CN114835822B

CN114835822B - Polymer vaccine of hog cholera virus and its preparing process

Info

Publication number: CN114835822B
Application number: CN202210425979.1A
Authority: CN
Inventors: 查银河; 盖其静; 余晓玉; 张稳涛
Original assignee: Zhejiang Hongsheng Biotechnology Co ltd
Current assignee: Zhejiang Hongsheng Biotechnology Co ltd
Priority date: 2022-04-22
Filing date: 2022-04-22
Publication date: 2022-11-04
Anticipated expiration: 2042-04-22
Also published as: CN114835822A

Abstract

The invention discloses a polymer vaccine of classical swine fever virus and a preparation method thereof, belonging to the field of biological products. The active component of the vaccine disclosed by the invention is a protein formed by connecting fusion protein (CSFV-E2) -SpyTag-HIgG and fusion protein CRM197-Spycatcher-LIgG. Also provides a preparation method of the protein, which comprises the step of expressing the coding genes of the fusion protein (CSFV-E2) -SpyTag-HIgG and the fusion protein CRM197-Spycatcher-LIgG in a biological cell to obtain the protein. The invention utilizes a protein engineering method to fuse the SpyTag in E2 and Fc protein, and fuse SpyCatcher with diphtheria toxin mutant CRM197 for co-expression, and finally forms immune stimulant polymer E2 protein, so that the state is more stable, and the immune effect is better.

Description

Polymer vaccine of hog cholera virus and its preparing process

Technical Field

The invention belongs to the field of biological products, and particularly relates to a polymer vaccine for preventing swine fever and a preparation method thereof.

Background

Hog cholera, commonly known as "gastrointestinal plague", is an acute, febrile, and contagious disease caused by hog cholera virus (Hogcholera virus, swine river virus, CSFV for short) of hog cholera genus of flaviviridae family, and has high infectivity and lethality. Pigs are the only natural host for the virus. The infectious disease was first discovered in 1833 in ohio, usa, and its prevalence has spread all over the world for centuries. The international animal epidemic department defines the infectious diseases as A class infectious diseases, and the infectious diseases are listed as a class infectious diseases by the animal epidemic prevention law in China, and are one of the main epidemic diseases which endanger the development of the pig industry in China at present. Chinese hog cholera lapinized attenuated vaccine plays a decisive role in preventing and treating hog cholera, but the immune effect is not ideal in recent years.

CSFV is enveloped virus, 40-60 nm, single strand positive strand RNA. The CSFV genome is about 123kb in length and contains only one large Open Reading Frame (ORF), which is translated into a polyprotein of 3898 amino acid residues with a molecular weight of about 438kD and further processed to mature protein by viral and host cell proteases. All structural and non-structural proteins of CSFV are encoded by this ORF, encoding 4 structural proteins (C, E) ^rns (E0) E1 and E2) and 8 non-structural proteins (N) ^pro P7, NS2, NS3, NS4A, NS4B, NS5A and NS 5B). Among the structural proteins, the most important proteins for the research of 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 the cyst membrane glycoprotein of CSFV and is the main antigen protein. In vitro, E2 induces neutralizing antibodies against the virus and in vivo, elicits challenge antibodies against CSFV. Due to the varying degree of glycosylation, the molecular weight of E2 can be between 51 and 58kDa. The 15 cysteine (Cys) residues in the E2 molecule are conserved within the genus, with the 6 Cys residues at the N-terminus participating in the formation of the antigenic domain and the 9 Cys residues at the C-terminus participating in the formation of homo-and heterodimers.

Diphtheria toxin is a protein consisting of 535 amino acids, and has two subunits connected by disulfide bonds. Has a molecular weight of 62kD, contains two disulfide bridges combined with a 14-amino acid ring, and has the characteristics of toxicity and specific immunity generation. Diphtheria toxin 197 is a mutant of diphtheria toxin, and amino acid position 52 is mutated from glycine to glutamic acid, so that the toxin loses activity and does not produce toxic effect on cells, but the antigenic activity and the immunological activity of diphtheria toxin are still consistent with those of natural toxin.

The IgG fusion protein is mainly characterized by comprising an Fc segment, and the function of the IgG fusion protein is similar to that of a monoclonal antibody Fc. The fusion protein can prolong the half-life of the protein, and when Fc is combined with Fc receptor (FcRn), the half-life can be prolonged to 21 days under the protection of the receptor. Meanwhile, the molecular weight is improved, the body clearance rate is reduced, fc is connected through a disulfide bond to form a dimer, the light chain immune stimulator is further modified to form an polymer compound, the molecular stability is improved, and meanwhile, the Fc structure is combined with an Fc receptor on the surface of an immune cell to play various biological functions of IgG and diphtheria toxin, wherein the biological functions mainly comprise the regulation of cytokine secretion, the regulation of B cell proliferation and differentiation, the improvement of antibody titer and the like. In addition, fc can be specifically combined with ProteinA, simplifies the purification steps and has important significance in biological products.

The SpyTag is a polypeptide segment, the Spycatcher is a protein corresponding to the SpyTag, and the SpyTag and the Spycatcher can be recombined and spontaneously form isopeptide bond coupling, so that the protein assembly and the chemical reaction are combined together and can be a gene-coded chemical reaction.

Disclosure of Invention

The technical problem to be solved by the invention is how to improve the stability and/or prolong the half-life of the classical swine fever E2 subunit vaccine and enhance the immune effect of the classical swine fever E2 subunit vaccine.

In order to solve the above technical problems, the present invention provides, in a first aspect, a protein comprising a fusion protein (CSFV-E2) -SpyTag-HIgG linked to a fusion protein CRM197-Spycatcher-LIgG,

the fusion protein (CSFV-E2) -SpyTag-HIgG is any one of the following A1) -A3):

a1 Protein of which the amino acid sequence is SEQ ID No.3;

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 CRM197-SpyCatcher-LIgG is any one of the following B1) -B3):

b1 Protein of which the amino acid sequence is SEQ ID No.4;

b2 B1) protein which has 80% or more identity with the protein shown in B1) and is related to a swine fever virus vaccine, and which is obtained by substituting and/or deleting and/or adding at least one amino acid residue in the amino acid sequence shown in B1);

b3 B1) or B2) to which a tag is attached at the N-terminus and/or C-terminus.

Wherein, the 1 st to 19 th sites in SEQ ID No.3 are the amino acid sequence of signal peptide, the 20 th to 379 th sites are the amino acid sequence of CSFV-E2, the 380 th to 402 th sites are the amino acid sequence of Linker and SpyTag polypeptide, the 403 th to 722 th sites are the amino acid sequence of pig IgG heavy chain constant region Fc, and can also contain partial sequence in the whole sequence; in SEQ ID No.4, the 1 st to 19 th sites are amino acid sequences of signal peptide, the 20 th to 554 th sites are amino acid sequences of CRM197 toxin, the 555 th to 678 th sites are amino acid sequences of Linker and Spycatcher polypeptide, the 679 th to 780 th sites are amino acid sequences of porcine IgG light chain constant region, and partial sequences of the whole sequences can also be contained.

The fusion protein (CSFV-E2) -SpyTag-HIgG is respectively a CSFV-E2, a SpyTag polypeptide and a porcine IgG heavy chain constant region from the N end to the C end, and can also contain partial sequences in the whole sequence. The fusion protein CRM197-SpyCatcher-LIgG is respectively a CRM197 polypeptide, a SpyCatcher polypeptide and a porcine IgG light chain constant region from the N end to the C end, and can also contain partial sequences in the whole sequence. Wherein the SpyTag and the SpyCatcher can recombine and spontaneously form isopeptide bond coupling, thereby connecting the fusion protein (CSFV-E2) -SpyTag-HIgG and the fusion protein CRM197-SpyCatcher-LIgG. The two porcine IgG heavy chain constant regions are linked by disulfide formation at the hinge region.

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 technical problems, in a second aspect, the present invention provides a nucleic acid molecule related to the above protein, wherein the nucleic acid molecule consists of a nucleic acid molecule encoding the fusion protein (CSFV-E2) -SpyTag-HIgG and a nucleic acid molecule encoding the fusion protein CRM 197-Spycatcher-LIgG;

the nucleic acid molecule for coding the fusion protein (CSFV-E2) -SpyTag-HIgG is any one of the following g 11) -g 13),

g11 The coding sequence of the coding chain is a DNA molecule shown in SEQ ID No. 1;

g12 The nucleotide sequence of the coding strand is a DNA molecule shown in SEQ ID No. 1;

g13 A DNA molecule having 80% or more of identity with the nucleotide sequence defined in g 11) or g 12) and encoding the above fusion protein (CSFV-E2) -SpyTag-HIgG;

the nucleic acid molecule encoding the fusion protein CRM197-SpyCatcher-LIgG is any one of g 21) -g 23),

g21 The coding sequence of the coding chain is a DNA molecule shown in SEQ ID No. 2;

g22 The nucleotide sequence of the coding strand is a DNA molecule shown in SEQ ID No. 2;

g23 A DNA molecule having 80% or more identity to the nucleotide sequence defined in g 21) or g 22) and encoding the above-mentioned fusion protein CRM197-Spycatcher-LIgG.

In SEQ ID No.1, the 1 st to 57 th sites are coding genes of signal peptides, the 58 th to 1137 th sites are coding sequences of CSFV-E2, the 1138 th to 1206 th sites are coding sequences of Linker and SpyTag polypeptides, and the 1207 th to 2169 th sites are coding sequences of porcine IgG heavy chain constant regions; in SEQ ID No.2, the 1 st to 57 th sites are coding sequences of signal peptides, the 58 th to 1662 th sites are CRM197 toxin genes, the 1663 th to 2034 th sites are coding sequences of Linker and Spycatcher polypeptides, and the 2035 th to 2343 th sites are coding sequences of porcine IgG light chain constant regions.

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

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

In order to achieve the above object, 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 achieve the above object, in a fifth aspect, the present invention provides a recombinant microorganism containing the above nucleic acid molecule or containing the above expression cassette or containing the above recombinant vector.

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

In order to achieve the above objects, 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.

In order to achieve the above object, in a seventh aspect, the present invention provides a swine fever vaccine, which comprises the protein.

The Swine fever is caused by Swine fever virus (CSFV), which is a Swine fever virus (Hogcholera virus, swine feeder virus, CSFV for short) of the genus CSFV of the family Flaviviridae.

In order to achieve the above object, according to an eighth aspect, the present invention provides a method for producing the above protein, comprising the steps of: expressing a gene encoding the fusion protein (CSFV-E2) -SpyTag-HIgG and a gene encoding the fusion protein CRM197-Spycatcher-LIgG in a biological cell to obtain the protein; the organism is a microorganism, a plant or a non-human animal.

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

In order to achieve the above objects, 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 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 Expect value to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, the Per residual Gap cost, and the Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation to search for 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.

SpyTag is a polypeptide segment, spycatcher is a protein corresponding to the SpyTag, spycatcher and SpyTag can be recombined and spontaneously form isopeptide bond coupling. The IgG fusion protein is mainly characterized by comprising an Fc segment which has similar functions with the Fc of a monoclonal antibody, the fusion protein can prolong the half-life of the protein, and when the Fc is combined with an Fc receptor (FcRn), the half-life can be prolonged to 21 days under the protection of the receptor. Meanwhile, the molecular weight is improved, the body clearance rate is reduced, fc is connected through a disulfide bond to form a dimer, the light chain immune stimulator is further modified to form an polymer compound, the molecular stability is improved, and meanwhile, the Fc structure is combined with an Fc receptor on the surface of an immune cell to play various biological functions of IgG and diphtheria toxin, wherein the biological functions mainly comprise the regulation of cytokine secretion, the regulation of B cell proliferation and differentiation, the improvement of antibody titer and the like. In addition, fc can specifically bind to ProteinA, simplifying purification steps. The invention utilizes a protein engineering method, uses swine fever E2 protein to replace a heavy chain variable region of a pig IgG antibody to be fused with a constant region sequence, adds SpyTag in the middle, fuses the SpyTag in E2 and Fc protein, and obtains fusion protein (CSFV-E2) -SpyTag-HIgG; the N-terminal of the constant region of the IgG light chain was replaced with the diphtheria toxin mutant CRM197, and Spycatcher was added in the middle to obtain the fusion protein CRM197-Spycatcher-LigG. The invention co-expresses the fusion protein (CSFV-E2) -SpyTag-HIgG gene and the fusion protein CRM197-Spycatcher-LigG gene, increases the IgG stability, finally forms an immune stimulant polymer E2 protein, and has more stable state.

The invention has the following beneficial effects:

by utilizing a protein engineering method, spyTag is fused in E2 and Fc protein and is fused with a diphtheria toxin mutant CRM197 to carry out co-expression, the IgG stability is increased, and finally an immune stimulator polymer E2 protein is formed, so that the state is more stable.

The swine fever E2 protein is used for replacing a swine IgG antibody heavy chain variable region and a constant region sequence for fusion, spyTag can be added in the middle, a diphtheria toxin mutant CRM197 is used for replacing the N end of a constant region of an LgG light chain, a fusion protein (CSFV-E2) -SpyTag-HIgG gene and a fusion protein CRM197-Spycatcher-LigG gene are respectively placed at the downstream of a mammalian cell promoter and connected in series with a double expression element, an expression vector is converted into CHO, a cultured product is purified through ProteiA to obtain a target fusion protein, and the target fusion protein is mixed with an adjuvant to prepare a vaccine. Experiments prove that the protein expression quantity is stable, and target animal pig experiments show that the method can stimulate the generation of neutralizing antibodies in pigs, can prevent swine fever, and can be prepared into spray drinking water type vaccines.

Drawings

FIG. 1 is a physical map of recombinant expression vector PEEGSE2197.

FIG. 2 shows the Western Blot detection result of protein expressed by positive cells.

FIG. 3 is a photograph of recombinant cells CHO/PEEGS-E2197-88 cultured in 10L fermentation feed.

FIG. 4 shows the Western Blot detection result of recombinant cell CHO/PEEGS-E2197-88 in 10L fermentation fed-batch culture expression product.

FIG. 5 is an electrophoretogram of the protein after purification of the expression product.

FIG. 6 is a liquid chromatogram of CHO/PEEGS-E2197-88 cell purified protein.

FIG. 7 shows the serum Joule reaction of fusion protein E219788-1 and E2.

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 experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Original gene synthesis and vector: the coding sequences of the recombinant fusion protein (CSFV-E2-SpyTag-HIgG) and the recombinant fusion protein CRM197-Spycatcher-LIgG are optimized and synthesized according to mammalian cell codons.

Enzymes and other biochemical reagents: the endonuclease and the ligase are purchased from TaKaRa company, the recombinase is purchased from holotype gold, the plasmid extraction kit is purchased from Tiangen biology, the ProteiA affinity filler is purchased from Jiangsu Qianjin biology company, and the LB culture medium, the inorganic salt and the like are all domestic reagents.

Example 1 fusion protein design and construction of recombinant expression vectors

1.1 design of fusion proteins

In order to improve the immune effect of the CSFV-E2 protein, the coding gene of CSFV-E2 is fused with the coding gene of pig IgG heavy chain constant region, and the coding gene of CRM19L is fused with the coding gene of pig IgG light chain constant region. The SpyTag is a polypeptide segment, the Spycatcher is a protein corresponding to the SpyTag, and the Spycatcher and the SpyTag can be recombined and spontaneously form isopeptide bond coupling.

The fusion protein (CSFV-E2) -SpyTag-HIgG encoding gene (SEQ ID No. 1) and the fusion protein CRM197-Spycatcher-LIgG encoding gene (SEQ ID No. 2) were synthesized by Beijing Optimalaceae organism Limited, wherein the fragment between HindIII and ECORI cleavage sites of the PEE12.4 vector (small fragment between HindIII and ECORI cleavage sites) was replaced with (CSFV-E2) -SpyTag-HIgG gene to obtain the recombinant expression vector PEE12.4- (CSFV-E2) -SpyTag-HIgG. The fragment between HindIII and ECORI cleavage sites of the PEE6.4 vector (a small fragment between HindIII and ECORI cleavage sites) was replaced with CRM197-Spycatcher-LIgG gene to obtain a recombinant expression vector PEE6.4-CRM197-Spycatcher-LIgG.

The nucleotide sequence of the fusion protein (CSFV-E2) -SpyTag-HIgG coding gene is SEQ ID No.1, and the coded amino acid sequence is SEQ ID No.3; the nucleotide sequence of the encoding gene of the fusion protein CRM197-SpyCatcher-LIgGIgG is SEQ ID No.2, and the amino acid sequence of the encoding fusion protein is SEQ ID No.4;

Wherein, the 1 st to 19 th sites in SEQ ID No.3 are the amino acid sequence of the signal peptide, the 20 th to 379 th sites are the amino acid sequence of CSFV-E2, the 380 th to 402 th sites are the amino acid sequence of Linker and SpyTag polypeptide, and the 403 th to 722 th sites are the amino acid sequence of the pig IgG heavy chain constant region Fc; in SEQ ID No.4, the 1 st to 19 th sites are amino acid sequences of signal peptide, the 20 th to 554 th sites are CRM197 toxin amino acid sequences, the 555 th to 678 th sites are amino acid sequences of Linker and SpyCatcher polypeptide, and the 679 th to 780 th sites are amino acid sequences of porcine IgG light chain constant regions.

1.2 construction of recombinant expression vectors

The recombinant expression vector PEE12.4- (CSFV-E2) -SpyTag-HIgG and the recombinant expression vector PEE6.4-CRM197-Spycatcher-LIgGIgG of 1.1 were double digested with Not I and Sal I restriction enzymes, respectively, and the digested products were subjected to agarose gel electrophoresis, and the fragments of corresponding sizes were recovered, respectively, and ligated for transformation.

The ligation product was transformed into DH 5. Alpha. Competent cells to obtain positive recombinant bacteria, and the positive clones were identified by selecting single clones and culturing in 1.5mL EP tube containing 500. Mu.L LB medium at 37 ℃ and 220rpm for 6 hours.

And (4) carrying out test tube enrichment on the positive clone, and then, upgrading the plasmid, and verifying the correctness by using Not I/Sal I double enzyme digestion. The correct recombinant expression vector is named as PEEGSE2197, the physical map of the recombinant expression vector is shown in figure 1, and the nucleotide sequence of the recombinant expression vector PEEGSE2197 is shown in SEQ ID No. 5.

Example 2 construction of Stable transgenic cell lines and protein expression

2.1 construction of stably transfected cell lines

The recombinant expression vector PEEGSE2197 of example 1 was digested and recovered with Pvu I to obtain a linearized expression vector PEEGSE2197. Transfecting a CHO cell with the linearized expression vector PEEGSE2197, the transfecting step comprising:

step 1: cell density was adjusted to 2.5-3X 10 24h before transfection ⁶ cells/mL, overnight culture.

Step 2: on the day of transfection, the cell density was diluted to 3X 10 with fresh medium ⁶ cells/mL, and the cell survival rate is more than or equal to 95 percent.

And step 3: preparing a DNA-PEI complex, respectively diluting DNA and PEI with Opti-MEM, wherein the final concentration of the DNA during transfection is 1-2 mug/mL, the mass ratio of PEI to DNA is 5.

And 4, step 4: the DNA-PEI complex is kept standing for 20min at room temperature and then slowly dripped into the cell suspension to be transfected.

And 5: the cell status was observed 48 hours after transfection to check whether the plasmid was expressed, and if fluorescence was visible, the culture broth was changed to a selection medium (IMDM medium (Gibco, cat # 12200036) containing 50 μ M MSX (methionine imino sulfone) for subsequent selection of high producing cell lines).

And 6: the cells were pressurized with IMDM medium (Gibco, cat # 12200036) containing 50. Mu.M MSXScreening for 1 month to obtain positive cells, diluting the positive cells into 96-well plates by limiting dilution, and microscopic examination to ensure that there is one cell per well and recording, at 5% CO ₂ Culturing at 37 deg.C for 15 days at 100rpm, adding IMDM medium containing 50 μ M MSX (Gibco, cat # 12200036) to the 48-well plate, and further processing at 5% CO ₂ And cultured at 37 ℃ for 15d at 100 rpm. Continuously subculturing after obtaining cells on a pressurized culture medium, continuously subculturing for 18 passages for 2 months, taking supernatant of culture wells, and detecting the protein expression quantity by a Western Blot method, wherein primary antibody used in the Western Blot method is polyclonal antibody obtained by immunizing a mouse by taking E2 protein (the amino acid sequence is 20-379 th site of SEQ ID No. 3) as immunogen, and HRP-goat anti-mouse IgG (reagent manufacturer Solarbio, product number SE 131) is taken as a secondary antibody. WB results are shown in FIG. 2, where MK denotes Marker in FIG. 2 and 72#, 88#, 96# and 102# denote cell numbers.

Obtaining two strains of 88# and 96# high expression fusion protein (CSFV-E2) -SpyTag-HIgG and fusion protein CRM197-Spycatcher-LIgGIgG stable cell strains in total, taking 88# and 96# as engineering cell strains, performing mass propagation, storing in liquid nitrogen at-80 ℃, and naming the strains as CHO/PEEGS-E2197-88 and CHO/PEEGS-E2197-96.

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

The fermentation tank adopts a 10L reaction system of Shanghai Bailun lactation fermentation tank, the liquid loading is 5L, the rotating speed is 80rpm, the pH value is 7.2, and the culture temperature is 37 ℃. Preparing 1L seed solution with cell density of 2.0 × 10 for CHO/PEEGS-E2197-88 ⁶ pieces/mL, added to the fermenter, counted daily, and observed for cell status. When the cell density reached 5.0X 10 at day 5 of fermentation ⁶ When the temperature is reduced to 33 ℃ while 400mL of the fed-batch culture medium is fed every day, continuously culturing for 6 days under the condition, stopping feeding the fed-batch culture medium when the protein concentration reaches the maximum, and stopping fermenting after the temperature is reduced to 30 ℃ and cultured for 1 day. Using O in the fermentation process ₂ Maintaining the fermentation tank at a dissolved oxygen level of not less than 10%, using CO ₂ And NaHCO ₃ The pH was controlled at 7.2 and the cell fermenter process was as shown in FIG. 3. The content of the expression product is detected by taking the fermentation liquid and using Western Blot, the result is shown in figure 4, and the result shows that the protein content is increased along with the increase of the fermentation time. Among them, westeThe primary antibody used in the rn Blot method was a polyclonal antibody obtained by immunizing a mouse with an E2 protein (amino acid sequence: positions 20 to 379 of SEQ ID No. 3) as an immunogen, and HRP-goat anti-mouse IgG (reagent manufacturer: solarbio, cat # SE 131) was used as the secondary antibody. The fermentation medium is GIBCO (A2910001); the feed medium was GIBCO (A2910001) +8% glucose.

2.3 purification of recombinant proteins

Collecting the fermentation liquor obtained in the step 2.2, and obtaining clear liquor through a national obstetrical Baite deep filtration system, wherein the first step is to select buffer A:20mM PBS,0.15M NaCl, pH7.2, conductivity 18.128ms/cm, equilibrium purification column (ProteiA affinity packing purchased from Kyosu pure biology Co.). Loading with clarified broth, buffer a washing the column for contaminating proteins, buffer B:4.5mM sodium citrate, 25mM citric acid pH3.0, conductivity 1.27ms/cm elution. The eluate is neutralized by 0.5M TIRS-CL to obtain a low pH purified solution, which is the purified protein, the SDS-PAGE detection result is shown in FIG. 5, the first lane from the left of FIG. 5 is Marker, and the second lane from the left and the third lane from the left are samples to be detected.

Protein concentration is measured by adopting a Coomassie Brilliant Blue (Coomassie Brilliant Blue) method, and results of fermentation of two cells show that the concentration of CHO/PEEGS-E2197-88 cell purified protein reaches 1.35mg/mL and the concentration of CHO/PEEGS-E2197-96 cell purified protein reaches 1.08mg/mL, wherein a detection curve Y =8.2037X; r is ² ＝0.9975。

The protein purity of the CHO/PEEGS-E2197-88 cell purified protein was determined by liquid chromatography, and the results are shown in FIG. 6, in which FIG. 6 shows the sample retention time (in min) on the abscissa and the wavelength signal intensity (in nm) on the ordinate. The results in FIG. 6 show that the protein purity reaches 93.82%, the effective protein concentration is 1.26mg/mL, and the batch of protein is used as the experimental protein sample. And detecting the endotoxin in the protein sample, wherein the endotoxin is detected according to a gel method, and the content of the endotoxin in the protein sample to be detected is 0.0075EU/mL and less than 0.015EU/mL. The results indicate that the endotoxin level of the protein sample meets the requirements of the biological product.

The whole purification process has simple steps. The product has high purity, the protein obtained by purification is named as fusion protein E219788-1, E219788-1 and swine fever E2 positive serum are subjected to an agar amplification test, the result is shown in figure 7, and agar amplification bands occur successfully in the purified protein and serum.

The fusion protein E219788-1 consists of (CSFV-E2) -SpyTag-HIgG and CRM197-Spycatcher-LIgGIgG, wherein the amino acid sequence of (CSFV-E2) -SpyTag-HIgG is SEQ ID No.3. In SEQ ID No.3, 1 to 19 sites are signal peptide, 20 to 379 sites are CSFV-E2, 380 to 402 sites are Linker and SpyTag polypeptide, and 403 to 722 sites are coding sequence of pig IgG heavy chain constant region. The amino acid sequence of CRM197-Spycatcher-LIgGIgG is SEQ ID No.4. In SEQ ID No.4, the 1 st to 19 th sites are amino acid sequences of signal peptide, the 20 th to 554 th sites are CRM197 toxin amino acid sequences, the 555 th to 678 th sites are amino acid sequences of Linker and SpyCatcher polypeptide, and the 679 th to 780 th sites are amino acid sequences of porcine IgG light chain constant regions.

Wherein the SpyTag in the (CSFV-E2) -SpyTag-HIgG and the SpyCatcher in the CRM197-SpyCatcher-LIgG can recombine and spontaneously form isopeptide bond coupling, thereby connecting the (CSFV-E2) -SpyTag-HIgG and the CRM197-SpyCatcher-LIgG. Porcine IgG heavy chain constant regions are interconnected by disulfide bond formation at the hinge region.

2.4 preparation of vaccines

2.4.1 preparation of Experimental vaccine for mice

500 μ g/mL vaccine A: 40mL of E219788-1 protein (concentration 1.35 mg/mL) prepared in 2.3 was placed in a sterile beaker in a clean bench, 35mL of sterile PBS and 25mL of Summit-350 (trial by Adjuvant Luffamide Biotech Co., ltd.) Adjuvant were added, and the manual homogenizer was adjusted to 4000rpm for emulsification for 10min, and the whole process was run on ice. After the operation is finished, the vaccine is aseptically subpackaged into penicillin bottles to obtain 500 mu g/mL E2 vaccine for later use.

2.4.2 preparation of Experimental vaccine for pig

50 μ g/mL vaccine B: the E219788-1 protein (concentration 1.35 mg/mL) prepared in 4mL 2.3 was placed in a sterile beaker in a clean bench, 71mL sterile PBS and 25mL Summit-350 (Adjuvant) Adjuvant were added, the manual homogenizer was adjusted to 4000rpm for emulsification for 10min, and the entire process was run on ice. After the operation is finished, the vaccine is aseptically subpackaged into penicillin bottles to obtain 50 mu g/mL E2 vaccine for later use.

2.4.3, 100. Mu.g/mL vaccine formulation

100 μ g/mL vaccine C: the E219788-1 protein (concentration 1.35 mg/mL) prepared in 8mL 2.3 was placed in a sterile beaker in a clean bench, 67mL sterile PBS was added and emulsified with 25mL Summit-350 (Adjuvant) Adjuvant for 10min with the manual homogenizer adjusted to 4000rpm, the entire process was operated on ice. After the operation is finished, the vaccine is aseptically subpackaged into penicillin bottles to obtain 100 mu g/mL E2 vaccine for later use.

Example 3 safety and efficacy validation of E2 vaccines

The E2 vaccine prepared in 2.4 of example 2 was used in this experiment and the commercial vaccine was classical swine fever virus (classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine (WH-09 strain)).

3.1 safety test of recombinant fusion proteins

The Balb/C mice (purchased from Shanghai Si Laike laboratory animal, ltd.) 6-8 weeks old and 18-22 g in weight were taken, 20 mice (male and female half) respectively were randomly divided into 5 groups, and 4 groups were used. Group 1 is the E2 vaccine 50 μ g group, group 2 is the E2 vaccine 100 μ g group, group 3 is the commercial vaccine group (positive control), group 4 is the PBS group (negative control).

Group 1: each mouse was first subcutaneously inoculated with 0.1mL of the E2 vaccine solution of 2.4.1 of example 2 (500. Mu.g/mL vaccine A) to give a dose of fusion protein E2 of 50. Mu.g/mouse, and a second inoculation was performed at the same dose after 2 weeks with 2 weeks of continuous observation after each injection.

Group 2: each mouse was first inoculated subcutaneously with 0.2mL of the E2 vaccine solution of 2.4.1 in example 2 (500. Mu.g/mL of vaccine A) to give a dose of fusion protein E2 of 100. Mu.g/mouse, and 2 weeks later, a second inoculation was performed at the same dose, and 2 weeks after each injection was continuously observed.

Group 3: the first inoculation is 0.2 mL/kewenjing, and the second inoculation is performed 2 weeks after the single-dose group inoculation at the same dose of 0.2 mL/kewenjing, with 2 weeks of continuous observation after each injection.

Group 4: the first inoculation of 0.2 mL/PBS solution, the second inoculation of 0.2 mL/PBS solution was performed 2 weeks after the single dose group inoculation in the same way, with 2 weeks of continuous observation after each injection.

During the experiment, the clinical symptom changes of the experimental animals, including spirit, food intake, activities, respiration, drinking water, excretion status, inflammatory reaction of injection sites, body temperature and the like, are observed and recorded every day. If the mouse dies, it is dissected, sampled and observed for gross and histopathological changes.

Through 2 weeks of continuous observation, clinical symptoms before and after injection of the E2 vaccine are compared, and it is found that 4 mice in groups all have normal diet, no adverse changes in spirit, no abnormal respiration and excretion, no inflammation phenomenon at the injection part, no adverse reaction of the mice during the experiment, and no death of the mice, and the E2 vaccine prepared in example 2 is a safe immunity protein.

3.2E 2 vaccine immunopotency test

3.2.1 animal immunization

50. Mu.g/mL of vaccine B and 100. Mu.g/mL of vaccine C prepared in example 2 were used as the experimental materials for this animal. The method comprises the steps of taking kewenjing as a positive control, taking PBS as a negative control, grouping according to a grouping mode shown in table 1, screening swine fever antibody negative pigs for animal tests, and carrying out a fusion protein vaccine immunization program according to a swine fever immunization program for 2 times, namely carrying out secondary immunization at the same dosage at an interval of 21 days after primary immunization.

The swine fever antibody ELISA detection kit is purchased from IDEXX company, and the batch number is as follows: REF:99-43220, LOT SN: g131 ); 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.

ELISA detection kit for hog cholera antigen, purchased from IDEXX company, with batch number: REF:99-40939, LOT SN: g351; and (3) judging standard: the S-N value of the sample to be detected 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 sample to be detected is larger than 0.1 and smaller than or equal to 0.3, and the sample is judged to be suspicious; the S-N value of the detected sample is more than 0.3, and the sample is judged to be positive by the hog cholera virus antigen.

The experimental instrument is a Bio-Rad Imark type enzyme-linked immunoassay analyzer.

Commercial 2-month-old piglets with the weight of about 40kg are selected, the detection of the swine fever antigen and the antibody is 25 piglets with double negative and long white, the average is 5 groups, 5 piglets are divided into each group, the grouping results are shown in the table 1, and immunization is carried out according to the grouping results. Intramuscular injection of 1 mL/pig, a second immunization with the same dose and the same way after 21 days, and 1 group of negative controls are shared in the comparative test with similar products; group 5 was raised in different groups without immunization as a control under the same conditions.

TABLE 1 groups of test animals

Group of	The medicine and dosage	Quantity (head)
			Group 1	50 ug/mL E2 vaccine	5
Group 2	100 ug/mL E2 vaccine	5
			Group 3	Medicine for treating disease	5
Group 4	PBS	5
			Group 5	Raising in different fields without any immunity	5

3.2.2 measurement of antibody titer

All 20 test pigs were bled in the anterior vena cava on the day of first-immunization, day 14, day 21 after first-immunization, day 14, day 21, and day 42 after second-immunization, and serum was isolated and tested for swine fever antibody blocking rate using IDEXX kit.

The results of the in vivo pig test are shown in Table 2, and the results of the in vivo pig serum antibody blocking rate are shown in Table 2: the immune dose of 100 mu g of the E2 vaccine is higher than the immune effect of the Kewenjing; the immune dose of 50 mu g of the E2 vaccine can achieve the immune effect of the commercial vaccine kewenjing, and the antibody level of the protective effect is achieved 21 days after secondary immunization, but the antibody blocking rate (82.35-95.45%) of the E2 vaccine is higher than that (76.51%) of the kewenjing swine fever inactivated vaccine along with time delay.

TABLE 2 values of antibody blocking rate after immunization of pigs with E2 vaccine

In Table 2, "/" indicates not measured.

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 within 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 examples, it will be appreciated that the invention may 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. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> Zhejiang Hongshan Cheng Biotechnology Ltd

<120> polymer vaccine of hog cholera virus and preparation method thereof

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2169

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggagttcg gcctgagctg ggtgttcctg gtggccatcc tgaagggcgt gcagtgcggc 60

aaggtgctgc gcggccagat cgtgcagggc gtggtgtggc tgctgctggt gaccggcgcc 120

cagggccgcc tggcctgcaa ggaggactac cgctacgcca tcagcagcac cgacgagatc 180

ggcctgctgg gcgccggcgg cctgaccacc acctggaagg agtacaacca cgacctgcag 240

ctgaacgacg gcaccgtgaa ggccagctgc gtggccggca gcttcaaggt gaccgccctg 300

aacgtggtga gccgccgcta cctggccagc ctgcacaaga aggccctgcc caccagcgtg 360

accttcgagc tgctgttcga cggcaccaac cccagcaccg aggagatggg cgacgacttc 420

cgcagcggcc tgtgcccctt cgacaccagc cccgtggtga agggcaagta caacaccacc 480

ctgctgaacg gcagcgcctt ctacctggtg tgccccatcg gctggaccgg cgtgatcgag 540

tgcaccgccg tgagccccac caccctgcgc accgaggtgg tgaagacctt ccgccgcgac 600

aagcccttcc cccaccgcat ggactgcgtg accaccaccg tggagaacga ggacctgttc 660

tactgcaagc tgggcggcaa ctggacctgc gtgaagggcg agcccgtggt gtacaccggc 720

ggcgtggtga agcagtgccg ctggtgcggc ttcgacttcg acggccccga cggcctgccc 780

cactacccca tcggcaagtg catcctggcc aacgagaccg gctaccgcat cgtggacagc 840

accgactgca accgcgacgg cgtggtgatc agcaccgagg gcagccacga gtgcctgatc 900

ggcaacacca ccgtgaaggt gcacgccagc gacgagcgcc tgggccccat gccctgccgc 960

cccaaggaga tcgtgagcag cgccggcccc gtgatgaaga ccagctgcac cttcaactac 1020

accaagaccc tgaagaaccg ctactacgag ccccgcgaca gctacttcca gcagtacatg 1080

ctgaagggcg agtaccagta ctggttcgac ctggacgcca ccgaccgcca cagcgacggc 1140

ggcggcggca gcgcccacat cgtgatggtg gacgcctaca agcccaccaa gggcggcggc 1200

ggcagctacc ccctggcccc ctgcggccgc gacaccagcg gccccaacgt ggccctgggc 1260

tgcctggcca gcagctactt ccccgagccc gtgaccatga cctggaacag cggcgccctg 1320

accagcggcg tgcacacctt ccccagcgtg ctgcagccca gcggcctgta cagcctgagc 1380

agcatggtga ccgtgcccgc cagcagcctg agcagcaaga gctacacctg caacgtgaac 1440

caccccgcca ccaccaccaa ggtggacaag cgcgtgggca ccaagaccaa gcccccctgc 1500

cccatctgcc ccggctgcga ggtggccggc cccagcgtgt tcatcttccc ccccaagccc 1560

aaggacaccc tgatgatcag ccagaccccc gaggtgacct gcgtggtggt ggacgtgagc 1620

aaggagcacg ccgaggtgca gttcagctgg tacgtggacg gcgtggaggt gcacaccgcc 1680

gagacccgcc ccaaggagga gcagttcaac agcacctacc gcgtggtgag cgtgctgccc 1740

atccagcacc aggactggct gaagggcaag gagttcaagt gcaaggtgaa caacgtggac 1800

ctgcccgccc ccatcacccg caccatcagc aaggccatcg gccagagccg cgagccccag 1860

gtgtacaccc tgcccccccc cgccgaggag ctgagccgca gcaaggtgac cgtgacctgc 1920

ctggtgatcg gcttctaccc ccccgacatc cacgtggagt ggaagagcaa cggccagccc 1980

gagcccgagg gcaactaccg caccaccccc ccccagcagg acgtggacgg caccttcttc 2040

ctgtacagca agctggccgt ggacaaggcc cgctgggacc acggcgagac cttcgagtgc 2100

gccgtgatgc acgaggccct gcacaaccac tacacccaga agagcatcag caagacccag 2160

ggcaagtaa 2169

<210> 2

<211> 2343

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggagttcg gcctgagctg ggtgttcctg gtggccatcc tgaagggcgt gcagtgcatg 60

ggcgccgacg acgtggtgga cagcagcaag agcttcgtga tggagaactt cagcagctac 120

cacggcacca agcccggcta cgtggacagc atccagaagg gcatccagaa gcccaagagc 180

ggcacccagg gcaactacga cgacgactgg aaggagttct acagcaccga caacaagtac 240

gacgccgccg gctacagcgt ggacaacgag aaccccctga gcggcaaggc cggcggcgtg 300

gtgaaggtga cctaccccgg cctgaccaag gtgctggccc tgaaggtgga caacgccgag 360

accatcaaga aggagctggg cctgagcctg accgagcccc tgatggagca ggtgggcacc 420

gaggagttca tcaagcgctt cggcgacggc gccagccgcg tggtgctgag cctgcccttc 480

gccgagggca gcagcagcgt ggagtacatc aacaactggg agcaggccaa ggccctgagc 540

gtggagctgg agatcaactt cgagacccgc ggcaagcgcg gccaggacgc catgtacgag 600

tacatggccc aggcctgcgc cggcaaccgc gtgcgccgca gcgtgggcag cagcctgagc 660

tgcatcaacc tggactggga cgtgatccgc gacaagacca agaccaagat cgagagcctg 720

aaggagcacg gccccatcaa gaacaagatg agcgagagcc ccaacaagac cgtgagcgag 780

gagaaggcca agcagtacct ggaggagttc caccagaccg ccctggagca ccccgagctg 840

agcgagctga agaccgtgac cggcaccaac cccgtgttcg ccggcgccaa ctacgccgcc 900

tgggccgtga acgtggccca ggtgatcgac agcgagaccg ccgacaacct ggagaagacc 960

accgccgccc tgagcatcct gcccggcatc ggcagcgtga tgggcatcgc cgacggcgcc 1020

gtgcaccaca acaccgagga gatcgtggcc cagagcatcg ccctgagcag cctgatggtg 1080

gcccaggcca tccccctggt gggcgagctg gtggacatcg gcttcgccgc ctacaacttc 1140

gtggagagca tcatcaacct gttccaggtg gtgcacaaca gctacaaccg ccccgcctac 1200

agccccggcc acaagaccca gcccttcctg cacgacggct acgccgtgag ctggaacacc 1260

gtggaggaca gcatcatccg caccggcttc cagggcgaga gcggccacga catcaagatc 1320

accgccgaga acacccccct gcccatcgcc ggcgtgctgc tgcccaccat ccccggcaag 1380

ctggacgtga acaagagcaa gacccacatc agcgtgaacg gccgcaagat ccgcatgcgc 1440

tgccgcgcca tcgacggcga cgtgaccttc tgccgcccca agagccccgt gtacgtgggc 1500

aacggcgtgc acgccaacct gcacgtggcc ttccaccgca gcagcagcga gaagatccac 1560

agcaacgaga tcagcagcga cagcatcggc gtgctgggct accagaagac cgtggaccac 1620

accaaggtga acagcaagct gagcctgttc ttcgagatca agagcggcgg cggcggcagc 1680

ggcggcggcg gcagcgtgga caccctgagc ggcctgagca gcgagcaggg ccagagcggc 1740

gacatgacca tcgaggagga cagcgccacc cacatcaagt tcagcaagcg cgacgaggac 1800

ggcaaggagc tggccggcgc caccatggag ctgcgcgaca gcagcggcaa gaccatcagc 1860

acctggatca gcgacggcca ggtgaaggac ttctacctgt accccggcaa gtacaccttc 1920

gtggagaccg ccgcccccga cggctacgag gtggccaccg ccatcacctt caccgtgaac 1980

gagcagggcc aggtgaccgt gaacggcaag gccaccaagg gcgacgccca catcggcttc 2040

atcttccccc ccagcaagga gcagctggcc acccccaccg tgagcgtggt gtgcctgatc 2100

aacaacttct tcccccgcga gatcagcgtg aagtggaagg tggacggcgt ggtgcagagc 2160

agcggccacc ccgacagcgt gaccgagcag gacagcaagg acagcaccta cagcctgagc 2220

agcaccctga gcctgcccac cagccagtac ctgagccaca acctgtacag ctgcgaggtg 2280

acccacaaga ccctggccag ccccctggtg accagcttca accgcaacga gtgcgaggcc 2340

taa 2343

<210> 3

<211> 722

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Gly Lys Val Leu Arg Gly Gln Ile Val Gln Gly Val Val

20 25 30

Trp Leu Leu Leu Val Thr Gly Ala Gln Gly Arg Leu Ala Cys Lys Glu

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Thr Phe Asn Tyr Thr Lys Thr Leu Lys Asn Arg Tyr Tyr Glu Pro Arg

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Gly Ser Tyr Pro Leu Ala Pro Cys Gly Arg Asp Thr Ser Gly Pro Asn

405 410 415

Val Ala Leu Gly Cys Leu Ala Ser Ser Tyr Phe Pro Glu Pro Val Thr

420 425 430

Met Thr Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

435 440 445

Ser Val Leu Gln Pro Ser Gly Leu Tyr Ser Leu Ser Ser Met Val Thr

450 455 460

Val Pro Ala Ser Ser Leu Ser Ser Lys Ser Tyr Thr Cys Asn Val Asn

465 470 475 480

His Pro Ala Thr Thr Thr Lys Val Asp Lys Arg Val Gly Thr Lys Thr

485 490 495

Lys Pro Pro Cys Pro Ile Cys Pro Gly Cys Glu Val Ala Gly Pro Ser

500 505 510

Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Gln

515 520 525

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Lys Glu His Ala

530 535 540

Glu Val Gln Phe Ser Trp Tyr Val Asp Gly Val Glu Val His Thr Ala

545 550 555 560

Glu Thr Arg Pro Lys Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

565 570 575

Ser Val Leu Pro Ile Gln His Gln Asp Trp Leu Lys Gly Lys Glu Phe

580 585 590

Lys Cys Lys Val Asn Asn Val Asp Leu Pro Ala Pro Ile Thr Arg Thr

595 600 605

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

610 615 620

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

625 630 635 640

Leu Val Ile Gly Phe Tyr Pro Pro Asp Ile His Val Glu Trp Lys Ser

645 650 655

Asn Gly Gln Pro Glu Pro Glu Gly Asn Tyr Arg Thr Thr Pro Pro Gln

660 665 670

Gln Asp Val Asp Gly Thr Phe Phe Leu Tyr Ser Lys Leu Ala Val Asp

675 680 685

Lys Ala Arg Trp Asp His Gly Glu Thr Phe Glu Cys Ala Val Met His

690 695 700

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Lys Thr Gln

705 710 715 720

Gly Lys

<210> 4

<211> 780

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Met Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe

20 25 30

Val Met Glu Asn Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val

35 40 45

Asp Ser Ile Gln Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly

50 55 60

Asn Tyr Asp Asp Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys Tyr

65 70 75 80

Asp Ala Ala Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys

85 90 95

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

100 105 110

Ala Leu Lys Val Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu

115 120 125

Ser Leu Thr Glu Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile

130 135 140

Lys Arg Phe Gly Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe

145 150 155 160

Ala Glu Gly Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala

165 170 175

Lys Ala Leu Ser Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys

180 185 190

Arg Gly Gln Asp Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly

195 200 205

Asn Arg Val Arg Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu

210 215 220

Asp Trp Asp Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu

225 230 235 240

Lys Glu His Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys

245 250 255

Thr Val Ser Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln

260 265 270

Thr Ala Leu Glu His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly

275 280 285

Thr Asn Pro Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn

290 295 300

Val Ala Gln Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr

305 310 315 320

Thr Ala Ala Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile

325 330 335

Ala Asp Gly Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser

340 345 350

Ile Ala Leu Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly

355 360 365

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

370 375 380

Ile Asn Leu Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr

385 390 395 400

Ser Pro Gly His Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val

405 410 415

Ser Trp Asn Thr Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly

420 425 430

Glu Ser Gly His Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro

435 440 445

Ile Ala Gly Val Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn

450 455 460

Lys Ser Lys Thr His Ile Ser Val Asn Gly Arg Lys Ile Arg Met Arg

465 470 475 480

Cys Arg Ala Ile Asp Gly Asp Val Thr Phe Cys Arg Pro Lys Ser Pro

485 490 495

Val Tyr Val Gly Asn Gly Val His Ala Asn Leu His Val Ala Phe His

500 505 510

Arg Ser Ser Ser Glu Lys Ile His Ser Asn Glu Ile Ser Ser Asp Ser

515 520 525

Ile Gly Val Leu Gly Tyr Gln Lys Thr Val Asp His Thr Lys Val Asn

530 535 540

Ser Lys Leu Ser Leu Phe Phe Glu Ile Lys Ser Gly Gly Gly Gly Ser

545 550 555 560

Gly Gly Gly Gly Ser Val Asp Thr Leu Ser Gly Leu Ser Ser Glu Gln

565 570 575

Gly Gln Ser Gly Asp Met Thr Ile Glu Glu Asp Ser Ala Thr His Ile

580 585 590

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

595 600 605

Met Glu Leu Arg Asp Ser Ser Gly Lys Thr Ile Ser Thr Trp Ile Ser

610 615 620

Asp Gly Gln Val Lys Asp Phe Tyr Leu Tyr Pro Gly Lys Tyr Thr Phe

625 630 635 640

Val Glu Thr Ala Ala Pro Asp Gly Tyr Glu Val Ala Thr Ala Ile Thr

645 650 655

Phe Thr Val Asn Glu Gln Gly Gln Val Thr Val Asn Gly Lys Ala Thr

660 665 670

Lys Gly Asp Ala His Ile Gly Phe Ile Phe Pro Pro Ser Lys Glu Gln

675 680 685

Leu Ala Thr Pro Thr Val Ser Val Val Cys Leu Ile Asn Asn Phe Phe

690 695 700

Pro Arg Glu Ile Ser Val Lys Trp Lys Val Asp Gly Val Val Gln Ser

705 710 715 720

Ser Gly His Pro Asp Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

725 730 735

Tyr Ser Leu Ser Ser Thr Leu Ser Leu Pro Thr Ser Gln Tyr Leu Ser

740 745 750

His Asn Leu Tyr Ser Cys Glu Val Thr His Lys Thr Leu Ala Ser Pro

755 760 765

Leu Val Thr Ser Phe Asn Arg Asn Glu Cys Glu Ala

770 775 780

<210> 5

<211> 11246

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gaattcattg atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa 60

cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt 120

gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa 180

agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca 240

tgtctggcgg ccgccgatat ttgaaaatat ggcatattga aaatgtcgcc gatgtgagtt 300

tctgtgtaac tgatatcgcc atttttccaa aagtgatttt tgggcatacg cgatatctgg 360

cgatagcgct tatatcgttt acgggggatg gcgatagacg actttggtga cttgggcgat 420

tctgtgtgtc gcaaatatcg cagtttcgat ataggtgaca gacgatatga ggctatatcg 480

ccgatagagg cgacatcaag ctggcacatg gccaatgcat atcgatctat acattgaatc 540

aatattggcc attagccata ttattcattg gttatatagc ataaatcaat attggctatt 600

ggccattgca tacgttgtat ccatatcata atatgtacat ttatattggc tcatgtccaa 660

cattaccgcc atgttgacat tgattattga ctagttatta atagtaatca attacggggt 720

cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc 780

ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag 840

taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc 900

acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg 960

gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc 1020

agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca 1080

atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca 1140

atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg 1200

ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc 1260

gtttagtgaa cccggtggag ggcagtgtag tctgagcagg acacgaagct tgccaccatg 1320

gagttcggcc tgagctgggt gttcctggtg gccatcctga agggcgtgca gtgcatgggc 1380

gccgacgacg tggtggacag cagcaagagc ttcgtgatgg agaacttcag cagctaccac 1440

ggcaccaagc ccggctacgt ggacagcatc cagaagggca tccagaagcc caagagcggc 1500

acccagggca actacgacga cgactggaag gagttctaca gcaccgacaa caagtacgac 1560

gccgccggct acagcgtgga caacgagaac cccctgagcg gcaaggccgg cggcgtggtg 1620

aaggtgacct accccggcct gaccaaggtg ctggccctga aggtggacaa cgccgagacc 1680

atcaagaagg agctgggcct gagcctgacc gagcccctga tggagcaggt gggcaccgag 1740

gagttcatca agcgcttcgg cgacggcgcc agccgcgtgg tgctgagcct gcccttcgcc 1800

gagggcagca gcagcgtgga gtacatcaac aactgggagc aggccaaggc cctgagcgtg 1860

gagctggaga tcaacttcga gacccgcggc aagcgcggcc aggacgccat gtacgagtac 1920

atggcccagg cctgcgccgg caaccgcgtg cgccgcagcg tgggcagcag cctgagctgc 1980

atcaacctgg actgggacgt gatccgcgac aagaccaaga ccaagatcga gagcctgaag 2040

gagcacggcc ccatcaagaa caagatgagc gagagcccca acaagaccgt gagcgaggag 2100

aaggccaagc agtacctgga ggagttccac cagaccgccc tggagcaccc cgagctgagc 2160

gagctgaaga ccgtgaccgg caccaacccc gtgttcgccg gcgccaacta cgccgcctgg 2220

gccgtgaacg tggcccaggt gatcgacagc gagaccgccg acaacctgga gaagaccacc 2280

gccgccctga gcatcctgcc cggcatcggc agcgtgatgg gcatcgccga cggcgccgtg 2340

caccacaaca ccgaggagat cgtggcccag agcatcgccc tgagcagcct gatggtggcc 2400

caggccatcc ccctggtggg cgagctggtg gacatcggct tcgccgccta caacttcgtg 2460

gagagcatca tcaacctgtt ccaggtggtg cacaacagct acaaccgccc cgcctacagc 2520

cccggccaca agacccagcc cttcctgcac gacggctacg ccgtgagctg gaacaccgtg 2580

gaggacagca tcatccgcac cggcttccag ggcgagagcg gccacgacat caagatcacc 2640

gccgagaaca cccccctgcc catcgccggc gtgctgctgc ccaccatccc cggcaagctg 2700

gacgtgaaca agagcaagac ccacatcagc gtgaacggcc gcaagatccg catgcgctgc 2760

cgcgccatcg acggcgacgt gaccttctgc cgccccaaga gccccgtgta cgtgggcaac 2820

ggcgtgcacg ccaacctgca cgtggccttc caccgcagca gcagcgagaa gatccacagc 2880

aacgagatca gcagcgacag catcggcgtg ctgggctacc agaagaccgt ggaccacacc 2940

aaggtgaaca gcaagctgag cctgttcttc gagatcaaga gcggcggcgg cggcagcggc 3000

ggcggcggca gcgtggacac cctgagcggc ctgagcagcg agcagggcca gagcggcgac 3060

atgaccatcg aggaggacag cgccacccac atcaagttca gcaagcgcga cgaggacggc 3120

aaggagctgg ccggcgccac catggagctg cgcgacagca gcggcaagac catcagcacc 3180

tggatcagcg acggccaggt gaaggacttc tacctgtacc ccggcaagta caccttcgtg 3240

gagaccgccg cccccgacgg ctacgaggtg gccaccgcca tcaccttcac cgtgaacgag 3300

cagggccagg tgaccgtgaa cggcaaggcc accaagggcg acgcccacat cggcttcatc 3360

ttccccccca gcaaggagca gctggccacc cccaccgtga gcgtggtgtg cctgatcaac 3420

aacttcttcc cccgcgagat cagcgtgaag tggaaggtgg acggcgtggt gcagagcagc 3480

ggccaccccg acagcgtgac cgagcaggac agcaaggaca gcacctacag cctgagcagc 3540

accctgagcc tgcccaccag ccagtacctg agccacaacc tgtacagctg cgaggtgacc 3600

cacaagaccc tggccagccc cctggtgacc agcttcaacc gcaacgagtg cgaggcctaa 3660

gaattcattg atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa 3720

cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt 3780

gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa 3840

agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca 3900

tgtctggatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt 3960

tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct 4020

catgagcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg 4080

cgccatctcc ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact 4140

actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgacctc gggccgcgtt 4200

gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 4260

tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 4320

cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 4380

ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 4440

cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 4500

atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 4560

agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 4620

gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 4680

gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 4740

tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 4800

agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 4860

gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 4920

aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 4980

aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 5040

ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 5100

gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 5160

aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 5220

ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 5280

tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 5340

ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 5400

cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 5460

agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 5520

gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 5580

gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 5640

acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 5700

acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 5760

agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 5820

aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 5880

gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 5940

tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa 6000

aaataggcgt atcacgaggc cctgatggct ctttgcggca cccatcgttc gtaatgttcc 6060

gtggcaccga ggacaaccct caagagaaaa tgtaatcaca ctggctcacc ttcgggtggg 6120

cctttctgcg tttataagga gacactttat gtttaagaag gttggtaaat tccttgcggc 6180

tttggcagcc aagctagatc cggctgtgga atgtgtgtca gttagggtgt ggaaagtccc 6240

caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt 6300

gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt 6360

cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg 6420

cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct 6480

cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca 6540

aaaagctagc ttggggccac cgctcagagc accttccacc atggccacct cagcaagttc 6600

ccacttgaac aaaaacatca agcaaatgta cttgtgcctg ccccagggtg agaaagtcca 6660

agccatgtat atctgggttg atggtactgg agaaggactg cgctgcaaaa cccgcaccct 6720

ggactgtgag cccaagtgtg tagaagagtt acctgagtgg aattttgatg gctctagtac 6780

ctttcagtct gagggctcca acagtgacat gtatctcagc cctgttgcca tgtttcggga 6840

ccccttccgc agagatccca acaagctggt gttctgtgaa gttttcaagt acaaccggaa 6900

gcctgcagag accaatttaa ggcactcgtg taaacggata atggacatgg tgagcaacca 6960

gcacccctgg tttggaatgg aacaggagta tactctgatg ggaacagatg ggcacccttt 7020

tggttggcct tccaatggct ttcctgggcc ccaaggtccg tattactgtg gtgtgggcgc 7080

agacaaagcc tatggcaggg atatcgtgga ggctcactac cgcgcctgct tgtatgctgg 7140

ggtcaagatt acaggaacaa atgctgaggt catgcctgcc cagtgggagt tccaaatagg 7200

accctgtgaa ggaatccgca tgggagatca tctctgggtg gcccgtttca tcttgcatcg 7260

agtatgtgaa gactttgggg taatagcaac ctttgacccc aagcccattc ctgggaactg 7320

gaatggtgca ggctgccata ccaactttag caccaaggcc atgcgggagg agaatggtct 7380

gaagcacatc gaggaggcca tcgagaaact aagcaagcgg caccggtacc acattcgagc 7440

ctacgatccc aaggggggcc tggacaatgc ccgtcgtctg actgggttcc acgaaacgtc 7500

caacatcaac gacttttctg ctggtgtcgc caatcgcagt gccagcatcc gcattccccg 7560

gactgtcggc caggagaaga aaggttactt tgaagaccgc cgcccctctg ccaattgtga 7620

cccctttgca gtgacagaag ccatcgtccg cacatgcctt ctcaatgaga ctggcgacga 7680

gcccttccaa tacaaaaact aattaaatga atgcaattgt tgttgttaac ttgtttattg 7740

cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 7800

tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga 7860

tctagcttcg tgtcaaggac ggtgactgca gtgaataata aaatgtgtgt ttgtccgaaa 7920

tacgcgtttt gagatttctg tcgccgacta aattcatgtc gcgcgatagt ggtgtttatc 7980

gccgatagag atggcgatat tggaaaaatc gatatttgaa aatatggcat attgaaaatg 8040

tcgccgatgt gagtttctgt gtaactgata tcgccatttt tccaaaagtg atttttgggc 8100

atacgcgata tctggcgata gcgcttatat cgtttacggg ggatggcgat agacgacttt 8160

ggtgacttgg gcgattctgt gtgtcgcaaa tatcgcagtt tcgatatagg tgacagacga 8220

tatgaggcta tatcgccgat agaggcgaca tcaagctggc acatggccaa tgcatatcga 8280

tctatacatt gaatcaatat tggccattag ccatattatt cattggttat atagcataaa 8340

tcaatattgg ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata 8400

ttggctcatg tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt 8460

aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta 8520

cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga 8580

cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt 8640

tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta 8700

ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg 8760

actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt 8820

tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc 8880

accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat 8940

gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct 9000

atataagcag agctcgttta gtgaaccgtc aggtggaggg cagtgtagtc tgagcagttg 9060

acacgaagct tgccaccatg gagttcggcc tgagctgggt gttcctggtg gccatcctga 9120

agggcgtgca gtgcggcaag gtgctgcgcg gccagatcgt gcagggcgtg gtgtggctgc 9180

tgctggtgac cggcgcccag ggccgcctgg cctgcaagga ggactaccgc tacgccatca 9240

gcagcaccga cgagatcggc ctgctgggcg ccggcggcct gaccaccacc tggaaggagt 9300

acaaccacga cctgcagctg aacgacggca ccgtgaaggc cagctgcgtg gccggcagct 9360

tcaaggtgac cgccctgaac gtggtgagcc gccgctacct ggccagcctg cacaagaagg 9420

ccctgcccac cagcgtgacc ttcgagctgc tgttcgacgg caccaacccc agcaccgagg 9480

agatgggcga cgacttccgc agcggcctgt gccccttcga caccagcccc gtggtgaagg 9540

gcaagtacaa caccaccctg ctgaacggca gcgccttcta cctggtgtgc cccatcggct 9600

ggaccggcgt gatcgagtgc accgccgtga gccccaccac cctgcgcacc gaggtggtga 9660

agaccttccg ccgcgacaag cccttccccc accgcatgga ctgcgtgacc accaccgtgg 9720

agaacgagga cctgttctac tgcaagctgg gcggcaactg gacctgcgtg aagggcgagc 9780

ccgtggtgta caccggcggc gtggtgaagc agtgccgctg gtgcggcttc gacttcgacg 9840

gccccgacgg cctgccccac taccccatcg gcaagtgcat cctggccaac gagaccggct 9900

accgcatcgt ggacagcacc gactgcaacc gcgacggcgt ggtgatcagc accgagggca 9960

gccacgagtg cctgatcggc aacaccaccg tgaaggtgca cgccagcgac gagcgcctgg 10020

gccccatgcc ctgccgcccc aaggagatcg tgagcagcgc cggccccgtg atgaagacca 10080

gctgcacctt caactacacc aagaccctga agaaccgcta ctacgagccc cgcgacagct 10140

acttccagca gtacatgctg aagggcgagt accagtactg gttcgacctg gacgccaccg 10200

accgccacag cgacggcggc ggcggcagcg cccacatcgt gatggtggac gcctacaagc 10260

ccaccaaggg cggcggcggc agctaccccc tggccccctg cggccgcgac accagcggcc 10320

ccaacgtggc cctgggctgc ctggccagca gctacttccc cgagcccgtg accatgacct 10380

ggaacagcgg cgccctgacc agcggcgtgc acaccttccc cagcgtgctg cagcccagcg 10440

gcctgtacag cctgagcagc atggtgaccg tgcccgccag cagcctgagc agcaagagct 10500

acacctgcaa cgtgaaccac cccgccacca ccaccaaggt ggacaagcgc gtgggcacca 10560

agaccaagcc cccctgcccc atctgccccg gctgcgaggt ggccggcccc agcgtgttca 10620

tcttcccccc caagcccaag gacaccctga tgatcagcca gacccccgag gtgacctgcg 10680

tggtggtgga cgtgagcaag gagcacgccg aggtgcagtt cagctggtac gtggacggcg 10740

tggaggtgca caccgccgag acccgcccca aggaggagca gttcaacagc acctaccgcg 10800

tggtgagcgt gctgcccatc cagcaccagg actggctgaa gggcaaggag ttcaagtgca 10860

aggtgaacaa cgtggacctg cccgccccca tcacccgcac catcagcaag gccatcggcc 10920

agagccgcga gccccaggtg tacaccctgc ccccccccgc cgaggagctg agccgcagca 10980

aggtgaccgt gacctgcctg gtgatcggct tctacccccc cgacatccac gtggagtgga 11040

agagcaacgg ccagcccgag cccgagggca actaccgcac cacccccccc cagcaggacg 11100

tggacggcac cttcttcctg tacagcaagc tggccgtgga caaggcccgc tgggaccacg 11160

gcgagacctt cgagtgcgcc gtgatgcacg aggccctgca caaccactac acccagaaga 11220

gcatcagcaa gacccagggc aagtaa 11246

Claims

1. A protein characterized by: the protein is formed by connecting a fusion protein (CSFV-E2) -SpyTag-HIgG and a fusion protein CRM 197-Spycatcher-LIgG;

the fusion protein (CSFV-E2) -SpyTag-HIgG is CSFV-E2, spyTag polypeptide and pig IgG heavy chain constant region from N terminal to C terminal, respectively, and the fusion protein CRM197-Spycatcher-LIgG is CRM197, spycatcher polypeptide and pig IgG light chain constant region from N terminal to C terminal, respectively, wherein SpyTag and Spycatcher can spontaneously form isopeptide bond coupling, thereby connecting the fusion protein (CSFV-E2) -SpyTag-HIgG and the fusion protein CRM 197-Spycatcher-LIgG;

the fusion protein (CSFV-E2) -SpyTag-HIgG is A1) or A2) as follows:

a1 Protein of which the amino acid sequence is SEQ ID No.3;

a2 A fusion protein obtained by connecting a label to the N-terminal and/or the C-terminal of A1);

the fusion protein CRM197-SpyCatcher-LIgG is B1) or B2) as follows:

b1 Protein of which the amino acid sequence is SEQ ID No.4;

b2 B1) to the N-terminus and/or C-terminus of B1).

2. A nucleic acid molecule characterized in that: the nucleic acid molecule consists of a nucleic acid molecule encoding the fusion protein (CSFV-E2) -SpyTag-HIgG and a nucleic acid molecule encoding the fusion protein CRM 197-Spycatcher-LIgG;

the nucleic acid molecule for coding the fusion protein (CSFV-E2) -SpyTag-HIgG is a DNA molecule shown in SEQ ID No. 1;

the nucleic acid molecule for coding the fusion protein CRM197-SpyCatcher-LIgG is a DNA molecule shown in SEQ ID No. 2.

3. An expression cassette characterized by: the expression cassette contains 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 in that: the recombinant microorganism contains the nucleic acid molecule of claim 2 or contains the expression cassette of claim 3 or contains 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 the steps of: a step of expressing a gene encoding the fusion protein (CSFV-E2) -SpyTag-HIgG according to claim 1 and a gene encoding the fusion protein CRM197-Spycatcher-LIgG according to claim 1 in a biological cell to obtain the protein; 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.