CN111393531A - Subunit fusion protein CD2V-Fc and preparation method and application thereof - Google Patents

Subunit fusion protein CD2V-Fc and preparation method and application thereof Download PDF

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CN111393531A
CN111393531A CN201910268140.XA CN201910268140A CN111393531A CN 111393531 A CN111393531 A CN 111393531A CN 201910268140 A CN201910268140 A CN 201910268140A CN 111393531 A CN111393531 A CN 111393531A
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钱泓
吴有强
张强
徐玉兰
吴素芳
车影
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Zhejiang Hailong Biotechnology Co ltd
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Abstract

The invention provides a subunit fusion protein CD2V-Fc and a preparation method and application thereof, wherein the subunit fusion protein CD2V-Fc contains an extracellular region of an African swine fever virus surface envelope protein CD2V and an antibody Fc protein of a pig, and the amino acid sequence of the subunit fusion protein CD2V-Fc is shown as SEQ ID No. 1. The invention can express CD2V-Fc in a large amount of soluble way, has stable protein, overcomes a plurality of problems in the prior art, and has simple preparation method and low cost.

Description

Subunit fusion protein CD2V-Fc and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological products for livestock. Relates to subunit fusion protein CD2V-Fc and a preparation method and application thereof.
Background
African Swine Fever (ASF) is an acute, febrile, highly contagious disease of swine caused by African Swine Fever Virus (ASFV). Pigs are the only mammalian host for natural infection of ASFV, including domestic pigs and wild pigs, especially domestic pigs, with extremely high susceptibility. After the swine is infected by African swine fever virus, the swine is clinically characterized by skin congestion, organ bleeding and high fever, and the morbidity and mortality rate are up to 100 percent. The world animal health organization classifies the zoonosis as a type A epidemic disease, and the world also classifies the zoonosis as a type infectious disease.
Since 1927, African swine fever was discovered in the African continent, there was a tremendous shock to the swine industry in both Africa and Europe. Since 8 months in 2018, outbreaks in a plurality of provinces of China bring serious economic losses to the pig industry in China. Although scholars at home and abroad do a lot of research work on African swine fever, the research finds that: the conventional African swine fever inactivated vaccine has an unobvious effect, and a weak virus vaccine has a poor protection effect and poor safety and is easy to cause virus dispersion. At present, no vaccine for effectively preventing the African swine fever and a medicament for treating the African swine fever are found in the world, and the development and production of a novel vaccine for preventing the African swine fever are urgently needed.
ASFV virus is an arbovirus DNA virus with an envelope. The virus particles are in an icosahedral symmetrical structure, the average diameter is 200nm, and the surfaces of the virus particles are covered by the sacculus membranes containing glycolipids. The viral genome is double-stranded linear DNA with the size of 170-190kb, and the whole genome has about 150 ORFs and encodes 150-200 proteins. The CD2V protein is encoded by the EP402R gene and has a signal peptide and a transmembrane region. The amino acid residues of the extracellular region are similar to those of the CD2V protein of a host, comprise 2 immunoglobulin-like domains, and can adsorb erythrocytes, thereby playing an important role in the process of virus spreading and lymphocyte damage. The research finds that CD2V is a virus envelope surface protein, and the antibody aiming at CD2V can well prevent the adsorption of the virus. Thus, CD2V is a very good protective antigen. In Ruiz-Gonzalvo, F., Rodriguez, F.and Escribano, J.M., Virology 218,285-289(1996), the expression of the extracellular region of CD2V was reported using a baculovirus system, and only results were obtained for western, without SDS-PAGE, with very low expression. However, it is not shown that the expression is large, which indicates that the stability of the extracellular domain of CD2V is poor, and therefore, it is difficult to apply the method in practical production. The antibody protein is abundant in blood, has a half-life of 21 days, and an Fc fragment of the antibody protein is a constant region of the antibody, has a homodimer structure, and has the function of stabilizing the protein. Therefore, the extracellular domain of the African swine fever virus CD2V protein and the Fc fragment of the antibody of the pig are creatively fused and expressed, and the invention finds that the homodimer structure of the CD2V can be maintained, the stability of the CD2V can be effectively maintained, and the invention can be produced and applied in a large scale. In the absence of a current possibility for large-scale production of inactivated or attenuated vaccines, it is of great interest to identify a method for producing an immunogenic protein of the virus, in order to study a vaccine capable of preventing the disease or to have subunit proteins capable of preventing the disease.
Disclosure of Invention
The invention aims to solve the technical problem of providing a fusion protein containing African swine fever surface CD2V subunit protein, a preparation method and application thereof, wherein the fusion protein can be industrially produced in a large scale;
in order to solve the problem, the inventor provides a subunit fusion protein CD2V-Fc which can stably and efficiently express the African swine fever CD2V antigen and pig immunoglobulin Fc and a construction and expression method thereof in a CHO or 293T cell system on the basis of fully analyzing and researching the currently available data of the African swine fever virus and analyzing the whole structure of the CD2V protein through a structure. The monoclonal cell strain capable of secreting and expressing the subunit fusion protein CD2V-Fc obtained by the invention has high expression yield, and a large amount of fusion proteins can be obtained by one-step affinity chromatography. The subunit fusion protein CD2V-Fc contains the extracellular region of African swine fever virus surface envelope protein CD2V and antibody Fc protein of pigs, and the amino acid sequence of the subunit fusion protein CD2V-Fc is shown in SEQ ID NO. 1.
According to the technical scheme of the subunit fusion protein CD2V-Fc, preferably, the amino acid sequence shown in SEQ ID NO1 comprises a derivative protein which is substituted, deleted or added with one amino acid or a plurality of amino acids and has immunogenicity.
According to the technical scheme of the subunit fusion protein CD2V-Fc, preferably, the amino acid sequence shown in SEQ ID NO1 comprises a derivative protein obtained by reversing the order of CD2V and Fc and placing Fc at the amino terminal.
According to the technical scheme of the subunit fusion protein CD2V-Fc, preferably, the amino acid sequence of the extracellular region of the African swine fever virus surface envelope protein CD2V is shown as SEQ ID No. 2.
According to the technical scheme of the subunit fusion protein CD2V-Fc, preferably, the porcine antibody Fc protein is the heavy chain constant region of porcine IgG, and the amino acid sequence of the porcine antibody Fc protein is shown as SEQ ID No. 3.
According to the technical scheme of the subunit fusion protein CD2V-Fc, one or more tag amino acids of poly-Arg, poly-His, flag, c-myc and HA are preferably connected to the amino terminal or the carboxyl terminal of the amino acid sequence shown in SEQ ID NO. 1.
According to the technical scheme of the subunit fusion protein CD2V-Fc, preferably, the expression system of the subunit fusion protein CD2V-Fc of the African swine fever CD2V comprises but is not limited to mammalian cells and insect cells. Preferably, the mammalian cell is a CHO cell, 293T cell. The mammalian cells are CHO cells and 293T cells. More preferably, the mammalian cell is a CHO cell.
According to another aspect of the present invention, there is provided a method for preparing a subunit fusion protein CD2V-Fc, the method comprising the steps of: 1) cloning the gene coded by the subunit fusion protein CD2V-Fc of the African swine fever CD2V shown in SEQ ID NO.4 into a eukaryotic expression vector to obtain a recombinant plasmid containing the gene coded by the subunit fusion protein CD2V-Fc of the African swine fever CD 2V; 2) transfecting a recombinant plasmid containing a gene coded by the subunit fusion protein of the African swine fever CD2V into a CHO cell to obtain a CHO cell strain; 3) culturing, screening and domesticating the CHO cell strain in the step 2) to obtain a highly expressed cell strain; 4) fermenting and culturing the highly expressed cell strain in the step 3), and purifying to obtain the subunit fusion protein CD2V-Fc of the recombinant African swine fever CD 2V.
According to the technical scheme of the preparation method, preferably, in the step 1), the eukaryotic expression vector is pEE6.4, pEE12.4, pG L4.13.13, pcDNA3.1, and pcDNA3.3, and preferably, the eukaryotic expression vector is pEE12.4.
According to the technical scheme of the preparation method, preferably, the CHO cells are DG44, DXB11, CHO-K1 and CHO-S cells. Preferably, the CHO cells are CHO-K1 cells.
According to the technical scheme of the preparation method, preferably, the expression system of the subunit fusion protein CD2V-Fc of the African swine fever CD2V is a mammalian cell, and the mammalian cell is a CHO cell or a 293T cell.
The invention constructs and screens a suspension stable high-efficiency secretion expression African swine fever virus CD2V subunit fusion protein CD2V-Fc CHO cell strain, the cell strain expresses the subunit fusion protein CD2V-Fc high yield (the yield is up to 1-2 g/L), is easy to purify (as shown in figure 4, the purity of the target protein in the cell culture supernatant can reach more than 70%, only one step of affinity chromatography is needed to ensure that the purity of the target protein can reach more than 90%, and the requirements of subunit vaccine and diagnostic reagent are met far, and the large-scale production is easy.
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FIG. 1 shows a map of the plasmid pEE12.4-OPTI-CD 2V-Fc.
FIG. 2 shows the results of the double-restriction enzyme identification of pEE12.4-OPTI-CD2V-Fc, wherein M is DNA Marker D L10000 Marker, and 1 is pEE12.4-OPTI-CD 2V-Fc.
FIG. 3 shows the SDS-PAGE detection of subunit fusion protein CD2V-Fc after purification: 1 is subunit fusion protein CD2V-Fc, 2 is Marker.
FIGS. 4A and B show SDS-PAGE of purified subunit fusion protein CD2V-Fc after 10 weeks at 4 ℃ and-20 ℃ respectively: m is Marker, 1 is subunit fusion protein CD2V-Fc at 4 ℃ after 10 weeks of storage; 2 is the subunit fusion protein CD2V-Fc at-20 ℃ and after 10 weeks of storage.
FIG. 5 shows the results of the Westernblot assay of subunit fusion protein CD2V-Fc after purification: 1 is Marker, 2 is subunit fusion protein CD2V-Fc
Detailed Description
The present invention will be further described with reference to the accompanying drawings and examples, which are only for illustrating the technical solutions of the present invention and are not to be construed as limiting the present invention.
The strains, plasmids and reagents used in the examples of the present invention are all commercially available products.
The sources of the reagent and the medicine of the invention are listed as follows:
the CHO-K1 cells are derived from cell banks of China academy of sciences type culture Collection cell banks, Shanghai Life sciences research institute of China academy of sciences;
cell culture medium and serum were purchased from gibco, usa;
the eukaryotic expression vector pEE12.4 is purchased from Shanghai Linyuan Biotech, Inc.;
l ipofectamine L TX from Thermo Fisher, USA;
methionine sulfoxide iminium ((L-methionine sulfoximine, MSX)) was purchased from Sigma;
BCA protein quantification kit was purchased from Thermo Fisher, USA.
Example 1 expression and preparation of CD2V-Fc protein
1.1 selection of African Swine fever CD2V-Fc protein
The African swine fever structural protein CD2V is a segment of polypeptide encoded by EP402R gene, and prediction analysis shows that a transmembrane region is arranged at 207-229aa, and research has shown that the CD2V protein can interact with erythrocytes and has important functions in the process of virus spreading and lymphocyte injury. Therefore, the CD2V protein is used as an antigen to well prevent and control the infection of African swine fever, and the protein can be obtained by large-scale expression and purification in a eukaryotic expression system at present, which is probably caused by the instability of the CD2V protein, so that the invention introduces the Fc fragment of the antibody of the pig at the carboxyl terminal of the CD2V to ensure the structure and the protein stability of the CD 2V.
1.2 African Swine fever CD2V-Fc protein codon optimization
In the laboratory, by taking an epidemic African swine fever strain subtype reported in 2018 in China and taking a Georgia 2007/1 complete gene sequence (GenBank: FR682468.1) as a template, a genome sequence 73369-. Further analysis showed that 1M-15S may be a secretion signal peptide of CD2V protein, and 16Q-206Y may be an extracellular region of CD2V protein. The predicted three-dimensional structure of the amino acid sequence is similar to that of human CD2 protein, and the three-dimensional structure pattern is shown in FIG. 1. From the structural point of view, the protein is a homodimer structure, and further in order to ensure that CD2V is stably expressed, a pig Fc sequence (GenBank: AK405774.1), namely CD2V-Fc is added at the carboxyl terminal of the extracellular region of CD2V to serve as an immunogenic protein. The amino acid sequence is shown as SEQ ID NO. 1.
In order to facilitate the purification of the subunit CD2V-Fc protein, a tag as shown in table one can be attached to the amino terminus or carboxy terminus of the amino acid sequence shown in SEQ ID No.1, specifically exemplified by Poly-His in this example, which is attached to the carboxy terminus of the amino acid sequence shown in SEQ ID No. 5.
TABLE-TAG AND ITS AMINO ACID SEQUENCE
Figure BDA0002017501170000051
The gene sequence of the sequence after the amino acid optimization of the segment is coded is shown as SEQ ID NO.6, and the sequence synthesis work is finished by Nanjing Kingsrey Biotech Co.
Example 2: construction of pEE12.4-OPTI-CD2V-Fc recombinant plasmid
2.1 PCR amplification of the fragment of interest OPTI-CD2V-Fc
2.1.1 PCR reaction
(1) Primer design and Synthesis
Upstream primer 5'-acgaAGCTTGCCGCCACCATGATCAT-3'
Downstream primer 5'-GCGGAATTGAATTCTTAATGGTGATG-3'
(2) Sample application system 50 μ L, as shown in the following table:
Figure BDA0002017501170000061
PCR amplification procedure:
Figure BDA0002017501170000062
2.1.2 PCR products for gel recovery
(1) Marking a sample collection EP tube, an adsorption column and a collection tube;
(2) weighing the weight of the marked empty EP pipe, and recording the numerical value;
(3) a single DNA band of interest was carefully cut from the agarose gel with a scalpel on a gel cutter into a clean 1.5m L centrifuge tube;
(4) adding 600 mu L PC buffer into the centrifugal tube of 1.5m L in the step (3), placing in a water bath at 50 ℃ for about 5min, and turning the centrifugal tube up and down continuously and gently to ensure that the gel block is fully dissolved;
(5) column equilibration, namely adding 500 mu L equilibration solution B L into an adsorption column CB2 (the adsorption column is put into a collection tube in advance), centrifuging at 12,000rpm/min for 1min, pouring off waste liquid in the collection tube, and putting the adsorption column back into the collection tube again;
(6) adding the solution obtained in the step (5) into an adsorption column CB2, standing for 2min at 10,000rpm/min, centrifuging for 30s, pouring out waste liquid in a collecting pipe, and then putting the adsorption column CB2 into the collecting pipe;
(7) adding a rinsing liquid PW buffer of 600 mu L into the adsorption column, standing for 3min, centrifuging at 10,000rpm/min for 30s, pouring off waste liquid in the collecting tube, and putting the adsorption column CB2 into the collecting tube;
(8) repeating the step (7);
(9) centrifuging with an empty adsorption column at 12,000rpm/min for 2min, removing rinsing liquid as much as possible, standing the adsorption column at room temperature for 10min, and completely air drying;
(10) placing adsorption column CB2 into a collecting tube, suspending and dropwise adding 50 μ L Elutionbuffer (preheated at 65 ℃) to the middle position of an adsorption film, standing for 3min, and centrifuging at 12,000rpm/min for 2 min;
(11) taking the centrifuge tube in the step (10) out of the centrifuge, discarding the middle adsorption column CB2, covering the centrifuge tube with a cover, and keeping the DNA sample in the centrifuge tube;
(12) and (3) storing the DNA sample in the step 11 at 4 ℃, and preparing an agarose gel electrophoresis identification gel to recover the DNA fragment.
2.2 double digestion of PCR products and vectors
(1) The required 1.5m L EP tube is marked, and the sample is added and mixed in the 1.5m L EP tube according to the following table, namely, a 50 mu L reaction system
Figure BDA0002017501170000071
(2) And (3) placing the 1.5m L EP pipe in the step (1) into a corresponding enzyme constant-temperature water bath kettle with the optimal temperature, and carrying out water bath for 2-3 h.
Recovering the double enzyme digestion product gel: taking out the double enzyme digestion system, and carrying out agarose gel electrophoresis to recover the DNA fragment in the double enzyme digestion system by the same method as that of the PCR product gel recovery in the 1.2.1.
2.3 ligation reaction
(1) A plurality of clean 1.5m L EP tubes are prepared, marked and placed on an EP tube rack for standby.
(2) The sample was loaded and mixed in a 1.5m L EP tube according to the following table.
Figure BDA0002017501170000072
Figure BDA0002017501170000081
(3) After sample adding is finished according to the table in the step (2), placing each 10 mu l reaction system in a low-temperature cooling liquid circulator at the temperature of 16 ℃ for water bath for 10-16 h;
(4) taking out the EP tube in the step (3), placing the EP tube in a water bath kettle at 65 ℃, and carrying out water bath for 15 min;
(5) taking out the EP tube in the step (4), and storing at 4 ℃.
2.4 conversion reaction
(1) Quickly adding 10 mu L ligation reaction liquid into 100 mu L competent cells, uniformly blowing and stirring, and carrying out ice bath for 30 min;
(2) taking out the sample tube, placing in water bath at 42 ℃ for 100s, and immediately carrying out ice bath for 2 min;
(3) taking out the sample tube, adding 600 mu L liquid L B culture medium into the sample tube in an ultra-clean workbench, then placing the sample tube on a constant temperature shaking table at 37 ℃, and culturing for 1h at 220 rpm/min;
(4) and (3) plate coating, namely taking out the sample tube in the step (3), centrifuging at room temperature for 8,000rpm/min for 2min, removing 600 mu L of supernatant liquid, re-suspending the thalli at the bottom of the tube by using the residual supernatant liquid, putting the re-suspended bacterial liquid into the center of a corresponding transformation plate, and uniformly spreading the bacterial liquid in the center of the transformation plate by using a bacteria coating rod.
(5) The flat plate in the transformation step (4) is placed in a biochemical constant-temperature incubator, and is cultured for 1h at 37 ℃, and then the transformation flat plate is inverted and cultured for 15 h;
(6) the transformation results were observed.
2.5 plasmid extraction and double restriction enzyme identification
2.5.1 plasmid extraction
(1) Single clones were picked from the transformation plates using a 10 μ L pipette tip into 5m L liquid medium containing ampicillin-resistant L B, shaken overnight at 37 ℃ and 220 rpm/min;
(2) transferring the bacterial liquid into a 1.5m L EP tube, centrifuging at room temperature at 12,000rpm/min for 2min, and removing the supernatant;
(3) adding 250 mu L plasmid extraction reagent P1buffer into the EP tube in the step (2), and completely suspending the thalli;
(4) adding 250 mu L P2 buffer into the solution in the step (3), immediately and gently inverting the centrifuge tube for 5-10 times, uniformly mixing, and standing for 2-4min at room temperature;
(5) adding 350 mu L P3 buffer into the solution in the step (4), immediately and gently inverting the centrifuge tube for 5-10 times, and uniformly mixing, standing at room temperature for 2-4 min;
(6) centrifuging the solution in the step (5) at room temperature, and carrying out centrifugation at 14,000rpm/min for 10 min;
(7) transferring the supernatant solution in the step (6) to the center of an adsorption column, centrifuging at room temperature for 30s at 12,000rpm/min, and pouring out liquid in a collecting pipe;
(8) adding 500 mu L buffer DW1 into the center of the adsorption column, centrifuging at room temperature for 30s at 12,000rpm/min, and pouring off the liquid in the collection tube;
(9) adding 500 mu L wash solution into the center of the adsorption column, centrifuging at room temperature, at 12,000rpm/min for 30s, pouring out the liquid in the collection tube, and repeating the steps once;
(10) the column was air-adsorbed, centrifuged at room temperature, 12,000rpm, 2 min.
(11) The adsorption column was placed in a clean 1.5m L centrifuge tube, 30. mu. L Elutionbuffer was added to the center of the adsorption membrane, left to stand at room temperature for 5min, centrifuged at room temperature, 12,000rpm, and the DNA solution in the tube was stored for 2 min.
2.5.2 double restriction enzyme identification
(1) The sample was loaded into the 1.5m L EP tube labeled as required according to the following table, 20. mu. L reaction system
Figure BDA0002017501170000091
(2) And (3) putting the EP tube 20 mu L reaction system in the step (1) into a constant-temperature water bath kettle at 37 ℃ and carrying out water bath for 2 h.
(3) Carrying out agarose gel electrophoresis on the double enzyme digestion system sample in the step (2), and checking whether the size of the inserted fragment is correct; the results are shown in FIG. 2: the construction is correct by enzyme digestion identification.
(4) Clones with correct inserts were selected for sequencing by the sequencing company.
2.6 Large extract of endotoxin-free plasmid
2.6.1 endotoxin-free plasmid extraction
(1) Inoculating the clone with correct sequencing into 100m L benzyl-containing resistant culture medium, shaking at 37 deg.C, and culturing at 220rpm/min for 15 h;
(2) transferring the bacterial liquid cultured in the step (1) into a 50m L centrifuge tube, centrifuging for 5min at room temperature of 8,000rpm/min, collecting thalli, and discarding a supernatant culture medium;
(3) adding 8m L solution P1 into the centrifuge tube in the step (2), and fully resuspending the thalli by a pipette;
(4) adding 8m L solution P2 into the centrifuge tube in the step (3), immediately and gently inverting the centrifuge tube for 6-8 times, and standing for 5min at room temperature;
(5) adding 8m L solution P4 into the centrifugal tube in the step (4), immediately turning upside down for 6-8 times, fully and uniformly mixing until white flocculent precipitate appears in the solution, standing at room temperature for about 10min, centrifuging at room temperature of 8,000rpm/min for 5-10min, and separating the white precipitate to the bottom of the tube;
(6) carefully transferring all the supernatant obtained in the step (5) into a filter CS1, slowly pushing the filter, and collecting the filtrate in a clean 50m L centrifuge tube;
(7) column equilibration, namely adding 2.5m L of equilibration liquid B L into an adsorption column CP6 (the adsorption column is placed into a 50m L collection tube), centrifuging for 2min at room temperature of 8,000rpm/min, pouring off waste liquid in the collection tube, and putting the adsorption column back into the collection tube again;
(8) adding isopropanol with the volume 0.3 times that of the filtrate in the step (6), turning upside down, mixing uniformly, and transferring to an adsorption column CP 6. Centrifuging at 8,000rpm/min for 2min at room temperature, pouring out liquid in the collecting tube, and putting the adsorption column CP6 into the same collecting tube again;
(9) adding 10m of L rinsing liquid PW into the adsorption column CP6 in the step (8), centrifuging for 2min at the room temperature of 8,000rpm/min, discarding the waste liquid in the collection tube, and putting the adsorption column back into the collection tube again;
(10) repeating the operation step (9) once;
(11) adding 3m L of absolute ethyl alcohol into the adsorption column CP6 in the step (10), centrifuging for 2min at the room temperature of 8,000rpm/min, and pouring off waste liquid;
(12) the adsorption column CP6 from step (11) was replaced in the collection tube and centrifuged at 8,000rpm/min for 5min at room temperature. Opening the adsorption column CP6, placing at room temperature, standing for several minutes, and air drying;
(13) and (3) putting the adsorption column in the step (12) into a clean 50m L centrifuge tube, adding 1-2m L buffer TB into the center of an adsorption film, standing for 5min at room temperature, centrifuging for 2min at room temperature of 8,000rpm/min, transferring all eluent in the 50m L centrifuge tube into a clean 1.5m L centrifuge tube, and measuring the concentration and storing at-20 ℃.
(14) The obtained plasmid DNA solution of 1-2. mu. L was subjected to agarose gel electrophoresis and the data of the electrophoresis results were saved.
Example 3: establishment of pEE12.4-OPTI-CD2V-Fc recombinant plasmid transfection CHO-K1 cell and monoclonal screening
3.1 CHO-K1 cell transfection
(1) Preparing: sterilizing the biological safety cabinet for 30min by ultraviolet; DMEM/F12 (containing 10% serum and 1% double antibody), DMEM/F12 and PBS were preheated to 37 ℃ in a 37 ℃ water bath.
(2) The cells (10cm cell culture dish) were removed from the 37 ℃ incubator, the supernatant medium was discarded, the cells were washed once with pre-warmed 8m L PBS, and the PBS was discarded.
(3) Adding 1-2m L0.25.25% trypsin-EDTA into each 10cm cell culture dish, digesting at room temperature for about 2min, observing the cells under a microscope to shrink and become round, and displaying the cells as single cells.
(4) The digestion reaction was stopped by adding 4m L DMEM/F12 (containing 10% serum, 1% double antibody) and the cells were blown off with a pipette.
(5) The digested cells were transferred to 15m L centrifuge tubes and centrifuged at room temperature for 5min at 200 g.
(6) Cells were resuspended in DMEM/F12 (10% serum, 1% double antibody) and counted.
(7) Dilute cells to 2 × 105M L, get2m L cells were mixed and added to a six well plate set at 37 ℃ with 5% CO2Incubate overnight in a cell incubator.
(8) And (5) taking out the cell culture dish in the step (7), observing the cell state, starting transfection when the cell intersection degree reaches 80-90%, and replacing the culture medium with antibiotic-free and serum-free DMEM/F12 and 2m L per hole before transfection.
(9) Diluting plasmid, the plasmid was diluted with OPTI-MEM, and 2.5. mu.g of the plasmid was added to 125. mu. L OPTI-MEM, followed by 2.5. mu. L plus, mixing, and standing at room temperature for 5 min.
(10) Dilute L ipofectamine L TX 125 mu L OPTI-MEM, add 9 mu L L ipofectamine L TX, then add 2.5 mu L plus, mix gently, and let stand at room temperature for 5 min.
(11) And (4) lightly mixing the mixture obtained in the step (10) and the step (11). Standing at room temperature for 5min, and then dropwise adding into a six-hole plate for uniform distribution.
(12) Placing the six-hole plate at 37 ℃ and 5% CO2Culturing in a cell culture box for 4-6 h.
(13) Changing the medium, removing the supernatant, adding 2m L DMEM/F12 (containing 10% serum and 1% double antibody), placing the six-well plate at 37 deg.C and 5% CO2Culturing in a cell culture box.
3.2 pressure screening
Pressurizing is started 24h after transfection, six-well plate cells are taken out from an incubator at 37 ℃, a supernatant medium is discarded, 2M L DMEM/F12 (containing 10% serum and 25 MuM MSX) is added, pressurizing is carried out for 7d, cells are observed in the middle, and a dead cell exchange solution is added.
3.3 monoclonal screening
(1) Monoclonal screening was initiated when the negative control cells were approximately 10-20% of their inventory, approximately 7days, by pressure screening.
(2) The six-well plate was removed, the medium was discarded, PBS was washed once, then 300. mu. L0.25.25% trypsin-EDTA was added, the digestion was carried out at room temperature for about 2min, 2M L DMEM/F12 (containing 10% serum + 25. mu.M MSX) was added to stop the digestion reaction, and the cells were blown off by a pipette.
(3) The digested cells were transferred to 15m L centrifuge tubes and centrifuged at room temperature for 5min at 200 g.
(4) Cells were resuspended in DMEM/F12 (containing 10% serum + 25. mu.M MSX) and counted.
(5) Plating, diluting the cells to 5/m L, adding the uniformly mixed cells of 200 mu L into a 96-well plate, placing the plate at 37 ℃ and 5% CO2And incubating for 4-6h in the cell incubator.
(6) Wells of individual cells were recorded.
(7) When the hole of a single cell in a 96-well plate grows up, the culture medium is discarded, PBS is washed once, 100 mu L0.25.25% of trypsin-EDTA is added, the digestion is carried out for about 2min at room temperature, 2M L DMEM/F12 (containing 10% serum and 25 mu M MSX) is added to stop the digestion reaction, a pipettor is used for blowing out the cell, the cell sap is transferred to a 12-well plate, when the 12-well plate is full, the supernatant is taken, E L ISA detects whether the clone is positive, and the high-efficiency expressed positive clone is continuously expanded, cultured and frozen.
Example 4: CHO-K1 cell strain acclimatized to suspension culture
(1) Preparing: sterilizing the biological safety cabinet for 30min by ultraviolet; DMEM/F12 (containing 10% serum, 25. mu.M MSX) was preheated to 37 ℃ in a 37 ℃ water bath.
(2) The cells (10cm cell culture dish) were removed from the 37 ℃ incubator, the supernatant medium was discarded, the cells were washed once with pre-warmed 8m L PBS, and the PBS was discarded.
(3) Adding 1-2m L0.25.25% trypsin-EDTA into each 10cm cell culture dish, digesting at room temperature for about 2min, observing the cells under a microscope to shrink and become round, and displaying the cells as single cells.
(4) Digestion was stopped by adding 4M L DMEM/F12 (containing 10% serum, 25. mu.M MSX) and cells were blown off with a pipette.
(5) The digested cells were transferred to 15m L centrifuge tubes and centrifuged at room temperature for 5min at 200 g.
(6) Cells were suspended in 100% DMEM/F12 (containing 10% serum, 25. mu.M MSX) and counted.
(7) Dilute cells to 5 × 105Cells/m L inoculated with 30m L medium in a 125m L shake flask, the cell culture flask was placed at 37 ℃ with 5% CO2Incubate overnight on an orbital shaker in a cell incubator at 120 rpm/min.
(8) Wiping the biological safety cabinet table top with 75% alcohol for sterilization, and irradiating with ultraviolet for 30 min.
(9) Cell density and viability were counted every 24 h.
(10) And performing second-generation culture when the cell survival rate reaches 94-97% after the first-generation cell culture is performed once.
(11) Preparation method comprises sterilizing biological safety cabinet with ultraviolet for 30min, placing 100% DMEM/F12 (containing 10% serum, 25 μ M MSX) and EX-CE LL 302 in CO2The cell incubator was preheated to 37 ℃.
(12) The cells were removed from the 37 ℃ incubator, transferred to a 50m L centrifuge tube, and centrifuged at 200g for 5min at room temperature.
(13) DMEM/F12 (containing 10% serum, 25. mu.M MSX) and EX-CE LL 302 were mixed at 1:1, the cells were resuspended and counted.
(14) Dilute cells to 5 × 105Cells/m L inoculated with 30m L medium in a 125m L shake flask, the cell culture flask was placed at 37 ℃ with 5% CO2Incubate overnight on an orbital shaker in a cell incubator at 120 rpm/min.
(15) Wiping the biological safety cabinet table top with 75% alcohol for sterilization, and irradiating with ultraviolet for 30 min.
(16) Cell density and viability were counted every 24 h.
(17) The cell survival rate obtained after the second generation culture is more than 95 percent after twice, the cell survival rate obtained after the third to sixth generation culture is more than 95 percent after three times, after 7 weeks, the cells are inoculated for 3 days and propagated for three generations, and the density reaches 1 × 106Individual cells/m L, with a 95% cell viability, which cells are considered to have been adapted to suspension culture, the seeding density was reduced to 3 × 105And each m L.
(18) After acclimation, the 1D11 strain and the 20A5 strain both meet the requirements, which indicates that the 1D11 strain and the 20A5 strain are successfully acclimated.
Example 5: cell shake flask fermentation
(1) Preparation of a subculture medium: 60% CD-CHO + 40% Ex-cell 302 was preheated to 37 ℃ in a 37 ℃ water bath.
(2) From CO2Taking out the shake flask cells by a constant temperature shaking table, and counting.
(3) The cells of 1D11 strain and 20A5 strain obtained in example 4 were diluted to 2.5-3.5 × 105Cells/m L inoculated with 30m L medium in a 125m L shake flask, the cell culture flask was placed at 37 ℃ with 5% CO2Incubate overnight in a constant temperature shaker at 100 rpm/min.
(4) Counting the cell density and activity every 24h, measuring the glucose, adding the glucose to 4 g/L when the blood sugar is lower than 2 g/L, taking 1m L samples every day, and using the supernatant for detecting the protein expression condition.
(5) Feed (about day four) 70 g/L CB5 was supplemented, adding 10% of the basal medium.
(6) Beginning on day 5, CO was added2The incubator temperature was adjusted to 32 ℃.
(7) On day nine, 70 g/L CB5 was supplemented and 10% of the basal medium was added.
(8) On day twelve, cells were harvested.
Example 6 construction and acclimatization of recombinant 293T cell line stably expressing CD2V-Fc protein
Following the procedures of examples 1-5, the inventors also readily constructed stable cell lines expressing CD2V-Fc protein, and thus, predicted common engineered mammalian cell lines. The method can be easily adopted to construct a stable cell strain for recombinant expression of CD2V-Fc, thereby producing the protein on a large scale. Therefore, the present invention is also within the scope of protection.
Example 7: protein purification
The cell culture fluid of example 5 (about 100ml per batch) was collected, centrifuged at 8,000g for 30min at 4 ℃, the supernatant was filtered through a 0.8 μm filter, loaded, and 80 μ L samples were reserved and added to 20 μ L of 5 × SDS-sample buffer for SDS-PAGE detection.
Column balancing: balancing 2-3 CV (column volume) with ultrapure water, and discharging ethanol preservation solution; and then balancing 5-10 CV by using 1 XPBS.
And (3) loading, namely combining 5m of L balanced ProteinA filler with a cell culture solution supernatant roller bottle for 1h, collecting Flowthrough (FT), taking an 80 mu L sample, adding 20 mu L of 5 × SDS-sample buffer solution, and using the sample for SDS-PAGE detection.
Washing: and (3) washing the protein which is not combined with the upper column and the hybrid protein with weaker combination ability by using 1 XPBS.
Elution was carried out by eluting the target protein with 0.1M glycine at pH 3.0buffer (adding an appropriate amount of 1M Tris-HCl to the collection tube to neutralize the final eluate pH to 7.4), collecting at 5M L/tube, mixing the collected samples (Elutethregh-ET), adding 80. mu. L samples to 20. mu. L of 5 × SDS-sample buffer for SDS-PAGE detection.
And (4) dialysis liquid change, namely pouring the eluent containing the target protein into a dialysis bag, dialyzing by 1 × PBS for at least 1,000 times, and taking 80 mu l of reserved sample for detection.
And (2) degerming and filtering, namely passing a low-protein binding needle filter of 0.22 mu m or a Nalgene filter of a 0.22 mu m filter membrane sterilized by a large amount of protein solution in a biological safety cabinet, storing a filtered protein solution sample in a refrigerator at the temperature of-80 ℃, measuring the protein concentration by using a BCA method, wherein the protein concentrations of the batches are respectively 2.5mg/ml and 2.8mg/ml, and the volumes are all about 40ml, and calculating (the protein yield is the protein concentration and the protein volume/the volume of the taken fermentation supernatant), and the protein yields of the 1D11 strain and the 20A5 strain are all about 1-1.12 g/L.
Example 8: nickel column protein purification
CD2V-Fc was prepared according to the method of examples 1-5, in which the amino acid sequence shown in SEQ ID NO.5 was replaced with the amino acid sequence shown in SEQ ID NO.1, and the gene sequence encoding the optimized sequence of this stretch of amino acids was shown in SEQ ID NO. 4.
The purification method was as follows.
The cell culture fluid of example 5 (about 100ml per batch) was collected, centrifuged at 8,000g for 30min at 4 ℃, the supernatant was filtered through a 0.8 μm filter, loaded, and 80 μ L samples were prepared and added to 20 μ L of 5 × SDS-sample buffer for SDS-PAGE detection.
Column balancing: balancing 2-3 CV (column volume) with ultrapure water, and discharging ethanol preservation solution; then using BufferA (20mM NaH)2PO4(pH 7.4) and 500mM NaCl, and balancing for 2-3 CV, 4-7 m L/min.
Loading, if 5m L pre-loaded nickel column one, loading at 1m L/min (adjusting loading Flow rate according to pre-loaded column volume, retention time 5min), collecting Flow Through (FT), taking 80 μ L sample and adding 20 μ L5 × SDS-sample buffer for SDS-PAGE detection.
Washing: with 4% bufferB (20mM NaH)2PO4(pH 7.4), 500mM NaCl, 20mM imidazole) washing the column, the flow rate is 4m L/min, the protein not combined with the column and the hybrid protein with weak combination ability are washed clean, until OD280nm base line is stable.
And (3) elution: 50% buffer B (20mM NaH)2PO4(pH 7.4), 500mM NaCl, 250mM imidazole) until the baseline was flat, 2m L/min, 10m L/tube, and after sample mixing (Elutethrogh-ET), 80 μ L samples were added to 20 μ L of 5 × SDS-sample buffer for SDS-PAGE detection.
Washing, namely washing 100% buffer B (20mM NaH2PO4(pH 7.4), 500mM NaCl, 500mM imidazole) and 4m L/min, washing 2-3 column volumes without collecting until the UV base line is leveled, balancing ultrapure water for 2-3 CV., and balancing the HisTrapexcel column by using 20% ethanol preservation solution for 2-3 CV.
And (4) dialysis liquid change, namely pouring the imidazole eluent containing the target protein into a dialysis bag, dialyzing by 1 × PBS for at least 1,000 times, and taking 80 mu l of reserved sample for detection.
And (3) degerming and filtering: in a biosafety cabinet, a 0.22 μm low protein binding needle filter, or a Nalgene filter with a 0.22 μm membrane sterilized with a large volume of protein solution was passed through, and the filtered protein solution sample was stored in a freezer at-80 ℃.
And (3) measuring the protein concentration by using a BCA method, wherein the protein concentrations of the batches are respectively 2.0mg/ml and 2.3mg/ml, the volumes of the batches are about 40ml, and the protein yields of the 1D11 strain and the 20A5 strain are about 0.8-0.92 g/L through calculation (protein yield is protein concentration and protein volume/volume of the obtained fermentation supernatant).
Example 9: detection and stability verification of CD2V-Fc protein
9.1 SDS-PAGE detection
The protein purified in example 6 was subjected to SDS-PAGE, and the concentration of CD2V-Fc protein in the sample was 2. mu.g/well, and the results are shown in FIG. 3: from the figure, it can be calculated that the purity of purified CD2V-Fc protein SDS-PAGE is 95%.
9.2 stability verification
The purified protein of example 6 was diluted to 0.8mg/ml with PBS and divided into 20 portions of 0.5ml each; ten portions are placed in a refrigerator at 4 ℃, and one portion is sampled every week and is continuously sampled for 10 times; ten portions are placed in a refrigerator at the temperature of 20 ℃ below zero, one portion is sampled every week, and 10 times of continuous sampling are carried out; protein concentration was measured with BCA after each sampling and the results are shown in the following table:
Figure BDA0002017501170000161
from the change in protein concentration, the protein remained essentially stable during both experiments. To further verify whether the treated protein was degraded, we performed SDS-PAGE with the tenth sample, and the specific results are shown in FIG. 4: m represents Marker; 1 is CD2V-Fc protein after 4 ℃ treatment, and the loading amount is 2 mug; 2 is CD2V-Fc protein after-20 ℃ treatment, and the loading amount is 2 mug; it can be seen from the figure that the treated sample (tenth sample) was still stable.
Example 10: vaccine preparation
10.1 vaccine preparation
Preparing an aqueous phase: according to the content of the CD2V-Fc protein in the vaccine, the CD2V-Fc protein is diluted by PBS (or physiological saline) to a proper concentration, namely a water phase;
preparing an oil phase: according to the total amount of the prepared vaccine, a proper amount of ISA 201 VG adjuvant is measured according to the weight ratio of 1:1 and the volume ratio of 46:54 of the antigen phase and the adjuvant;
emulsification: preheating the water phase and the oil phase to 33 ℃, slowly adding the water phase into the oil phase, stirring at 200-500rpm for 20-30min, standing at 20 ℃ for 1h, and standing at 4 ℃ overnight;
subpackaging and storing: subpackaging as required, and storing at 4 deg.C for use after qualified inspection.
10.2 vaccine quality testing
Observing the physical properties by adopting an eye-watching method to observe the appearance (whether the emulsion is milky white or not);
sucking a small amount of vaccine by using a clean straw and dripping the vaccine into cold water, observing (except for the 1 st drop), wherein the vaccine is dispersed in a cloud form and is judged to be a water-in-oil-in-water dosage form;
adding 5ml of vaccine into a centrifuge tube, centrifuging for 15min at 3000r/min, and judging the vaccine to be stable if the water phase separated out from the tube bottom is less than or equal to 0.25m L;
and (4) performing viscosity detection on the vaccine by using a viscometer, wherein the viscosity detection is required to be within 20-50cp, and the vaccine is judged to be qualified.
Example 11: safety experiment and immunogenicity experiment of recombinant CD2V-Fc fusion protein
11.1 safety test
20 healthy pigs (purchased from a Shaoxing pig farm) at 35 days of age (half of male and female) were randomly divided into 4 groups, and 5 groups were used for each group, and safety tests were conducted in the following manner.
Single dose one-time immunization group: each group was inoculated 1ml (50 ug/head) by intramuscular injection into the neck at 5 heads and observed continuously for 2 weeks.
Single dose secondary immunization group: each group was inoculated 1ml (50 ug/head) by intramuscular injection into the neck at 5 heads and observed continuously for 2 weeks. After 2 weeks, another vaccination was performed at the same dose for 2 additional weeks.
Overdose one-time immunization group: each group was inoculated 1ml (500 ug/head) by intramuscular injection into the neck at 5 heads and continuously observed for 2 weeks. Control group: each group was inoculated 1ml (PBS) of vaccine by intramuscular injection into the neck at 5 heads for 2 weeks.
During the experiment, the animal's spirit, feeding, activity, drinking, inflammation change at injection site, excretion and other clinical changes were observed every day, and the abnormal conditions of the animal were recorded.
Through continuous observation, clinical symptoms of the pigs injected with the CD2V-Fc fusion protein are compared, and a single dose, a secondary immune dose, an overdose immune group and a control group are normal in diet, normal in excretion and no adverse reaction in inoculated animals, no mental change and no inflammation phenomenon is found at the injection part, and no dead pig appears. The vaccine protein prepared by the invention has no obvious side effect even if injected at high dose (500ug/ml), and is a safe immune protein.
10.2 immunogenicity experiments
The purified protein of example 6 was subjected to the Werstern Blot assay using a sample having a concentration of CD2V-Fc protein (labeled 1 in the figure) of 2. mu.g/well; the primary anti-CD 2V-Fc immune pig serum is diluted at a ratio of 1: 100; the secondary antibody is a goat anti-pig IgG secondary antibody marked by HRP, and the dilution ratio is 1: 5000. The results are shown in FIG. 5: the serum can be specifically combined with CD2V-Fc protein.
The invention is illustrated by the above examples, but it should be understood that the invention is not limited to the particular examples and embodiments described herein. These specific examples and embodiments are included to assist those skilled in the art in practicing the present invention. Further modifications and improvements will readily occur to those skilled in the art without departing from the spirit and scope of the invention and, accordingly, it is intended that the invention be limited only by the terms of the appended claims, along with the full scope of equivalents to which such terms are entitled.
Figure BDA0002017501170000191
Figure BDA0002017501170000201
Figure BDA0002017501170000211
Sequence listing
<110> Zhejiang Hilon Biotechnology Ltd
<120> preparation method and application of African swine fever virus CD2V and pig Fc fusion protein
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<170>SIPOSequenceListing 1.0
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<213> CD2V-Fc protein amino acid sequence (PRT)
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1 5 10 15
Asp Tyr Trp Val Ser Phe Asn Lys Thr Ile Ile Leu Asp Ser Asn Ile
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Thr Asn Asp Asn Asn Asp Ile Asn Gly Val Ser Trp Asn Phe Phe Asn
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Asn Ser Phe Asn Thr Leu Ala Thr Cys Gly Lys Ala Gly Asn Phe Cys
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Glu Cys Ser Asn Tyr Ser Thr Ser Ile Tyr Asn Ile Thr Asn Asn Cys
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Ser Leu Thr Ile Phe Pro His Asn Asp Val Phe Asp Thr Thr Tyr Gln
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Val Val Trp Asn Gln Ile Ile Asn Tyr Thr Ile Lys Leu Leu Thr Pro
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Ala Thr Pro Pro Asn Ile Thr Tyr Asn Cys Thr Asn Phe Leu Ile Thr
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Cys Lys Lys Asn Asn Gly Thr Asn Thr Asn Ile Tyr Leu Asn Ile Asn
130 135 140
Asp Thr Phe Val Lys Tyr Thr Asn Glu Ser Ile Leu Glu Tyr Asn Trp
145 150 155 160
Asn Asn Ser Asn Ile Asn Asn Phe Thr Ala Thr Cys Ile Ile Asn Asn
165 170 175
Thr Ile Ser Thr Ser Asn Glu Thr Thr Leu Ile Asn Cys Thr Tyr Leu
180 185 190
Thr Leu Ser Ser Asn Tyr Phe Tyr Thr Phe Phe Lys Leu Tyr Gly Gly
195 200 205
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Cys Pro
210 215 220
Ala Cys Glu Ser Pro Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro
225 230 235 240
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Gln Val Thr Cys Val Val
245 250 255
Val Asp Val Ser Gln Glu Asn Pro Glu Val Gln Phe Ser Trp Tyr Val
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Asp Gly Val Glu Val His Thr Ala Gln Thr Arg Pro Lys Glu Glu Gln
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Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gln His Gln
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Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp
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Leu Pro Ala Pro Ile Thr Arg Ile Ile Ser Lys Ala Lys Gly Pro Ser
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Arg Glu Pro Gln Val Tyr Thr Leu Ser Pro Ser Ala Glu Glu Leu Ser
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Arg Ser Lys Val Ser Ile Thr Cys Leu Val Thr Gly Phe Tyr Pro Pro
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Asp Ile Asp Val Glu Trp Lys Ser Asn Gly Gln Pro Glu Pro Glu Gly
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Asn Tyr Arg Thr Thr Pro Pro Gln Gln Asp Val Asp Gly Thr Tyr Phe
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Leu Tyr Ser Lys Leu Ala Val Asp Lys Ala Ser Trp Gln Arg Gly Asp
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Gln Lys Ser Ile Ser Lys Thr Pro Gly Lys
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<212>PRT
<213> amino acid sequence (PRT) of extracellular domain protein of CD2V
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Met Ile Ile Leu Ile Phe Leu Ile Phe Ser Asn Ile Val Leu Ser Ile
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Asp Tyr Trp Val Ser Phe Asn Lys Thr Ile Ile Leu Asp Ser Asn Ile
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Ser Leu Thr Ile Phe Pro His Asn Asp Val Phe Asp Thr Thr Tyr Gln
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Thr Leu Ser Ser Asn Tyr Phe Tyr Thr Phe Phe Lys Leu Tyr
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<212>PRT
<213> amino acid sequence (PRT) of porcine Fc protein
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Ile Cys Pro Ala Cys Glu Ser Pro Gly Pro Ser Val Phe Ile Phe Pro
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Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Gln Val Thr
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Cys Val Val Val Asp Val Ser Gln Glu Asn Pro Glu Val Gln Phe Ser
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Trp Tyr Val Asp Gly Val Glu Val His Thr Ala Gln Thr Arg Pro Lys
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Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Ile
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Gln His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Lys Val Asn
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Asn Lys Asp Leu Pro Ala Pro Ile Thr Arg Ile Ile Ser Lys Ala Lys
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<212>PRT
<213> CD2V-Fc protein amino acid sequence with HIS tag (PRT)
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Met Ile Ile Leu Ile Phe Leu Ile Phe Ser Asn Ile Val Leu Ser Ile
1 5 10 15
Asp Tyr Trp Val Ser Phe Asn Lys Thr Ile Ile Leu Asp Ser Asn Ile
20 25 30
Thr Asn Asp Asn Asn Asp Ile Asn Gly Val Ser Trp Asn Phe Phe Asn
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Asn Ser Phe Asn Thr Leu Ala Thr Cys Gly Lys Ala Gly Asn Phe Cys
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Ala Thr Pro Pro Asn Ile Thr Tyr Asn Cys Thr Asn Phe Leu Ile Thr
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Asp Thr Phe Val Lys Tyr Thr Asn Glu Ser Ile Leu Glu Tyr Asn Trp
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Asn Asn Ser Asn Ile Asn Asn Phe Thr Ala Thr Cys Ile Ile Asn Asn
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Thr Ile Ser Thr Ser Asn Glu Thr Thr Leu Ile Asn Cys Thr Tyr Leu
180 185 190
Thr Leu Ser Ser Asn Tyr Phe Tyr Thr Phe Phe Lys Leu Tyr Gly Gly
195 200 205
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Cys Pro
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Ala Cys Glu Ser Pro Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro
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Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Gln Val Thr Cys Val Val
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Val Asp Val Ser Gln Glu Asn Pro Glu Val Gln Phe Ser Trp Tyr Val
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Asp Gly Val Glu Val His Thr Ala Gln Thr Arg Pro Lys Glu Glu Gln
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Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gln His Gln
290 295 300
Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp
305 310 315 320
Leu Pro Ala Pro Ile Thr Arg Ile Ile Ser Lys Ala Lys Gly Pro Ser
325 330 335
Arg Glu Pro Gln Val Tyr Thr Leu Ser Pro Ser Ala Glu Glu Leu Ser
340 345 350
Arg Ser Lys Val Ser Ile Thr Cys Leu Val Thr Gly Phe Tyr Pro Pro
355 360 365
Asp Ile Asp Val Glu Trp Lys Ser Asn Gly Gln Pro Glu Pro Glu Gly
370 375 380
Asn Tyr Arg Thr Thr Pro Pro Gln Gln Asp Val Asp Gly Thr Tyr Phe
385 390 395 400
Leu Tyr Ser Lys Leu Ala Val Asp Lys Ala Ser Trp Gln Arg Gly Asp
405 410 415
Pro Phe Gln Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr
420 425 430
Gln Lys Ser Ile Ser Lys Thr Pro Gly Lys His His His His His His
435 440 445
<210>6
<211>1365
<212>DNA
<213> nucleotide sequence encoding CD2V-Fc with HIS tag (DNA)
<400>6

Claims (10)

1. A subunit fusion protein CD2V-Fc, wherein the subunit fusion protein CD2V-Fc comprises the extracellular region of African swine fever virus surface envelope protein CD2V and an antibody Fc protein of a pig.
2. The subunit fusion protein CD2V-Fc according to claim 1, wherein the amino acid sequence of the extracellular domain of the African swine fever virus surface envelope protein CD2V is shown in SEQ ID No.2, wherein the antibody Fc protein of swine is the heavy chain constant region of IgG of swine and the amino acid sequence is shown in SEQ ID No. 3.
3. The subunit fusion protein CD2V-Fc of claim 2, wherein the subunit fusion protein CD2V-Fc has the amino acid sequence set forth in SEQ ID No. 1.
4. The subunit fusion protein CD2V-Fc of claim 3, wherein the subunit fusion protein CD2V-Fc further comprises a protein derived from the amino acid sequence of SEQ ID NO1 by substitution, deletion or addition of one or more amino acids and having immunogenicity.
5. The subunit fusion protein CD2V-Fc of claim 4, wherein the subunit fusion protein CD2V-Fc further comprises a derivative protein in which the amino acid sequence as set forth in SEQ ID NO1 comprises the amino acid sequence of CD2V and Fc reversed and the Fc is placed amino-terminal.
6. The subunit fusion protein CD2V-Fc of claim 5, wherein the subunit fusion protein CD2V-Fc further comprises one or more tag amino acids selected from the group consisting of poly-Arg, poly-His, flag, c-myc, and HA linked at the amino terminus or carboxy terminus of the amino acid sequence set forth in SEQ ID No. 1.
7. The subunit fusion protein CD2V-Fc of claim 3, wherein the subunit fusion protein CD2V-Fc of African swine fever CD2V encodes the gene sequence shown in SEQ ID No. 4.
8. A method for preparing the subunit fusion protein CD2V-Fc according to any one of claims 1 to 7, comprising the steps of:
1) cloning the gene coded by the subunit fusion protein CD2V-Fc of the African swine fever CD2V shown in SEQ ID NO.4 into a eukaryotic expression vector to obtain a recombinant plasmid containing the gene coded by the subunit fusion protein CD2V-Fc of the African swine fever CD2V, wherein the eukaryotic expression vector comprises pEE6.4, pEE12.4, pG L4.13.13, pcDNA3.1 and pcDNA3.3;
2) transfecting a recombinant plasmid containing a gene coded by the subunit fusion protein of the African swine fever CD2V into a CHO cell to obtain a CHO cell strain;
3) culturing, screening and domesticating the CHO cell strain in the step 2) to obtain a highly expressed cell strain;
4) fermenting and culturing the highly expressed cell strain in the step 3), and purifying to obtain the subunit fusion protein CD2V-Fc of the recombinant African swine fever CD 2V.
9. The preparation method of claim 8, wherein the expression system of the subunit fusion protein CD2V-Fc of the African swine fever CD2V is a mammalian cell, the mammalian cell is a CHO cell, 293T cell, the CHO cell is DG44, DXB11, CHO-K1, CHO-S cell.
10. Use of the subunit fusion protein CD2V-Fc according to any one of claims 1 to 7 in the manufacture of a vaccine for the diagnosis, prevention and treatment of african swine fever.
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CN113136400B (en) * 2020-01-17 2022-09-09 普莱柯生物工程股份有限公司 Construction method and application of CHO cell strain expressing foreign protein
CN113136400A (en) * 2020-01-17 2021-07-20 普莱柯生物工程股份有限公司 Construction method and application of CHO cell strain for efficiently expressing foreign protein
CN111849922A (en) * 2020-07-20 2020-10-30 华中农业大学 Monoclonal antibody prepared from African swine fever virus truncated protein p54 and application thereof
CN111848748B (en) * 2020-07-20 2021-06-29 华中农业大学 African swine fever virus truncated protein and application thereof in preparation of ELISA (enzyme-linked immunosorbent assay) detection kit
CN111849922B (en) * 2020-07-20 2021-08-03 华中农业大学 Monoclonal antibody prepared from African swine fever virus truncated protein p54 and application thereof
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CN114426974A (en) * 2020-10-29 2022-05-03 洛阳普泰生物技术有限公司 African swine fever virus CD2v protein, kit and antibody prepared from same
CN114426974B (en) * 2020-10-29 2023-11-21 洛阳普泰生物技术有限公司 Antibodies or antibody fragments specifically binding to the CD2v protein of African swine fever virus
CN114644714A (en) * 2022-01-11 2022-06-21 中国农业科学院兰州兽医研究所 African swine fever virus recombinant fusion protein CPE, preparation and application thereof
CN114644714B (en) * 2022-01-11 2023-11-10 中国农业科学院兰州兽医研究所 African swine fever virus recombinant fusion protein CPE, preparation and application thereof
CN115073609A (en) * 2022-04-18 2022-09-20 湖南派智生物科技有限公司 Recombinant protein based on extracellular domain of African swine fever virus CD2v protein, and construction method and application thereof
CN116284260A (en) * 2023-03-15 2023-06-23 中国科学院微生物研究所 African swine fever multicomponent subunit vaccine and preparation method and application thereof
CN116284260B (en) * 2023-03-15 2023-11-17 中国科学院微生物研究所 African swine fever multicomponent subunit vaccine and preparation method and application thereof

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