctgaaggac ccccccctga accccctcga gcaccaccac caccaccact 5760

gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc 5820

aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag 5880

gaggaactat atccggat 5898

<210> 2

<211> 228

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Lys Asn Gly Ile Phe Asn Thr Arg Leu Ser Arg Thr Ile Gly Tyr

1 5 10 15

Thr Val Lys Lys Thr Thr Val Arg Thr Pro Ser Trp Asn Val Asp Met

20 25 30

Met Arg Phe Asn Ile Asn Asp Phe Leu Pro Pro Gly Gly Gly Ser Arg

35 40 45

Trp Arg Leu Glu Gly Thr Asp Asp Lys Glu Glu Pro Glu Ser Gln Arg

50 55 60

Arg Ile Gly Arg Phe Gly Gly Gly Ser Asn Pro Leu Thr Val Pro Phe

65 70 75 80

Glu Tyr Tyr Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser

85 90 95

Pro Ile Thr Gln Gly Asp Arg Gly Val Gly Ser Thr Ala Val Ile Leu

100 105 110

Asp Asp Asn Phe Val Thr Lys Ala Asn Ala Leu Thr Tyr Asp Pro Tyr

115 120 125

Val Asn Tyr Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr His

130 135 140

Ser Arg Tyr Phe Thr Pro Lys Pro Val Leu Asp Arg Thr Ile Asp Tyr

145 150 155 160

Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr

165 170 175

Thr Gly Asn Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser

180 185 190

Ile Tyr Asp Gln Asp Tyr Asn Ile Arg Ile Thr Met Tyr Val Gln Phe

195 200 205

Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu Asn Pro Leu Glu His His

210 215 220

His His His His

225

<210> 3

<211> 5820

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340

caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400

ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460

gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760

acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820

ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180

catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240

ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300

gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360

gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420

ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480

atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600

tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780

atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc 4980

gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa 5040

ataattttgt ttaactttaa gaaggagata tacatatgaa gaacggcatc ttcaacaccc 5100

gcctgagccg caccatcggc tacaccgtga agaagaccac cgtgcgcacc cccagctgga 5160

acgtggacat gatgcgcttc aacatcaacg acttcctgcc ccccggcggc ggcagcaacc 5220

ccctgaccgt gcccttcgag tactaccgca tccgcaaggt gaaggtggag ttctggccct 5280

gcagccccat cacccagggc gaccgcggcg tgggcagcac cgccgtgatc ctggacgaca 5340

acttcgtgac caaggccaac gccctgacct acgaccccta cgtgaactac agcagccgcc 5400

acaccatcac ccagcccttc agctaccaca gccgctactt cacccccaag cccgtgctgg 5460

accgcaccat cgactacttc cagcccaaca acaagcgcaa ccagctgtgg ctgcgcctgc 5520

agaccaccgg caacgtggac cacgtgggcc tgggcaccgc cttcgagaac agcatctacg 5580

accaggacta caacatccgc atcaccatgt acgtgcagtt ccgcgagttc aacctgaagg 5640

acccccccct gaaccccctc gagcaccacc accaccacca ctgagatccg gctgctaaca 5700

aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc 5760

ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat 5820

<210> 4

<211> 202

<212> PRT

<213> Porcine circovirus

<400> 4

Met Lys Asn Gly Ile Phe Asn Thr Arg Leu Ser Arg Thr Ile Gly Tyr

1 5 10 15

Thr Val Lys Lys Thr Thr Val Arg Thr Pro Ser Trp Asn Val Asp Met

20 25 30

Met Arg Phe Asn Ile Asn Asp Phe Leu Pro Pro Gly Gly Gly Ser Asn

35 40 45

Pro Leu Thr Val Pro Phe Glu Tyr Tyr Arg Ile Arg Lys Val Lys Val

50 55 60

Glu Phe Trp Pro Cys Ser Pro Ile Thr Gln Gly Asp Arg Gly Val Gly

65 70 75 80

Ser Thr Ala Val Ile Leu Asp Asp Asn Phe Val Thr Lys Ala Asn Ala

85 90 95

Leu Thr Tyr Asp Pro Tyr Val Asn Tyr Ser Ser Arg His Thr Ile Thr

100 105 110

Gln Pro Phe Ser Tyr His Ser Arg Tyr Phe Thr Pro Lys Pro Val Leu

115 120 125

Asp Arg Thr Ile Asp Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu

130 135 140

Trp Leu Arg Leu Gln Thr Thr Gly Asn Val Asp His Val Gly Leu Gly

145 150 155 160

Thr Ala Phe Glu Asn Ser Ile Tyr Asp Gln Asp Tyr Asn Ile Arg Ile

165 170 175

Thr Met Tyr Val Gln Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu

180 185 190

Asn Pro Leu Glu His His His His His His

195 200

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cacggatatt gtagtcctgg t 21

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cgcaccttcg gatatactgt c 21

Claims

1. A protein characterized by: the protein is a recombinant protein Cap286W6, and the recombinant protein Cap286W6 is the following A1) or A2):

a1 Protein with the amino acid sequence of 1 st to 220 th position of SEQ ID No. 2;

a2 A) and a tag is linked to the N-terminus and/or C-terminus of A1).

2. A biomaterial, characterized by: the biological material is any one of the following B1) -B4):

b1 Nucleic acid molecule which is a nucleic acid molecule encoding a recombinant protein Cap286W6, and the nucleic acid molecule is g 1) or g 2),

g1 A DNA molecule shown as 5076-5735 th site of SEQ ID No. 1;

b2 A) an expression cassette comprising the nucleic acid molecule of B1);

b4 A recombinant microorganism containing the nucleic acid molecule of B1) or containing the expression cassette of B2) or containing the recombinant vector of B3).

3. A virus-like particle, comprising: the virus-like particle comprises the protein of claim 1.

4. A vaccine of porcine circovirus type 2 comprising the protein of claim 1 or the virus-like particle of claim 3.

5. A method for producing the protein according to claim 1, comprising: expressing a gene encoding the recombinant protein Cap286W6 according to claim 1 in a biological cell to obtain the recombinant protein Cap286W6.

6. The method of claim 5, wherein: the organism is a microorganism, a plant or a non-human animal.

7. A method of making the virus-like particle of claim 3, wherein: the method comprising the step of mineralizing the protein of claim 1 to obtain virus-like particles.

8. Use of a protein according to claim 1 for the preparation of a product for the prevention of porcine circovirus type 2 infection.

9. Use of the biomaterial of claim 2 in the manufacture of a product for the prevention of porcine circovirus type 2 infection.

10. Use of the virus-like particle of claim 3 in the preparation of a product for the prevention of porcine circovirus type 2 infection.