CN114230677A

CN114230677A - Recombinant protein containing Cap of hog cholera E2 and circovirus, preparation method and application thereof

Info

Publication number: CN114230677A
Application number: CN202210164172.7A
Authority: CN
Inventors: 伏显华
Original assignee: Beijing Zhonghai Biotech Co Ltd
Current assignee: Beijing Zhonghai Biotech Co Ltd
Priority date: 2022-02-23
Filing date: 2022-02-23
Publication date: 2022-03-25
Anticipated expiration: 2042-02-23
Also published as: CN114230677B

Abstract

The invention discloses a recombinant protein containing Cap of classical swine fever E2 and circovirus, a preparation method and application thereof, and belongs to the field of vaccines. The active component of the vaccine disclosed by the invention is recombinant protein, the recombinant protein is formed by connecting fusion proteins CSFVE2-Spycatcher and Cap-SpyTag, and the fusion proteins CSFVE2-Spycatcher is protein with an amino acid sequence of SEQ ID No. 2; the fusion protein Cap-SpyTag is a protein with an amino acid sequence of SEQ ID No. 3. The invention also provides a construction method for co-expressing the swine fever E2 in the CHO cell strain, which utilizes circular VLPs to display E2, achieves the conversion of E2 VLPs, can improve the immunogenicity of E2 antigen, achieves mutual stimulation immunity, and improves the protective effects of swine fever and circular immunity. The invention improves the defects that the expressing product of the swine fever E2 protein in a mammalian cell has poor immune effect, and the expressing product of the swine fever E2 protein in a prokaryotic expression system can lead the swine to generate short antibody time and cause immune death by prokaryotic endotoxin residue after the swine is immunized.

Description

Recombinant protein containing Cap of hog cholera E2 and circovirus, preparation method and application thereof

Technical Field

The invention belongs to the field of vaccines, and particularly relates to a recombinant protein containing Cap of classical swine fever E2 and circovirus, and a preparation method and application thereof.

Background

Hog cholera, commonly known as "gastrointestinal plague", is an acute, febrile, contagious disease caused by Classical Swine Fever Virus (CSFV) of the genus hog cholera of the family Flaviviridae, and is highly contagious and lethal. Pigs are the only natural host for the virus. CSFV is enveloped virus, 40-60 nm, single strand positive strand RNA. The CSFV genome is about 123 kb in length and contains only one large Open Reading Frame (ORF) that is translated into a polyprotein of 3898 amino acid residues with a molecular weight of about 438 kDa and further processed to the mature protein by viral and host cell proteases. All structural and non-structural proteins of CSFV are encoded by the ORF, which is flanked by a 5 'untranslated region (5' UTR) and a3 'untranslated region (3' UTR), and has no cap structure at the 5 'end and no poly (A) tail at the 3' end. This large precursor protein is processed in a co-and post-translated form by cellular and virus-specific proteases into structural and nonstructural proteins, which are encoded in the viral RNA in the sequence Npro, c, Erns (E0), E1, E2, P7, NS2-3, NS4A, NS4B, NS5A and NS 5B. NS2-3 can be processed into NS2, NS3(P80), and all except c, E0, E1 and E2 as structural proteins are non-structural proteins. Among the structural proteins, the most important proteins for the research on immune control are E0 and E2, and particularly, the E2 protein is the first choice protein of the subunit vaccine at present.

The E2 protein is also called gp55, is envelope glycoprotein of CSFV, is the main antigen protein of virus, and is the molecule with lowest conservation and most variable in three virus glycoproteins. gp55 is often present on the surface of virions and CSFV-infected cells in 2 forms: one is the formation of a homodimer of 100 kDa and the second is the formation of a heterodimer of 75 kDa with gp 33. In vitro, E2 induced the production of neutralizing antibodies against the virus and in vivo, the production of attacking antibodies against CSFV. The E2 biphasic water-in-oil emulsion is used for immunizing pigs, and can resist the virulent attack of the 100 LD50 CSFV Brescia strain. The protein backbone of E2 consisted of 371 amino acids (690-1060 amino acid residues encoded by the ORF) and was anchored to the membrane with 40 hydrophobic amino acids at its C-terminus. Due to the different glycosylation degree, the molecular weight of E2 can be 51-58 kDa. The 15 cysteine (Cys) residues in the E2 molecule are conserved within a genus, with the 6 Cys residues at the N-terminus involved in the formation of the antigenic domain and the 9 Cys residues at the C-terminus involved in the formation of homo-and heterodimers.

Porcine Circovirus (PCV) belongs to the genus circovirus of the family circoviridae, the virion has a size of 14-25nm and an average diameter of 17 nm, is symmetrical icosahedron, is free of cyst membrane, has a single-stranded circular DNA genome, consists of deoxyribonucleic acid, and has a suspension density in tissues of 1.37 cm³. Porcine circovirus type 2 (PCV-2) is an important pathogen of Postweaning Multisystemic Wasting Syndrome (PMWS), porcine respiratory syndrome (PRDC), Porcine Dermatitis and Nephrotic Syndrome (PDNS), can also cause diseases such as various enteritis diseases, piglet congenital tremor (piglet tremor disease), sow dysreproduction and the like, and nucleocapsid protein (Cap) of the porcine circovirus type 2 ORF2 gene encoding virus belongs to virus protective antigen and has important immune function.

The preparation of the swine fever subunit vaccine by using a genetic engineering method is mainly focused on the E2 antigen region, and the E2 antigen vaccine is also one of the important ways for controlling the swine fever subunit vaccine at present. Although the supply of subunit vaccines expressing hog cholera E2 antigen and Cap genetic engineering such as Escherichia coli and baculovirus is available in the market, people still need a new preparation method of long-acting antigen vaccine with low cost. Having perfect post-translational modification functions is the main reason that mammalian cells are selected as hosts for expression of most biopharmaceutical proteins. Among them, Chinese Hamster Ovary cells (CHO for short) are the most successful host cells for eukaryotic foreign gene expression, and more medicinal proteins have been efficiently expressed therein, and many human recombinant protein drugs have been on the market. Compared with other expression systems, the system has many advantages, such as complete post-translational processing processes including glycosylation and hydroxylation, enables the expressed exogenous eukaryotic gene product to maintain the natural structure and activity, enables the expression product to be secreted to the extracellular space, and is beneficial to the separation and purification of exogenous proteins.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to improve the poor immune effect of the expression product of the swine fever E2 protein in mammalian cells and/or how to overcome the defects that the time for producing antibodies by pigs is short and the prokaryotic endotoxin residue causes the immune death after the swine is immunized by the expression product of the swine fever E2 protein in a prokaryotic expression system.

In order to solve the above-mentioned problems, the present invention provides, in a first aspect, a protein comprising a fusion protein CSFVE2-Spycatcher and a fusion protein Cap-SpyTag linked together,

the fusion protein CSFVE2-Spycatcher can be any one of the following A1) -A3):

A1) a protein having the amino acid sequence of SEQ ID No. 2;

A2) a protein which is obtained by substituting and/or deleting and/or adding more than one amino acid residue in the amino acid sequence shown in A1), has more than 80% of identity with the protein shown in A1), and is related to the classical swine fever virus vaccine;

A3) a fusion protein obtained by connecting a label to the N-terminal and/or the C-terminal of A1) or A2);

the fusion protein Cap-SpyTag can be any one of the following B1) -B3):

B1) a protein having the amino acid sequence of SEQ ID No. 3;

B2) b1) is obtained by substituting and/or deleting and/or adding more than one amino acid residue of the amino acid sequence shown in B1) to obtain a protein which has more than 80 percent of identity with the protein shown in B1) and is related to the classical swine fever virus vaccine;

B3) a fusion protein obtained by connecting a label to the N-terminal and/or the C-terminal of B1) or B2).

Wherein the SpyTag and the SpyCatcher can recombine and spontaneously form isopeptide bond coupling, thereby connecting the fusion protein CSFVE2-SpyCatcher and the fusion protein Cap-SpyTag.

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

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 order to solve the above-mentioned technical problems, the present invention provides, in a second aspect, a nucleic acid molecule related to the above-mentioned protein, said nucleic acid molecule consisting of a nucleic acid molecule encoding said fusion protein CSFVE2-Spycatcher and a nucleic acid molecule encoding said fusion protein Cap-SpyTag,

the nucleic acid molecule encoding the fusion protein CSFVE2-Spycatcher can be any one of g11) -g13),

g11) the coding sequence of the coding strand is a DNA molecule shown by 5776-bit 7263 of SEQ ID No. 1;

g12) the nucleotide sequence of the coding strand is a DNA molecule shown in the 5776-bit 7263-bit of SEQ ID No. 1;

g13) a DNA molecule which has 80% or more than 80% of identity with the nucleotide sequence limited by g11) or g12) and codes the fusion protein CSFVE 2-Spycatcher;

the nucleic acid molecule encoding the fusion protein Cap-SpyTag can be any one of g21) -g23),

g21) the coding sequence of the coding strand is a DNA molecule shown by nucleotides 9337-10029 of SEQ ID No. 1;

g22) the nucleotide sequence of the coding strand is a DNA molecule shown in the 9337-10029 site of SEQ ID No. 1;

g23) a DNA molecule which has 80% or more than 80% of identity with the nucleotide sequence limited by g21) or g22) and encodes the fusion protein Cap-SpyTag.

The DNA molecule may be synthesized according to codon optimization of mammalian cells.

In order to solve the above technical problem, in a third aspect, the present invention provides an expression cassette comprising the above nucleic acid molecule.

In order to solve the above technical problems, in a fourth aspect, the present invention provides a recombinant vector comprising the above nucleic acid molecule or comprising the above expression cassette.

In order to solve the above technical problems, in a fifth aspect, the present invention provides a recombinant microorganism comprising the above nucleic acid molecule or comprising the above expression cassette or comprising the above recombinant vector.

Further, the recombinant microorganism may be Escherichia coli or yeast.

In order to solve the above technical problems, in a sixth aspect, the present invention provides a recombinant transgenic cell line comprising the above nucleic acid molecule or comprising the above expression cassette or comprising the above recombinant vector.

Further, the transgenic cell line can be a mammalian cell.

Further, the transgenic cell line may be a non-human animal cell.

Further, the transgenic cell line may be a CHO cell.

Further, the transgenic cell line may be CHO/dhFr-cells.

In order to solve the above technical problems, in a seventh aspect, the present invention provides a swine fever vaccine comprising the above protein.

The swine fever is caused by swine fever virus, and the swine fever virus is swine fever virus of flaviviridae (the name of Latin university swine river virus, CSFV for short).

In order to solve the above technical problems, in an eighth aspect, the present invention provides a method for producing the above protein, which may comprise the steps of: expressing a gene encoding the fusion protein CSFVE2-Spycatcher and genes encoding the fusion protein and the fusion protein Cap-SpyTag in a biological cell to obtain the protein; the organism is a microorganism, a plant or a non-human animal.

Further, in the above method, the biological cell may be a non-human mammalian cell.

In order to solve the above technical problems, in a ninth aspect, the present invention provides the use of the above protein or the above nucleic acid molecule or the above expression cassette or the above recombinant vector or the above recombinant microorganism or the above recombinant transgenic cell line in the preparation of a product for preventing swine fever.

Further, the product for preventing swine fever may be a swine fever vaccine.

In the present invention, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. 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.

The nanoparticle vaccine is a better body-delivered vaccine at present, can obtain higher immune antibody, the SpyTag is a polypeptide segment, and the Spycatcher is a protein corresponding to the SpyTag. They can recombine and spontaneously form isopeptide bond coupling. This combines protein assembly with chemical reactions, which are genetically encodable. The invention utilizes a protein engineering method to fuse SpyTag in Cap protein to be co-expressed with E2 fused Spycatcher, and VLPs are finally formed.

The monoclonal cell strain capable of secreting and expressing E2 protein and Cap covalent VLPs obtained by the invention has high expression quantity of fusion protein, the fusion protein obtained by His affinity separation and purification can be combined with a monoclonal antibody, the generation of a neutralizing antibody of an immune animal is higher than that of a product on the market at present, and the fusion protein can be used for preventing a vaccine for swine fever circular rings, so that the production cost and the immune failure loss are reduced.

The above-mentioned nucleic acid molecules encoding the recombinant fusion protein E2 antigen (CSFVE2) fused to the SpyCatcher polypeptide and Cap fused to the spyTag polypeptide can be easily mutated in the nucleotide sequence of the nucleic acid molecule encoding the recombinant fusion protein E2 antigen (CSFVE2) fused to the SpyCatcher polypeptide and Cap fused to the spyTag polypeptide by the ordinary skilled person in the art by using 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 encoding the recombinant fusion protein E2 antigen (CSFVE2) fused SpyCatcher polypeptide and Cap fused SpyTag polypeptide isolated in the present invention and encoding the recombinant fusion protein E2 antigen (CSFVE2) fused SpyCatcher polypeptide and Cap fused SpyTag polypeptide are both 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 polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with an initiation codon, e.g., ATG, GTG, or TTG, and ends with a stop codon, e.g., TAA, TAG, or TGA. The coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

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 is a DNA capable of expressing a recombinant fusion protein E2 antigen (CSFVE2) fused SpyCatcher polypeptide and Cap fused SpyTag polypeptide in a recombinant cell, and the DNA may include not only a promoter for initiating the transcription of the gene of E2 antigen (CSFVE2) fused SpyCatcher polypeptide and Cap fused spycaag polypeptide, but also a terminator for terminating the transcription of the gene of E2 antigen (CSFVE2) fused SpyCatcher polypeptide and Cap fused spycaag polypeptide. Further, the expression cassette may also include an enhancer sequence. The recombinant expression vector containing the nucleic acid molecule of the recombinant fusion protein E2 antigen (CSFVE2) fusion Spycatcher polypeptide and the Cap fusion SpyTag polypeptide gene can be specifically a recombinant expression vector obtained by inserting the E2 antigen (CSFVE2) fusion Spycatcher polypeptide and the Cap fusion SpyTag polypeptide gene into the multiple cloning site of the vector PCHO 1.0. The recombinant microorganism can be specifically mammalian cells and can also be other expression systems such as yeasts, bacteria, algae and plants. The mammalian cell may specifically be a CHO cell.

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 restriction enzyme 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 preparing an expression vector, a coding sequence is placed in the vector so that the coding sequence is operably linked with the appropriate regulatory 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 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, when introduced into a recombinant cell, the vector may be one which 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 that mediates autonomous replication and 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 term "recombinant cell" means any cell type that is susceptible to transformation, transfection, transduction, and the like using a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "recombinant cell" encompasses the progeny of any parent cell that differs from the parent cell due to mutations that occur during replication.

The recombinant cell comprising a polynucleotide of the invention operably linked to one or more control sequences that direct the production of a polypeptide of the invention. The construct or vector comprising the polynucleotide is introduced into a recombinant cell and the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described previously. The term "recombinant cell" includes any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication. The choice of cell will depend to a large extent on the gene encoding the polypeptide and its source. The recombinant cell may be any cell useful in the recombinant production of a polypeptide of the invention, e.g., a prokaryotic or eukaryotic cell.

The fusion protein is secreted and expressed in CHO cells; expression is preferentially in CHO/dhFr-cells. The fusion protein is in CHO/dhFr-cells are expressed in suspension culture. The eukaryotic plasmid vector can be specifically selected from PCHO1.0 plasmid, but is not limited to the vector, and can be any mammalian cell expression vector.

The beneficial technical effects produced by the invention are as follows: the invention improves the defects that the expressing product of the swine fever E2 protein in a mammalian cell has poor immune effect, and the expressing product of the swine fever E2 protein in a prokaryotic expression system can lead the swine to generate short antibody time and cause immune death by prokaryotic endotoxin residue after the swine is immunized. The invention also provides a construction method for co-expressing the swine fever E2 in the CHO cell strain, which utilizes circular VLPs to display E2, achieves the conversion of E2 VLPs, can improve the immunogenicity of E2 antigen, achieves mutual stimulation immunity, and improves the protective effects of swine fever and circular immunity.

Drawings

FIG. 1 shows the PCR identification results of bacterial liquid of PMD18-E2 and PMD 18-Cap.

FIG. 2 is a physical map of recombinant vector PCHO 1.0-E2-Cap.

FIG. 3 shows the electron microscope results of E2-Cap-VLPs.

FIG. 4 shows the result of detection of 20 selected piglet plague antigen antibodies.

FIG. 5 shows the results of measurement of antibody levels after 20 piglets had been immunized.

FIG. 6 shows the body temperature measurements and clinical observations of 20160608a vaccine batches after challenge.

FIG. 7 shows the body temperature measurements and clinical observations of 20160608b vaccine batches after challenge.

FIG. 8 shows the body temperature measurement and clinical observation results of 20160303c vaccine immunized pigs after challenge.

Fig. 9 shows the body temperature measurement results and clinical observation results of control pigs after challenge.

FIG. 10 shows the results of determination of onset of swine fever after immunization and challenge and protection with 3 batches of the subunit vaccine of swine fever E2 protein.

FIG. 11 is a chart of the clinical symptoms and anatomical changes recorded in pigs after challenge.

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 indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

CHO/dhFr-cells (cell Bank, national academy of sciences, catalogue number GNHa identifier: 4CSTR:19375.09.3101HAMGNHa 4).

The fusion gene was synthesized according to the preference of murine codons, and the PCHO1.0 expression vector was provided by Anhui general biology, Inc.

Enzymes and other biochemical reagents: the endonuclease and ligase are purchased from TaKaRa company, the plasmid extraction kit is purchased from Tiangen organisms, and the others are all made reagents.

Example 1 expression of CSFVE2-Spycatcher and Cap-SpyTag fusion proteins

1.1 obtaining of coding Gene

In order to improve the immunogenicity of wild E2, the protein sequence of hog cholera E2 is fused with a SpyCatcher fragment to obtain a fusion protein CSFVE2-SpyCatcher, and the amino acid sequence of the fusion protein is shown in SEQ ID No. 2; meanwhile, the Cap and the SpyTag are fused to obtain a fusion protein Cap-SpyTag, and the amino acid sequence of the fusion protein Cap-SpyTag is shown in SEQ ID No. 3. The encoding gene of the fusion protein is synthesized according to the preference of murine codons and is synthesized by Anhui general biology company, wherein the encoding gene of the fusion protein CSFVE2-Spycatcher is named as CSFVE2-Spycatcher gene, and the CSFVE2-Spycatcher gene is constructed to PMD18 to obtain a recombinant vector PMD 18-E2; the coding gene of the fusion protein Cap-SpyTag is named Cap-SpyTag gene, and the Cap-SpyTag gene is constructed to PMD18 to obtain a recombinant vector PMD 18-Cap.

1.2 construction of recombinant expression vectors

And (3) carrying out PCR amplification by using a recombinant vector PMD18-E2 as a template and a primer pair F1/R1 as PCR amplification primers to obtain a PCR product containing a CSFVE2-Spycatcher gene segment. Collecting a PCR product and an AVRII enzyme-digested eukaryotic expression vector PCHO1.0(-), carrying out recombinant connection by using a recombinant connection system, transforming DH5 alpha competent cells by using the recombinant connection product to obtain a positive recombinant bacterium, and identifying positive clone by using PCR by using a primer pair IF1/IR1 as primers, wherein the result shows that: the positive clones all contained the CSFVE2-SpyCatcher gene. The obtained recombinant expression vector containing the CSFVE2-SpyCatcher gene is named as PCHO 1.0-E2.

Wherein, the nucleotide sequence of the primer pair F1 (SEQ ID No. 4)/R1 (SEQ ID No. 5) is as follows:

F1：5'-GGTTCCGGGCCGCCTAGGGCCACCATGGGCAAGGTGCTGAGGGGCCAGATC-3'；

R1：5'-GTATAATATAGAGTATACAGTtcaGATGTGGGCGTCGCCCTTGGTGG-3'。

the recombinant connector system is 1 mu L of AVRII enzyme digestion PCHO1.0(-), 1 mu L of PCR recovery fragment of PMD18-E2 and 2 mu L of 2x recombinant ligase, and the reaction condition is 50 ℃ for 15 min.

The nucleotide sequence of the primer pair IF1 (SEQ ID No. 6)/IF 2 (SEQ ID No. 7) is as follows:

IF1：5'-GCATGCTGGTTCGTTTGGACTGA-3'；

IR1：5'-AAACGAATTCGAGCTCGGGAGTAGCGGATGCCCCGG-3'。

and (3) performing PCR amplification by using PMD18-Cap as a template and a primer pair F2/R2 as PCR amplification primers to obtain a PCR product containing a Cap-SpyTag gene fragment. And (3) collecting the PCR product and an ECORV enzyme-digested eukaryotic expression vector PCHO1.0-E2, carrying out recombinant connection by using a recombinant connection system, and transforming DH5 alpha competent cells by using the recombinant connection product to obtain the positive recombinant bacteria.

The primer pairs IF1/IR1 and IF2/IR2 are respectively used as primers, positive clone results are identified by PCR (polymerase chain reaction) as shown in figure 1, M in figure 1 represents Marker, the sizes of the bands from top to bottom are respectively 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp, the right lane 1-7 of the Marker is the PCR identification result of the positive clone IF2/IR2 on Cap-SpyTag gene, the left lane 1-7 of the Marker is the PCR identification result of the positive clone IF1/IR1 on CSFVE2-SpyCatcher gene, and the results show that: 1. the positive clones of the bacteria in

lanes

2, 3, 4 and 6 all contain Cap-SpyTag and CSFVE2-Spycatcher genes.

Wherein the nucleotide sequence of the primer pair F2 (SEQ ID No. 8)/R2 (SEQ ID No. 9) is as follows:

F2：5'-TCTCGAGCTCAAGCTTGATGCCACCATGGAGACCGACACCCTGCTGCTGTGGG-3'；

R2：5'-AAGCTTAATTAACGCCGATTCAGGGGTTCAGGGGGGGGTCCTTCA-3'。

the recombinant connector system is 1 mu L of ECORV enzyme digestion PCHO1.0-E2, 1 mu L of PCR recovery fragment of PMD18-E2 and 2uL of 2x recombinant ligase, and the reaction condition is 15min at 50 ℃.

The nucleotide sequence of the primer pair IF2 (SEQ ID No. 10)/IR 2 (SEQ ID No. 11) is as follows:

IF2：5'-CAGGTACTAGCGCTACCGGAC-3'；

IR2：5'-AAGCTTAATTAACGCCGATTCAGGGGTTCAGGGGGGGGTCCTTC-3'。

extracting plasmid with Tiangen non-endotoxin kit and recovering to obtain recombinant expression vector. The obtained recombinant expression vector containing the CSFVE2-Spycatcher gene and the Cap-SpyTag gene is named as PCHO 1.0-E2-Cap.

Sequencing results show that the nucleotide sequence of the recombinant expression vector PCHO1.0-E2-Cap is shown in SEQ ID No. 1. The physical map is shown in figure 2.

Wherein, the nucleotide sequence of the CSFVE2-SpyCatcher gene is shown as the 5776-7266 th site of SEQ ID No.1, the 5776-6861 th site of SEQ ID No.1 is a CSFVE2 coding gene, the 6895-6912 th site of SEQ ID No.1 is a 6XHis coding gene, and the 6925-7263 th site of SEQ ID No.1 is a SpyCatcher coding gene. The protein coded by the CSFVE2-Spycatcher gene is named as CSFVE2-Spycatcher, and the amino acid sequence of the protein is shown as SEQ ID No. 2.

The nucleotide sequence of the Cap-SpyTag gene is shown in the 9337-10029 of SEQ ID No.1, the 9337-9585 site of the SEQ ID No.1 is the N-terminal gene of the Cap coding gene, and the 9535-9573 site of the SEQ ID No.1 is the SpyTag coding gene. The No.1 position 9586-9602 is 6XHis coding gene, the No.1 position 9604-10029 is C end gene of Cap coding gene, the protein coded by Cap-SpyTag gene is named Cap-SpyTag, and the amino acid sequence is shown in SEQ ID No. 3.

Example 2 construction of Stable transgenic cell lines and protein expression

2.1 construction of stably transfected cell lines

The recombinant expression vector PCHO1.0-E2-Cap is cut by pUul enzyme and then is recovered by a glue recovery kit, and the recovered product is ready to transfect mammalian cells CHO/dhFr-，CHO/dhFr-was purchased from the cell bank of the culture Collection of type culture of Chinese academy of sciences (website address https:// www.cellbank.org.cn/index. php), catalog number GNHa4 with identification number CSTR:19375.09.3101HAMGNHa 4.

Step 1: the day before transfection, 6-well culture plates were seeded with appropriate cell density; when transfection is carried out, the cells are fused by 90-95%;

step 2: preparing a solution 1 and a solution 2, mixing the solution 1 and the solution 2, and standing at room temperature for 20 min;

solution 1: 240 μ L serum-free medium (Gibco, cat # 12200036) +10 μ L Lipofectamine 2000 Transfection Reagent (Invitrogen, cat # 11668019)/well, incubate for 5min (250 μ L total volume per well);

solution 2: 225 μ L serum free medium (Gibco, cat # 12200036) +25 μ L (4 μ g) plasmid per well (250 μ L total volume per well);

and step 3: washing the cells in the 6-hole plate in the step 1 with a serum-free culture medium twice, and adding 2mL of the serum-free culture medium;

and 4, step 4: dropping the mixed solution of the solution 1 and the solution 2 into the holes, shaking the culture plate, and gently mixing the solution and the culture plate; 5% CO at 37 deg.C₂Preserving heat for 6 hours;

and 5: serum-containing whole medium (IMDM Medium 90% Gibco, cat # 12200036, fetal bovine serum 10%) was changed at 37 ℃ with 5% CO₂The transfection level was measured in 48-72 h.

Step 6: stable transfection was performed. Dropping the mixed solution of the solution 1 and the solution 2 into the holes, shaking the culture plate, and gently mixing the solution and the culture plate; 5% CO at 37 deg.C₂Preserving heat for 6 hours; 24h after changing to serum-containing whole medium (IMDM medium 90% Gibco, cat # 12200036, fetal bovine serum 10%), the culture was repeated at 1: 10 to a new plate containing whole serum medium (IMDM medium 90% Gibco, cat # 12200036, fetal bovine serum 10%), antibiotics (10 ug/mL puromycin) were added for selection to obtain stably transfected monoclonal cells.

2.2 cloning and screening

After the stably transfected monoclonal cells grow for two days and adhere to the wall, the cells are cultured under pressure by changing a liquid (IMDM culture medium (Gibco, cat # 12200036) 90% +10% FBS +10ug/mL puromycin) until a large number of cells die, and then the liquid is directly changed without pressure until the clones grow out. Cells were grown in single colonies to the appropriate size and ready for selection. All clones were picked into 96-well plates. Culturing in incubator until the clone grows to cover 80% of the bottom of the hole, taking supernatant SDS-PAGE to screen cell strain with high expression, and remaining to continue screening, wherein the positive clone is named as CHO/PCHO1.0-E2-Cap, and the expressed fusion protein is named as protein E2-Cap.

2.3 cell suspension acclimatization

CHO/PCHO1.0-E2-Cap cells are transferred to a 6-well plate of a serum-free medium CD DG44 (medium cargo number: 12610010) for culture, continuous culture and passage are carried out for 2 months, and a cell strain which is finally suitable for suspension culture and has high yield is selected as an engineering strain and is named as CHO/PCHO 1.0-E2-Cap-96. The protein expression is detected by a Western Blot method (a specific antibody is an anti-HIS antibody).

2.4 fermentation fed-batch culture of Stable cell lines of 10L

The fermentation tank adopts a 10L reaction system of Shanghai Bailun mammalian fermentation tank, the liquid loading is 5L, the culture medium is CD DG44 (cat No.: 12610010), the rotating speed is 80rpm, the pH is controlled to be 7.2, and the culture temperature is 37 ℃. 1L of seed solution of CHO/PCHO1.0-E2-Cap-96 with a density of 2.0X 10 was prepared⁶Adding into a fermenter, counting every day, observing cell state, and allowing cell density to reach 5.0 × 10 at 5 days of fermentation⁶Each/mL of the cells was supplemented with 400mL of a CHO CD efficient feed ^ A nutrient additive (cat # A1023401) per day while the temperature was reduced to 33 ℃ and the cells were cultured continuously for 6 days under the conditions that the protein concentration reached the maximum, the supplement of the feed medium was stopped, and the cells were cultured at 30 ℃ for 1 day and then the fermentation was stopped. Using O in the fermentation process₂Maintaining the dissolved oxygen level of the fermentation tank at not less than 10%, CO₂And NaHCO₃The pH was controlled at 7.2 and the cell fermentation changes are shown in Table 1.

TABLE 1 fermentation parameters for the production of protein E2-Cap by fermentation of CHO/PCHO1.0-E2-Cap-96 cells

Days of fermentation	Fermentation Density (one/mL)	Mortality rate	Volume of feed supplement
				Day.1	0.5×10⁶	1%
Day.3	1.6×10⁶	1%
				Day.5	4.5×10⁶	2%	400 ml/day
			400 ml/day
				Day.7	3.12×10⁷	4%	400 ml/day
			400 ml/day
				Day.9	6.23×10⁷	6%	400 ml/day
			400 ml/day
				Day.11	3.14×10⁸	11.3%	400 ml/day
			Stopping feeding
				Day.13	5.26×10⁸	27%	Stop pot

2.5 purification of recombinant proteins

The fermentation broth was collected, centrifuged at 5000rpm for 10min to obtain a supernatant, and filtered through a 0.22 μm membrane. In the first step, buffer A is selected: the solvent is water, and the solute is: 20mM Tris, 0.2M NaCl, 10mM imidazole; pH7.5, equilibrated purification column (NI-TED purose 6FF, from thousand pure organisms, cat # A41002-06100 mL), loaded with broth, equilibrated by buffer A elution with buffer B of the following composition: the solvent is water, and the solute is: 20mM Tris, 0.15M NaCl, 250MM imidazole. The eluate was transferred to a PCV2 virus-like particle assembly system (6% PEG3350, 2.5% isopropanol, 0.3M ammonium citrate, 10% glycerol overnight at 4 ℃). The electrophoresis purity is achieved through SDS-PAGE detection. The whole purification process steps are well connected, and the eluent of the previous step of chromatography can be used as the next treatment solution only by slight adjustment. The intermediate treatment steps are reduced, the industrial operation is facilitated, and the pollution caused by the intermediate operation is reduced. The sample is diluted by 100 times and then is subjected to electron microscope detection, and the electron microscope result is shown in figure 3. The results show that the size of E2-Cap-VLPs (virus-like particles, VLPs) is between 38 and 45nm, and is larger than that of prokaryotic expression circular ring Cap2-VLPs particles (the size is about 20 nm).

2.6 preparation of Swine fever E2-VLPs vaccine

One or more components such as a preservative, a protein protective solution, a stabilizer and the like are mixed with the E2-Cap-VLPs, the pH value of the mixture is adjusted to a physiological value, namely pH 7.0-7.5, and an adjuvant is added to form the swine fever E2-VLPs vaccine. The concentration of the effective components of the protein E2-Cap-VLPs prepared into the vaccine is not lower than 100 mu g/mL, and each dose is 50 mu g.

Preparation of Water-in-oil-in-Water (W/O/W) vaccine: taking Monanide from Spiranski ^TM1 part of ISA201 VG adjuvant, 1 part of qualified antigen, and the weight ratio of the antigen: adjuvant 50: 50 (vv) are mixed in an emulsifying cylinder, stirred and emulsified for 30 minutes at low speed, and the subunit vaccine of the water-in-oil-in-water type of the protein of the swine fever E2-VLPs is prepared.

The specific formula is as follows:

20160608a vaccine preparation: the batch protein was diluted to a final concentration of 100. mu.g/mL with PBS, 250mL was taken and Monanide was added^TMMixing 250ml of ISA201 VG adjuvant in an emulsifying cylinder, stirring at low speed for emulsifying for 30 min, and controlling temperature at 4 deg.C to obtain subunit vaccine 20160303a of water-in-oil-in-water type of protein of swine fever E2-VLPs

20160608b vaccine preparation: the batch protein was diluted to a final concentration of 100. mu.g/mL with PBS and 500mL was taken and Monanide was added^TM500ml of ISA201 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 swine fever E2-VLPs protein water-in-oil-in-water dosage form subunit vaccine 20160303 b.

20160303c vaccine preparation: for batch proteinPBS was diluted to a final concentration of 100. mu.g/mL, and 1L of Monanide was added^TMMISA201 VG adjuvant 1L is mixed in an emulsification cylinder, stirred and emulsified at low speed for 30 minutes, and the temperature is controlled to be 4 ℃, so that the swine fever E2-VLPs protein water-in-oil-in-water type subunit vaccine 20160303c is prepared.

Example 3 functional verification of classical swine fever E2-VLPs vaccine

3.1 Experimental materials

(1) The 4-6-week-old piglets of the test animals come from non-immune healthy swinery, are detected by a hog cholera antigen and antibody ELISA kit, are negative in antigen and antibody detection, and are tested under strict isolation conditions.

(2) Vaccine: E2-VLPs vaccine prepared at 2.6 in example 2,

lot numbers

20160303c, 20160608a and 20160608 b.

(3) Test strains: the hog cholera shimen system blood virus AV1411 strain (F115 generation, production date: 2011 month 6) is provided by animal experiment company Jinyubao Ling biopharmaceutical GmbH, and the strain is purchased from strain collection center of China veterinary medicine inspection institute by Jinyubao Ling biopharmaceutical GmbH.

(4) The hog cholera antibody ELISA detection kit is purchased from IDEXX company, and has the batch number of: REF: 99-43220, LOT SN: G131) (ii) a And (3) judging standard: the blocking rate is more than or equal to 40 percent and the test result is positive; the blocking rate is equal to or less than 30 percent, and the test result is negative; the blocking rate is judged to be suspicious between 30 percent and 40 percent.

(5) The hog cholera antigen ELISA detection kit is purchased from IDEXX company, and has the batch number of: REF: 99-40939, LOT SN: g351; and (3) judging standard: the S-N value of the detected sample is equal to or less than 0.100, and the sample is judged to be hog cholera virus antigen negative; the S-N value of the detected sample is larger than 0.100 and smaller than or equal to 0.300, and the detected sample is judged to be suspicious; the S-N value of the detected sample is more than 0.300, and the sample is judged to be positive by the hog cholera virus antigen.

(6) The laboratory apparatus is a Bio-Rad Imark type enzyme linked immunoassay analyzer.

3.2 test methods

3.2.1 screening and grouping of test animals

20 piglets (Du-Chang-Dai-Sanyuan hybrid, purchased from local farmers) with 4-6 weeks old are selected for detection, divided into 4 groups with 5 heads in each group, wherein the 1 st group and the 3 rd group are immunized with 3 batches of hog cholera E2 antigen (CSFVE2) fused with Spycatcher polypeptide and Cap fused with SpyTag polypeptide respectively, and the 4 th group is used as a control for immunization.

3.2.2 immunization

The pigs of groups 1-3 are inoculated with swine fever E2-VLPs vaccine of 20160303c batch, 20160608a batch and 20160608b batch through neck muscle respectively, each 1mL, the second immunization is carried out in the same dose and the same way after 21 days, and the group 4 is a control group; pigs of 4 experimental groups were housed in columns under the same conditions.

3.2.3 measurement of antibody titer

All 20 test pigs were subjected to vena cava blood collection on the day of first-immunization, 14 days, 21 days after first-immunization, 14 days after second-immunization, and 20 days before first-immunization, and serum was separated and tested for swine fever antibody blocking rate by using an IDEXX kit.

3.2.4 counteracting toxic pathogen and observing

The immunized group of 15 pigs and 5 negative control pigs were challenged with hog cholera-shimen system blood virus AV1411 strain (F115 generation) in a dose of 2mL per head (containing 1mL of blood virus, 106.0 MLD) 21 days after the second immunization.

Observation after toxin attack: the observation is carried out for 16 days after the toxin is attacked. All pigs were subjected to temperature measurements 1 time each day in the morning and afternoon, and the clinical symptoms of the test pigs were observed and recorded.

3.3, results of the experiment

3.1 detection of antigen and antibody of preimmune piglet

The detection result of the selected 20 piglet swine fever antigen antibodies is shown in figure 4, wherein negative is represented by 'minus' in figure 4, and the result shows that the selected 20 piglet swine fever antigen antibodies are all negative.

3.2 post-immunization piglet antibody levels

The measurement result of the antibody level after immunization is shown in figure 5, the antibody of part of pigs turns positive 21 days after the first immunization, and the antibody blocking rate is more than 70% 14 days after the second immunization; the blocking rate of 12 pigs in 20 days after the secondary immunization is more than 80 percent; negative control porcine antibodies were all negative.

3.3 post-challenge observations

Body temperature measurement: the pigs were observed day by day for 16 days after challenge. The temperature was measured 2 times a day, 1 time each in the morning and afternoon, starting two days before the challenge. From the temperature measurement result, the body temperature of the immunized pig after toxin attack is basically below 40 ℃ except that the temperature of individual pigs reaches 40 ℃ respectively; after the control pigs are challenged, the body temperature of 5 pigs is raised by more than 1.5 ℃ compared with the basal body temperature in different time periods.

And (3) clinical observation: all pigs in the immunity group after the challenge had good appetite and no adverse reaction.

Control pigs developed clinical symptoms at different times: at first, the spirit is low, aversion to cold and cold, then the rising and lying are difficult, and the gait is unstable; after toxin attack, 5 pigs all have inappetence on day 2, and then have anorexia and finally waste food; 5, diarrhea of the control pigs before death; conjunctivitis; control pigs were all ill and died.

The results of body temperature measurements and clinical observations of immunized pigs immunized with 20160608a batches of vaccines are shown in FIG. 6. The results of body temperature measurements and clinical observations of immunized pigs immunized with 20160608b vaccine batches are shown in FIG. 7. 20160303 the body temperature measurement results and clinical observation results after challenge of vaccine batches are shown in FIG. 8. The body temperature measurement results and clinical observation results of the control vaccine immunized pigs after challenge are shown in fig. 9, the growth state of the pigs is normal, the average temperature represents the basal body temperature, and the average temperature of the first 5 time points 3 days before challenge; "-" represents spirit, appetite, no abnormalities in stool, no conjunctivitis; in fig. 9, the average temperature represents the basal body temperature, the average temperature at the first 5 time points 3 days before challenge; "-" represents spirit, appetite, no abnormalities in stool, no conjunctivitis; "+" indicates cachexia or gait instability, loss of appetite or eating, constipation or diarrhea, conjunctivitis. Disease occurrence judgment standard: 1) the body temperature after the toxin attack is increased by 1.5 ℃ compared with the basal body temperature and kept for 48 hours; 2) listlessness, intolerance of cold, difficulty in rising and lying, and unstable gait; 3) loss of appetite, anorexia and even eating waste; 4) constipation, diarrhea, or alternans; 5) conjunctivitis. Death or compliance with any of the 3 clinical symptoms above is considered to be onset.

The results of disease determination and virus attack protection after 3 batches of the swine fever E2 protein subunit vaccine are shown in figure 10, wherein in figure 10, "+" represents listlessness or unstable gait, inappetence or waste food, constipation or diarrhea, conjunctivitis, and body temperature rise over 1.5 ℃, and stays for 48 hours; "-" indicates mental, appetite, absence of abnormalities in stool, absence of ocular conjunctivitis, no increase in body temperature above 1.5 deg.C, and no remaining for 48 hours. Disease occurrence judgment standard: 1) the body temperature after the toxin attack is increased by 1.5 ℃ compared with the basal body temperature and kept for 48 hours; 2) listlessness, intolerance of cold, difficulty in rising and lying, and unstable gait; 3) loss of appetite, anorexia and even eating waste; 4) constipation, diarrhea, or alternans; 5) conjunctivitis. Death or compliance with any of the 3 clinical symptoms above is considered to be onset.

Histological and pathological anatomy dissection results after toxin attack: performing dissection examination on the control dead pigs and healthy live pigs at the experimental observation period of the immune pigs after the challenge, collecting tonsil, inguinal lymph node, submaxillary lymph node, mesenteric lymph node, spleen, kidney, liver and ileocaecal valve tissue organs, and observing and recording pathological changes; the records of clinical symptoms and anatomical changes of the pigs after the challenge are shown in figure 11, and the "-" in figure 11 indicates that no abnormality exists; "+" indicates a lesion, i.e.: swelling lymph nodes, ulcer or suppuration of tonsil, spleen infarction, bleeding point of kidney and liver, bleeding point of ileocecal valve or blood stasis, etc.

The results of the immune efficacy test show that: carrying out secondary immunization on 4-6 weeks old piglets by 3 batches of swine fever E2-VLPs vaccine, carrying out swine fever phylum virulent virus attack 21 days after secondary immunization, and leading 5 control pigs to be completely attacked and die; all 15 pigs immunized by 3 batches of vaccines are healthy and alive, and the protection rate is 100%. The swine fever E2-VLPs vaccine has good immune protection effect on the attack of the virulent swine fever phylum virus.

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 Zhonghai Biotechnology Ltd

<120> recombinant protein containing Cap of hog cholera E2 and circovirus, preparation method and application thereof

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 15184

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca gattggctat 60

tggccattgc atacgttgta tccatatcat aatatgtaca attcgccctt catttaattc 120

taccgggtag gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg 180

ctgggcactt ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt 240

aggcgccaac cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta 300

gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta 360

gcacgtctca ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc 420

ctttggggca gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga 480

aggggtgggt ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg 540

tcctccggag gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc 600

tcttcctcat ctccgggcct ttctcgagct caagcttggg gggggggaca gctcagggct 660

gcgatttcgc gccaaacttg acggcaatcc tagcgtgaag gctggtagga ttttatcccc 720

gctgccatca tggttcgacc attgaactgc atcgtcgccg tgtcccaaaa tatggggatt 780

ggcaagaacg gagacctacc ctggcctccg ctcaggaacg agttcaagta cttccaaaga 840

atgaccacaa cctcttcagt ggaaggtaaa cagaatctgg tgattatggg taggaaaacc 900

tggttctcca ttcctgagaa gaatcgacct ttaaaggaca gaattaatat agttctcagt 960

agagaactca aagaaccacc acgaggagct cattttcttg ccaaaagttt ggatgatgcc 1020

ttaagactta ttgaacaacc ggaattggca agtaaagtag acatggtttg gatagtcgga 1080

ggcagttctg tttaccagga agccatgaat caaccaggcc acctcagact ctttgtgaca 1140

aggatcatgc aggaatttga aagtgacacg tttttcccag aaattgattt ggggaaatat 1200

aaacttctcc cagaataccc aggcgtcctc tctgaggtcc aggaggaaaa aggcatcaag 1260

tataagtttg aagtctacga gaagaaagac taacaggaag atgctttcaa gttctctgct 1320

cccctcctaa agctatgcat ttttataaga ccatgggact tttgctggct ttagatcgta 1380

caagtaaagc ggccgcgact ctagatcata atcagccata ccacatttgt agaggtttta 1440

cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt 1500

gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca 1560

aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc 1620

aatgtatctt aaggcgtaaa ttgtaagcgt taataaatga agttcctata ctatttgaag 1680

aataggaact tcggaatagg aacttctcgg ccgatggccg cgaattgcag cggagcttct 1740

ctgcagaggt gaattttttt attggtcccc accctaaaag acccagcgta tatttattat 1800

tgtacttatt acagctgacc tggttgtggc tgcttgtggt actccaggat tctgctcttc 1860

atggatgtgg ggatgacagt gggagagatc cttccctgcc aacacctggc tccatccctg 1920

cagttatggg gttgaaaatt cccctttttc ccttaagctc tccccctggt tctttaaatt 1980

acatgatgtt agaatttttc aataagcaac ccatgcactt gagaagaaat tcaacccctc 2040

ttttattaag ttccgggata cctgtgaaca gccccccctc cttttttttt tttttttttt 2100

tttgagacag agtcttactc tcagtcgcac aggctggagt gcatgcagtg ccgtgatctc 2160

ggctcactgc aacctctgcc tctcgggttc aagcggttct cctgcctcag cctccaaagt 2220

agctgggatt tcaggcgttc actaccacgc cgggctaatt tttttatttt tagtaaagac 2280

ggggtttctc catattgggt aggctggtct cgaactcctg acttcaggtg atttgcctgc 2340

ctcggcctcc caaagtgctg ggattacagg cgtgacccac cgcaagcagc ctaacccctc 2400

tttaattttt aaaattttta aaaatttttt tgagacggaa tctcgctctg tcgccaggct 2460

ggagagcagt ggcgcgatct cggctaactg caacctccgc ctctcgcctg acacctcttt 2520

taatgaaaac ctttccatta catctctgaa atgctcccac tttgcacgga aaaaacggct 2580

gcgaagcccc caataggaaa cgcaacacgt agcaggtgct cagtgcattt aaaaattttg 2640

aagaatttta ttattaataa tgcgaaactg actacccctc tgctaaaaac tcatgcccca 2700

aatggcgcat gcgcttgact tgcaacttaa ccaaacgcgt cacaggtaca ggtgtaggtg 2760

gttagggacc ccgggcgctc ccatctgctc cctgccgcgc cctctgcctt tcagagcctc 2820

aatcctttcg gaagatactt ttcagttttc cactgggtta aatggtgata ggaagagcaa 2880

tcatccgtta aatgaaaccc aaggttgaga cagcgctttg taaagcaaag tgcccggagc 2940

attcgtgagg gggaggagtg tacaggttta ttttagagtc caggctggaa tttgcattgc 3000

tttttgtgga tccacacctc cccctctccc agcccaggaa aagcactgca gtggctctcc 3060

tgtttcacaa agtgagttta aggaaagatg taccgcaggg ggttcatttg cactgtgaga 3120

acatcattca tgcaagaaat gctgagtacc tactatgtgc taaacagcgg gttgggcttt 3180

tcataaggga tagagttaca caattgaatg ccaattcatg agatatgaaa tacattcaac 3240

tgtttcttag ggtcagatta ggacacaaat gctaaacagt cttcctgcgc ctccccctgg 3300

agaggaaatg aaatagaaaa tgaaattgtg ccagtaaacc gcttagccgg gtgcctggga 3360

catggcaaac cagtcctgaa ataaataact gttttattaa tagcaatctt agctaattaa 3420

aatagatagc gtttattgag cgttgggtat caggcacggt cctaattctt ttagatgtct 3480

ttagttcgtt tcactctccc ccaaacaata gggtggtatt gatcacctcc gagcaggtga 3540

aattgaggca cagagaaatc ctagtagctg gtagaagaac acgcagtgtg gtcaagctag 3600

caaggtgttt ggtccactgc tatatctaca aaacccctaa caatgcctgg tgtatagatg 3660

ctcagtatgc atttgtggga tcagtgattc cgatgcctgc ttcttataaa gtttttattt 3720

agaaataatt acaggtaagg agttgcaaaa acagtatagt gggatcgagt gtcctttttc 3780

cctcggcttc tcccaggggt atcgtcttac gtaacaatgt ccaaacaggg aaattgactt 3840

gggtataatc cacagactct attcaccttg tagattggtt ttaatagaag tatatcctac 3900

ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 3960

catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 4020

cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 4080

ctccgcccca ttgacgcaaa tgggcggtag ggatactgga caacttgtaa gctaatatcg 4140

ttgctatggt tctcgttctc agctaaaacg gcgctcttta ctttgtgcac ctgaacactg 4200

cacaccgagg gcgaccaccg cccccgagat gcccagcttc tattctagag cgccgcgccg 4260

gcgccgaatg ggttaacggg cggggggaca cgcctccgtg cgcttgcgcg gcgtcccttc 4320

gccccgcctt cgcagcgcag tcacatgacc cgcccaaccg gcgtccgcct ataaaaagct 4380

gagtgttgac gtcagcgttc tcttccgccg tcgtcgccgc catcctcggc gcgactcgct 4440

tctttcggtt ctacctggga gaatccaccg ccatccgcca acgcgtcagg tgagtaaccc 4500

tgcctcgctg gtgccccgga gcggcggcgg ggcctccgcg gggcctacgc tgcctagcct 4560

gggacgctgg ggccccccgt gccaggagga gacgtagcgg cggcggggcc ggaggacccg 4620

gggctgggga agcggccgcc gccatgtctg tgcgccgcgc tgttcaccga gccctttctc 4680

cgtttccgag ggcgccataa ccttgcccag acctggttgg agctgggttg ttagcgggga 4740

tgggggtggg gaatgatgat gtggcaggcg tagtaatggc gggcaccgcg gtggaagcgg 4800

ggagacaggg aggcgcctta tgtaacccgc gggccgcgag tttgagatcg attttctgcc 4860

gggggactag gggcggcagg gaaatggcag aacgagcaaa gcgacacctg aaaggctccc 4920

cttttccttc caaatacctt ctcctgatgc tgttaatcgt cgaccttagg cacttgtcct 4980

tcctatgact cccagatgta caaagactct tattgagaca cgaggtgtag gcttgtggca 5040

gttagggccg ttccggctcc ccgttatttc ctggggtggg tggccttgtc tgatcccgtt 5100

tgccaagggg tgaccttgca ttttatgatg aagcttcttg ctcggggaag tttgggtggc 5160

ccggtgacaa cgtggagggg gctttaggag aggattcatc ccttacgtgt ttggccccaa 5220

atgagacttt agtatttgta cctggtatca aggaaatctg ttgacactca gttttattcc 5280

tgagcacttt tatttctggg ttgtcaatca tgaatgacac ctattaacgg tggttccttg 5340

gagattgcgg ggtggctctt aaggggtgtt aggctggcaa gattcggtgg gcttggggca 5400

ccccagcacc aaccccctcc tggccttggg catgctggtt cgtttggact gaacgctccg 5460

tgccatgctg tgttcctgga aatcacgagc tggtctgagc ctccttgtct tgcccaggtg 5520

aactagatcc cctacctgcg cctctcttcc ccagacctgc gcgctactgc ggctcgggcg 5580

gtcgctcgcc tggctctgct ccatttgact gtctgtgtgc agtcgcagaa cttcgaagag 5640

ggttttgcgc tccatccgtg gcgtttcgct tttgttcggt tttgttgttt atttcatttt 5700

ttttttccgg agagaggcga ggcggtggtc cacacccgcc cgaggaggaa cggttccggg 5760

ccgcctaggg ccaccatggg caaggtgctg aggggccaga tcgtgcaggg cgtggtgtgg 5820

ctgctgctgg tgaccggcgc ccagggcagg ctggcctgca aggaggacta caggtacgcc 5880

atcagcagca ccgacgagat cggcctgctg ggcgccggcg gcctgaccac cacctggaag 5940

gagtacaacc acgacctgca gctgaacgac ggcaccgtga aggccagctg cgtggccggc 6000

agcttcaagg tgaccgccct gaacgtggtg agcaggaggt acctggccag cctgcacaag 6060

aaggccctgc ccaccagcgt gaccttcgag ctgctgttcg acggcaccaa ccccagcacc 6120

gaggagatgg gcgacgactt caggagcggc ctgtgcccct tcgacaccag ccccgtggtg 6180

aagggcaagt acaacaccac cctgctgaac ggcagcgcct tctacctggt gtgccccatc 6240

ggctggaccg gcgtgatcga gtgcaccgcc gtgagcccca ccaccctgag gaccgaggtg 6300

gtgaagacct tcaggaggga caagcccttc ccccacagga tggactgcgt gaccaccacc 6360

gtggagaacg aggacctgtt ctactgcaag ctgggcggca actggacctg cgtgaagggc 6420

gagcccgtgg tgtacaccgg cggcgtggtg aagcagtgca ggtggtgcgg cttcgacttc 6480

gacggccccg acggcctgcc ccactacccc atcggcaagt gcatcctggc caacgagacc 6540

ggctacagga tcgtggacag caccgactgc aacagggacg gcgtggtgat cagcaccgag 6600

ggcagccacg agtgcctgat cggcaacacc accgtgaagg tgcacgccag cgacgagagg 6660

ctgggcccca tgccctgcag gcccaaggag atcgtgagca gcgccggccc cgtgatgaag 6720

accagctgca ccttcaacta caccaagacc ctgaagaaca ggtactacga gcccagggac 6780

agctacttcc agcagtacat gctgaagggc gagtaccagt actggttcga cctggacgcc 6840

accgacaggc acagcgacgg ccccgggggc ggcggcagcg gcggcggcgg cagccaccac 6900

caccaccacc acggcggcgg cagcgtggac accctgagcg gcctgagcag cgagcagggc 6960

cagagcggcg acatgaccat cgaggaggac agcgccaccc acatcaagtt cagcaagcgc 7020

gacgaggacg gcaaggagct ggccggcgcc accatggagc tgcgcgacag cagcggcaag 7080

accatcagca cctggatcag cgacggccag gtgaaggact tctacctgta ccccggcaag 7140

tacaccttcg tggagaccgc cgcccccgac ggctacgagg tggccaccgc catcaccttc 7200

accgtgaacg agcagggcca ggtgaccgtg aacggcaagg ccaccaaggg cgacgcccac 7260

atctgaactg tatactctat attatactct atgttatact ctgtaatcct actcaataaa 7320

cgtgtcacgc ctgtgaaacc gtactaagtc tcccgtgtct tcttatcacc atcaggtgac 7380

atcctcgccc aggctgtcaa tcatgccggt atcgattcca gtagcaccgg ccccacgctg 7440

acaacccact cttgcagcgt tagcagcgcc cctcttaaca agccgacccc caccagcgtc 7500

gcggttacta acactcctct ccccggggca tccgctactc ccgagctcga attcgtttat 7560

cggccaccga ggccaagatc tggccgccat atactgagtc attagggact ttccaatggg 7620

ttttgcccag tacataaggt caataggggt gaatcaacag gaaagtccca ttggagccaa 7680

gtacactgag tcaataggga ctttccattg ggttttgccc agtacaaaag gtcaataggg 7740

ggtgagtcaa tgggtttttc ccattattgg cacgtacata aggtcaatag gggtgagtca 7800

ttgggttttt ccagccaatt tataaaacgc catgtacttt cccaccattg acgtcaatgg 7860

gctattgaaa ctaatgcaac gtgaccttta aacggtactt tcccatagct gattaatggg 7920

aaagtaccgt tctcgagcca atacacgtca atgggaagtg aaagggcagc caaaacgtaa 7980

caccgccccg gttttcccct ggaaattcca tattggcacg cattctattg gctgagctgc 8040

gttctacgtg ggtataagag gcgcgaccag cgtcggtagg gaccggtttg ccgccagaac 8100

acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg 8160

cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg agcttcgggt 8220

tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg 8280

agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc 8340

ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac 8400

gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc 8460

ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag 8520

gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag atggccggcc 8580

tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc 8640

ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag 8700

ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa 8760

aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc 8820

caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg 8880

gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg 8940

gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct 9000

caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt gaggaatttc 9060

aggtactagc gctaccggac tcagatcccc tacctgcgcc tctcttcccc agacctgcgc 9120

gctactgcgg ctcgggcggt cgctcgcctg gctctgctcc atttgactgt ctgtgtgcag 9180

tcgcagaact tcgaagaggg ttttgcgctc catccgtggc gtttcgcttt tgttcggttt 9240

tgttgtttat ttcatttttt ttttccggag agaggcgagg cggtggtcca cacccgcccg 9300

aggaggaagg atctcgagct caagcttgat gccaccatgg agaccgacac cctgctgctg 9360

tgggtgctgc tgctgtgggt gcccggcagc accggcgacg tgcacccccg ccaccactac 9420

cgcagccgcc gcaagaacgg catcttcaac acccgcctga gccgcaccat cggctacacc 9480

gtgaagaaga ccaccgtgcg cacccccagc tggaacgtgg acatgatgcg cttcgcccac 9540

atcgtgatgg tggacgccta caagcccacc aagggggggg ggagccatca tcatcatcat 9600

catttcgagt actaccgcat ccgcaaggtg aaggtggagt tctggccctg cagccccatc 9660

acccagggcg accgcggcgt gggcagcacc gccgtgatcc tggacgacaa cttcgtgacc 9720

aaggccaacg ccctgaccta cgacccctac gtgaactaca gcagccgcca caccatcacc 9780

cagcccttca gctaccacag ccgctacttc acccccaagc ccgtgctgga ccgcaccatc 9840

gactacttcc agcccaacaa caagcgcaac cagctgtggc tgcgcctgca gaccaccggc 9900

aacgtggacc acgtgggcct gggcaccgcc ttcgagaaca gcatctacga ccaggactac 9960

aacatccgca tcaccatgta cgtgcagttc cgcgagttca acctgaagga cccccccctg 10020

aacccctgaa tcggcgttaa ttaagcttcg atccagacat gataagatac attgatgagt 10080

ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg 10140

ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca 10200

ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc 10260

tctacaaatg tggtatggct gattatgatc cggctgcctc gcgcgtttcg gtgatgacgg 10320

tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc 10380

cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc 10440

catgacccag tcacgtagcg atagcggagt gtattcgagc tcggacattg attattgact 10500

agactggcca ccgaggccgt cgactggggc tcgagatcca ctagttctag aatggtgggg 10560

ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc tgggcgtggt 10620

tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc 10680

gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc tgctccgccc 10740

ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca 10800

cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc gcgccgaccg 10860

cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag cggccgggaa 10920

ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg 10980

tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct cgttctcgag 11040

ttgagacgac cttccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac 11100

gtcccccggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac 11160

accgtcgacc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg 11220

cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc 11280

tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg 11340

gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg 11400

caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag 11460

ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg 11520

gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc 11580

ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc 11640

aagcccggtg cctgacgccc gccccacgac ccgcagcgcc cgaccgaaag gagcgcacga 11700

ccccatggct ccgaccgaag ccacccgggg cggccccgcc gaccccgcac ccgcccccga 11760

ggcccaccga ctctagagga tcataatcag ccataccaca tttgtagagg ttttacttgc 11820

tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt 11880

tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt 11940

cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt 12000

atcttatcat gtctggatcg gtaccggtcg acgagcgtca cagcacaacc tgcaaaaacg 12060

gagctgggct gcagctgggg ctggcatgga ctttcatttc agagattcgg tttttaagaa 12120

gatgcatgcc taacgtgttc tttttttttt ccaatgattt gtaatataca ttttatgact 12180

ggaaactttt ttgtacaaca ctccaataaa cattttgatt ttaggttctg cctctgagtt 12240

tattcctgag gggaagctcg agccggggcc tctgccctaa tgaagcggat gtctaagaaa 12300

gatccctcca cccccaagga aaaaggtcac tggctagtgt agctagtgta aacaggaccc 12360

aggcgatgca tgggaccctg cccttttttt tctagtgagc ctccgacgct gttgcacaag 12420

ctgactcttc gtcacgtgat gcgaccgggc tccgccccgg cggcaacacg ctgtatagac 12480

gcgccgggtg cctcgtgcgt gacgaagagt cagcttgtgc aacagcgtcg caggctcaca 12540

ttaccctgtt atccctagtt gacctcgtct tccgcttcct cgctcactga ctcgctgcgc 12600

tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 12660

acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 12720

aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 12780

cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 12840

gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 12900

tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 12960

tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 13020

cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 13080

gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 13140

ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 13200

ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 13260

ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 13320

agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 13380

aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 13440

atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 13500

tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 13560

tcatccatag ttgcctgact cggggggggg gggcgctgag gtctgcctcg tgaagaaggt 13620

gttgctgact cataccaggc ctgaatcgcc ccatcatcca gccagaaagt gagggagcca 13680

cggttgatga gagctttgtt gtaggtggac cagttggtga ttttgaactt ttgctttgcc 13740

acggaacggt ctgcgttgtc gggaagatgc gtgatctgat ccttcaactc agcaaaagtt 13800

cgatttattc aacaaagccg ccgtcccgtc aagtcagcgt aatgctctgc cagtgttaca 13860

accaattaac caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat 13920

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 13980

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 14040

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 14100

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc 14160

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 14220

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 14280

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 14340

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 14400

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 14460

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 14520

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 14580

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 14640

tgttggaatt taatcgcggc ctcgagcaag acgtttcccg ttgaatatgg ctcataacac 14700

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat 14760

cttgtgcaat gtaacatcag agattttgag acacaacgtg gctttccccc cccccccatt 14820

attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 14880

aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag 14940

aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc 15000

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 15060

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 15120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 15180

acca 15184

<210> 2

<211> 496

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Gly Lys Val Leu Arg Gly Gln Ile Val Gln Gly Val Val Trp Leu

1 5 10 15

Leu Leu Val Thr Gly Ala Gln Gly Arg Leu Ala Cys Lys Glu Asp Tyr

20 25 30

Arg Tyr Ala Ile Ser Ser Thr Asp Glu Ile Gly Leu Leu Gly Ala Gly

35 40 45

Gly Leu Thr Thr Thr Trp Lys Glu Tyr Asn His Asp Leu Gln Leu Asn

50 55 60

Asp Gly Thr Val Lys Ala Ser Cys Val Ala Gly Ser Phe Lys Val Thr

65 70 75 80

Ala Leu Asn Val Val Ser Arg Arg Tyr Leu Ala Ser Leu His Lys Lys

85 90 95

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

100 105 110

Pro Ser Thr Glu Glu Met Gly Asp Asp Phe Arg Ser Gly Leu Cys Pro

115 120 125

Phe Asp Thr Ser Pro Val Val Lys Gly Lys Tyr Asn Thr Thr Leu Leu

130 135 140

Asn Gly Ser Ala Phe Tyr Leu Val Cys Pro Ile Gly Trp Thr Gly Val

145 150 155 160

Ile Glu Cys Thr Ala Val Ser Pro Thr Thr Leu Arg Thr Glu Val Val

165 170 175

Lys Thr Phe Arg Arg Asp Lys Pro Phe Pro His Arg Met Asp Cys Val

180 185 190

Thr Thr Thr Val Glu Asn Glu Asp Leu Phe Tyr Cys Lys Leu Gly Gly

195 200 205

Asn Trp Thr Cys Val Lys Gly Glu Pro Val Val Tyr Thr Gly Gly Val

210 215 220

Val Lys Gln Cys Arg Trp Cys Gly Phe Asp Phe Asp Gly Pro Asp Gly

225 230 235 240

Leu Pro His Tyr Pro Ile Gly Lys Cys Ile Leu Ala Asn Glu Thr Gly

245 250 255

Tyr Arg Ile Val Asp Ser Thr Asp Cys Asn Arg Asp Gly Val Val Ile

260 265 270

Ser Thr Glu Gly Ser His Glu Cys Leu Ile Gly Asn Thr Thr Val Lys

275 280 285

Val His Ala Ser Asp Glu Arg Leu Gly Pro Met Pro Cys Arg Pro Lys

290 295 300

Glu Ile Val Ser Ser Ala Gly Pro Val Met Lys Thr Ser Cys Thr Phe

305 310 315 320

Asn Tyr Thr Lys Thr Leu Lys Asn Arg Tyr Tyr Glu Pro Arg Asp Ser

325 330 335

Tyr Phe Gln Gln Tyr Met Leu Lys Gly Glu Tyr Gln Tyr Trp Phe Asp

340 345 350

Leu Asp Ala Thr Asp Arg His Ser Asp Gly Pro Gly Gly Gly Gly Ser

355 360 365

Gly Gly Gly Gly Ser His His His His His His Gly Gly Gly Ser Val

370 375 380

Asp Thr Leu Ser Gly Leu Ser Ser Glu Gln Gly Gln Ser Gly Asp Met

385 390 395 400

Thr Ile Glu Glu Asp Ser Ala Thr His Ile Lys Phe Ser Lys Arg Asp

405 410 415

Glu Asp Gly Lys Glu Leu Ala Gly Ala Thr Met Glu Leu Arg Asp Ser

420 425 430

Ser Gly Lys Thr Ile Ser Thr Trp Ile Ser Asp Gly Gln Val Lys Asp

435 440 445

Phe Tyr Leu Tyr Pro Gly Lys Tyr Thr Phe Val Glu Thr Ala Ala Pro

450 455 460

Asp Gly Tyr Glu Val Ala Thr Ala Ile Thr Phe Thr Val Asn Glu Gln

465 470 475 480

Gly Gln Val Thr Val Asn Gly Lys Ala Thr Lys Gly Asp Ala His Ile

485 490 495

<210> 3

<211> 230

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Val His Pro Arg His His Tyr Arg Ser Arg Arg

20 25 30

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

35 40 45

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

50 55 60

Arg Phe Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys Gly

65 70 75 80

Gly Gly Ser His His His His His His Phe Glu Tyr Tyr Arg Ile Arg

85 90 95

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

100 105 110

Arg Gly Val Gly Ser Thr Ala Val Ile Leu Asp Asp Asn Phe Val Thr

115 120 125

Lys Ala Asn Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr Ser Ser Arg

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile Tyr Asp Gln Asp Tyr

195 200 205

Asn Ile Arg Ile Thr Met Tyr Val Gln Phe Arg Glu Phe Asn Leu Lys

210 215 220

Asp Pro Pro Leu Asn Pro

225 230

<210> 4

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggttccgggc cgcctagggc caccatgggc aaggtgctga ggggccagat c 51

<210> 5

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gtataatata gagtatacag ttcagatgtg ggcgtcgccc ttggtgg 47

<210> 6

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gcatgctggt tcgtttggac tga 23

<210> 7

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aaacgaattc gagctcggga gtagcggatg ccccgg 36

<210> 8

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tctcgagctc aagcttgatg ccaccatgga gaccgacacc ctgctgctgt ggg 53

<210> 9

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aagcttaatt aacgccgatt caggggttca ggggggggtc cttca 45

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

caggtactag cgctaccgga c 21

<210> 11

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

aagcttaatt aacgccgatt caggggttca ggggggggtc cttc 44

Claims

1. A protein characterized by: the protein is formed by connecting a fusion protein CSFVE2-Spycatcher and a fusion protein Cap-SpyTag,

the fusion protein CSFVE2-Spycatcher is any one of the following A1) -A3):

A1) a protein having the amino acid sequence of SEQ ID No. 2;

the fusion protein Cap-SpyTag is any one of the following B1) -B3):

B1) a protein having the amino acid sequence of SEQ ID No. 3;

2. A nucleic acid molecule characterized by: the nucleic acid molecule consists of a nucleic acid molecule coding the fusion protein CSFVE2-Spycatcher and a nucleic acid molecule coding the fusion protein Cap-SpyTag,

the nucleic acid molecule encoding the fusion protein CSFVE2-Spycatcher is any one of g11) -g13),

g11) the coding sequence of the coding strand is a DNA molecule shown by 5776-7266 nucleotides in SEQ ID No. 1;

g12) the nucleotide sequence of the coding strand is a DNA molecule shown in the 5776-bit 7266 site of SEQ ID No. 1;

g13) a DNA molecule having 80% or more than 80% identity with the nucleotide sequence defined in g11) or g12) and encoding the fusion protein CSFVE2-Spycatcher according to claim 1;

the nucleic acid molecule for coding the fusion protein Cap-SpyTag is any one of g21) -g23),

g23) a DNA molecule having 80% or more 80% identity with the nucleotide sequence defined in g21) or g22) and encoding the Cap-SpyTag fusion protein of claim 1.

3. An expression cassette characterized by: the expression cassette comprising the nucleic acid molecule of claim 2.

4. A recombinant vector characterized by: the recombinant vector contains the nucleic acid molecule of claim 2 or contains the expression cassette of claim 3.

5. A recombinant microorganism characterized by: the recombinant microorganism contains the nucleic acid molecule of claim 2 or the expression cassette of claim 3 or the recombinant vector of claim 4.

6. A recombinant transgenic cell line characterized by: the recombinant transgenic cell line containing the nucleic acid molecule of claim 2 or containing the expression cassette of claim 3 or containing the recombinant vector of claim 4.

7. A swine fever vaccine comprising the protein of claim 1.

8. A method for producing the protein of claim 1, comprising: a step of expressing a gene encoding the fusion protein CSFVE2-Spycatcher according to claim 1 and a gene encoding the fusion protein Cap-SpyTag according to claim 1 in a biological cell to obtain the protein according to claim 1; the organism is a microorganism, a plant or a non-human animal.

9. The method of claim 8, wherein: the biological cell is a non-human mammalian cell.

10. Use of the protein of claim 1 or the nucleic acid molecule of claim 2 or the expression cassette of claim 3 or the recombinant vector of claim 4 or the recombinant microorganism of claim 5 or the recombinant transgenic cell line of claim 6 for the preparation of a product for the prevention of swine fever.