CN111393531B - 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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CN111393531B
CN111393531B CN201910268140.XA CN201910268140A CN111393531B CN 111393531 B CN111393531 B CN 111393531B CN 201910268140 A CN201910268140 A CN 201910268140A CN 111393531 B CN111393531 B CN 111393531B
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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 a 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). The pigs are the only mammalian hosts of natural infection of the ASFV, and comprise domestic pigs and wild pigs, particularly the domestic pigs, and the susceptibility is extremely high. After the swine is infected with African swine fever virus, the swine is clinically characterized by skin congestion, internal 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 animal infectious disease.
African swine fever has been discovered in the African continent since 1927, and has caused a tremendous shock to the swine industry in Africa and Europe. Since 8 months in 2018, outbreaks in a plurality of provinces of China bring serious economic loss to the pig raising 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 a non-obvious effect, and the weak virus vaccine has a poor protection effect and 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 diffusion and lymphocyte injury. The research finds that CD2V is the surface protein of the viral envelope, and the antibody aiming at the 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 the results of western were obtained, and the expression was very low without SDS-PAGE. However, the expression of the protein in a large amount is not observed, which indicates that the stability of the extracellular domain of CD2V is poor, and therefore, the protein is difficult to be applied in actual 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 discovery shows that the homodimer structure of the CD2V can be maintained, the stability of the CD2V can be effectively maintained, and the invention can also 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 through one-step affinity chromatography. The subunit fusion protein CD2V-Fc contains an extracellular region of 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.
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 a 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, comprising the steps of: 1) Cloning a gene coded by a subunit fusion protein CD2V-Fc of the African swine fever CD2V shown as 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 a subunit fusion protein of 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, pGL4.13, pcDNA3.1 or pcDNA3.3. 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 CHO cell strain of a subunit fusion protein CD2V-Fc which can stably and efficiently secrete and express the African swine fever virus CD2V in a suspended manner, the cell strain expresses the subunit fusion protein CD2V-Fc with high yield (the yield is up to 1-2 g/L), the purification is easy (as shown in figure 4, the purity of target protein in cell culture supernatant can reach more than 70 percent, only one step of affinity chromatography is needed to ensure that the purity of the target protein can reach more than 90 percent, the requirements of subunit vaccine and diagnostic reagent are far met), and the large-scale production is easy. Therefore, the problem that CD2V cannot be stably expressed is solved and the production cost is reduced. In addition, the CHO cell strain for production has high controllability during culture, easy quality control, stable production protein batch-to-batch, and high biosafety (no virus, no risk of virus dispersion).
Drawings
FIG. 1 shows a map of pEE12.4-OPTI-CD2V-Fc plasmid.
FIG. 2 shows the results of the double-restriction enzyme identification of pEE12.4-OPTI-CD 2V-Fc: m is a DNA Marker: DL10000Marker;1 is the result of pEE12.4-OPTI-CD2V-Fc double-restriction electrophoresis.
FIG. 3 shows SDS-PAGE detection of subunit fusion protein CD2V-Fc after purification: 1 is subunit fusion protein CD2V-Fc,2 is Marker.
FIG. 4A, B shows SDS-PAGE detection 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 after 10 weeks of storage at 4 ℃;2 is the subunit fusion protein CD2V-Fc at-20 ℃ 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 used for illustrating the technical solutions of the present invention and are not meant to limit 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 from cell banks of China academy of Sciences (SCEMC) cell banks of China academy of sciences Shanghai Life sciences research institute;
cell culture medium and serum were purchased from gibco, usa;
the eukaryotic expression vector pEE12.4 is purchased from Shanghai Linyuan Biotech, inc.;
lipofectamine LTX was purchased from Thermo Fisher, USA;
methionine sulfoxide ammonium sulfite (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 polypeptide encoded by an EP402R gene, a transmembrane region is formed at 207-229aa in prediction analysis, and research shows that the CD2V protein can interact with erythrocytes and plays an important role 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 a pig at the carboxyl terminal of the CD2V to ensure the structure and the protein stability of the CD2V in order to solve the important technical problem.
1.2 codon optimization of African Swine fever CD2V-Fc protein
The laboratory finds a genome sequence 73369-74451 encoding African swine fever CD2V protein sequence by taking an epidemic African swine fever strain subtype reported in China in 2018 and referring to a Georgia 2007/1 complete gene sequence (GenBank: FR 682468.1) as a template. Further analysis of 1M-15S may be the secretion signal peptide of CD2V protein, 16Q-206Y may be the 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 figure 1. The structure is a homodimer structure, and further in order to ensure that the CD2V is stably expressed, a pig Fc sequence (GenBank: AK 405774.1), namely CD2V-Fc, is added at the carboxyl terminal of the extracellular region of the 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 in this example by Poly-His, 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 target fragment OPTI-CD2V-Fc
2.1.1 PCR reaction
(1) Primer design and Synthesis
Upstream primer of 5
Downstream primer of 5' GCGGAATTGAATTCTTAATGGTGATG-3
(2) Sample loading system 50 μ L, as shown in the following table:
Figure BDA0002017501170000061
PCR amplification procedure:
Figure BDA0002017501170000062
2.1.2 PCR product is subjected to gel recovery
(1) Marking a sample collection EP tube, an adsorption column and a collection tube;
(2) Weighing the marked empty EP tube, and recording the numerical value;
(3) A single target DNA band was carefully cut from the agarose gel on a gel cutter with a scalpel into a clean 1.5mL centrifuge tube;
(4) Adding 600 mu L of PC buffer into the 1.5mL of centrifuge tube in the step (3), placing in a water bath at 50 ℃ for about 5min, and turning the centrifuge tube up and down continuously and gently during the process to ensure that the gel block is fully dissolved;
(5) Column balancing: adding 500 μ L of balance liquid BL into adsorption column CB2 (the adsorption column is placed into the collection tube in advance), centrifuging at 12,000rpm/min for 1min, pouring off waste liquid in the collection tube, and placing the adsorption column back into the collection tube;
(6) Adding the solution obtained in the step (5) into an adsorption column CB2, standing for 2min, standing for 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 600 mu L of rinsing liquid PW buffer into the adsorption column, standing for 3min, centrifuging at 10,000rpm/min for 30s, pouring out waste liquid in the collecting pipe, and putting the adsorption column CB2 into the collecting pipe;
(8) Repeating the step (7);
(9) Centrifuging with air 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 air drying completely;
(10) Placing adsorption column CB2 into a collecting tube, suspending and dropwise adding 50 μ L of Elutionbuffer (preheated at 65 ℃) to the middle position of an adsorption film, standing for 3min, centrifuging for 12,000rpm/min, and standing for 2min;
(11) Taking the centrifuge tube in the step (10) out of the centrifuge, discarding the middle adsorption column CB2, covering the centrifuge tube 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 PCR product and vector double enzyme digestion reaction
(1) The required 1.5mL EP tube was labeled, and the sample was loaded and mixed in the 1.5mL EP tube according to the following table: 50 μ L reaction System
Figure BDA0002017501170000071
(2) And (3) placing the 1.5mL EP tube in the step (1) into a corresponding enzyme constant-temperature water bath kettle with the optimal temperature, and carrying out water bath for 2-3h.
Recovering the double enzyme digestion product glue: taking out the double enzyme digestion system, and carrying out agarose gel electrophoresis to recover the DNA fragment in the double enzyme digestion system, wherein the method is the same as that for recovering the PCR product gel in 1.2.1.
2.3 ligation reaction
(1) A plurality of clean 1.5mL EP tubes are prepared, marked and placed on an EP tube frame for standby.
(2) The sample was loaded and mixed in a 1.5mL EP tube as described in 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 10-16h;
(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 15min;
(5) Taking out the EP tube in the step (4), and storing at 4 ℃.
2.4 conversion reaction
(1) Quickly adding 10 μ L of the ligation reaction solution into 100 μ L of competent cells, uniformly mixing by blowing, and carrying out ice bath for 30min;
(2) Taking out the sample tube, placing in water bath at 42 ℃ for 100s, and immediately carrying out ice bath for 2min;
(3) Taking out the sample tube, adding 600 mu L of liquid LB culture medium into the sample tube in an ultra-clean workbench, and then placing the sample tube on a constant temperature shaking table at 37 ℃ at 220rpm/min for culturing for 1h;
(4) Coating a plate: and (4) 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, suspending thalli at the bottom of the tube by the residual supernatant liquid, putting the suspended bacterium liquid into the center of a corresponding transformation plate, and uniformly spreading the bacterium liquid in the center of the transformation plate by using a bacterium 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 15h;
(6) The transformation results were observed.
2.5 plasmid extraction and double restriction enzyme identification
2.5.1 plasmid extraction
(1) Picking the monoclonals from the conversion plate by using a 10-microliter pipette tip to 5mL of LB liquid culture medium containing the benzyl resistance, and shaking the bacteria at 37 ℃ and 220rpm/min overnight;
(2) Transferring the bacterial liquid into a 1.5mL EP tube, centrifuging at room temperature at 12,000rpm/min for 2min, and discarding the supernatant;
(3) Adding 250 mu L of plasmid extraction reagent P1buffer into the EP tube in the step (2) to completely suspend the thalli;
(4) Adding 250 mu L of P2 buffer into the solution in the step (3), immediately and gently inverting the centrifuge tube for 5-10 times, uniformly mixing, and standing at room temperature for 2-4min;
(5) Adding 350 mu L of P3 buffer into the solution in the step (4), immediately and gently reversing the centrifuge tube for 5-10 times, and uniformly mixing; standing at room temperature for 2-4min;
(6) Centrifuging the solution in the step (5) at room temperature for 10min at 14,000rpm;
(7) Transferring the supernatant solution in the step (6) to the center of an adsorption column, centrifuging at room temperature for 12,000rpm/min for 30s, and pouring out liquid in a collecting pipe;
(8) Adding 500 μ L buffer DW1 into the center of the adsorption column, centrifuging at room temperature for 12,000rpm/min for 30s, and pouring off the liquid in the collection tube;
(9) Adding 500 μ L wash solution into the center of the adsorption column, centrifuging at room temperature for 12,000rpm/min for 30s, pouring off the liquid in the collection tube, and repeating once;
(10) Empty adsorption column, centrifuge at room temperature, 12,000rpm,2min.
(11) The adsorption column was placed in a clean 1.5mL centrifuge tube, 30. Mu.L of Elution buffer was added to the center of the adsorption membrane, and the mixture was allowed to stand at room temperature for 5min, centrifuged at room temperature, 12,000rpm, and 2min. The DNA solution in the tube was preserved.
2.5.2 double enzyme digestion identification
(1) The 1.5mL EP tubes were labeled for use and loaded as follows: 20 μ L reaction System
Figure BDA0002017501170000091
(2) And (2) putting the EP tube 20 mu L of reaction system in the step (1) into a constant-temperature water bath kettle at 37 ℃ for water bath for 2h.
(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 of the experiment are shown in FIG. 2: the enzyme digestion identification construction is correct.
(4) The correct insert clone was selected and sequenced 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 100mL of a culture medium containing benzyl amine resistance, and culturing for 15h by a constant temperature shaking table at 37 ℃ at 220 rpm/min;
(2) Transferring the bacterial liquid cultured in the step (1) into a 50mL centrifuge tube, centrifuging for 5min at the room temperature of 8,000rpm/min, collecting thalli, and discarding a supernatant culture medium;
(3) Adding 8mL of solution P1 into the centrifuge tube in the step (2), and fully resuspending the thalli by using a pipette;
(4) Adding 8mL of 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) And (5) adding 8mL of solution P4 into the centrifuge 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, and standing at room temperature for about 10 min. Centrifuging at 8,000rpm/min for 5-10min at room temperature to separate 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 handle filter, and collecting the filtrate in a clean 50mL centrifuge tube;
(7) Column balancing: adding 2.5mL of equilibrium liquid BL into adsorption column CP6 (the adsorption column is placed into a 50mL collection tube), centrifuging at room temperature of 8,000rpm/min for 2min, pouring off waste liquid in the collection tube, and placing the adsorption column back into the collection tube again;
(8) And (4) adding isopropanol with the volume of 0.3 time of the filtrate into the filtrate in the step (6), turning upside down, uniformly mixing, and transferring to an adsorption column CP 6. Centrifuging at 8,000rpm/min for 2min, pouring out liquid in the collecting pipe, and putting the adsorption column CP6 into the same collecting pipe again;
(9) Adding 10mL of rinsing liquid PW into the adsorption column CP6 in the step (8), centrifuging at the room temperature of 8,000rpm/min for 2min, discarding waste liquid in the collection pipe, and replacing the adsorption column in the collection pipe again;
(10) Repeating the operation step (9) once;
(11) Adding 3mL of absolute ethyl alcohol into the adsorption column CP6 in the step (10), centrifuging at room temperature of 8,000rpm/min for 2min, and pouring off waste liquid;
(12) And (5) putting the adsorption column CP6 in the step (11) back into the collection tube, and centrifuging for 5min at the room temperature of 8,000rpm. Opening the adsorption column CP6, placing at room temperature, standing for several minutes, and air drying;
(13) And (4) putting the adsorption column in the step (12) into a clean 50mL centrifuge tube, adding 1-2mL 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 50mL centrifuge tube into a clean 1.5mL centrifuge tube, measuring the concentration, and storing at-20 ℃.
(14) 1-2. Mu.L of the obtained plasmid DNA solution was subjected to agarose gel electrophoresis and the data of the electrophoresis results were preserved.
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 (10 cm cell culture dish) were removed from the 37 ℃ incubator, the supernatant medium was discarded, the cells were washed once with pre-warmed 8mL PBS, and the PBS was discarded.
(3) Adding 0.25% of trypsin-EDTA per 10cm cell culture dish, digesting at room temperature for about 2min, observing under microscope that the cells shrink and become round and present as single cells.
(4) The digestion was stopped by adding 4mL of DMEM/F12 (10% serum, 1% double antibody) and the cells were pipetted off.
(5) The digested cells were transferred to a 15mL centrifuge tube and centrifuged at room temperature for 200g,5min.
(6) Cells were resuspended in DMEM/F12 (10% serum, 1% double antibody) and counted.
(7) Dilute cells to 2 × 10 5 cell/mL, 2mL of the mixed cells were added to a six-well plate, the six-well plate was set to 37 ℃,5% 2 Incubate overnight in cell incubator.
(8) Taking out the cell culture dish in the step (7), and observing the cell state: transfection was initiated when cell confluence reached 80% -90%, and the medium was changed to antibiotic-free and serum-free DMEM/F12,2 mL/well before transfection.
(9) Plasmid dilution: the plasmid was diluted with OPTI-MEM, and 2.5. Mu.g of the plasmid was added to 125. Mu.L of OPTI-MEM, followed by 2.5. Mu.L of plus, followed by mixing, and the mixture was allowed to stand at room temperature for 5min.
(10) Dilution of Lipofectamine LTX: mu.L of OPTI-MEM was added with 9. Mu.L of Lipofectamine LTX, followed by 2.5. Mu.L of plus, gently mixed, and allowed to stand at room temperature for 5min.
(11) And (5) 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 well plates at 37 ℃ 5% CO 2 Culturing in a cell culture box for 4-6h.
(13) Liquid changing: discarding supernatant medium, adding 2mL DMEM/F12 (containing 10% serum 1% diabody), placing the six well plates at 37 ℃,5% 2 Culturing in a cell culture box.
3.2 pressure screening
Pressurization was started 24h after transfection: six well plate cells were removed from the 37 ℃ incubator, supernatant medium was discarded, 2mL of DMEM/F12 (containing 10% serum + 25. Mu.M MSX) was added, pressurized for 7days, cells were observed in the middle, and the dead cells were replaced with more fluid.
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.L of 0.25% trypsin-EDTA was added, the mixture was digested at room temperature for about 2min, 2mL of DMEM/F12 (containing 10% serum + 25. Mu.M MSX) was added to terminate the digestion reaction, and the cells were blown off by a pipette.
(3) The digested cells were transferred to a 15mL centrifuge tube and centrifuged at room temperature for 200g,5min.
(4) Cells were resuspended in DMEM/F12 (containing 10% serum + 25. Mu.M MSX) and counted.
(5) Plate paving: diluting the cells to 5/mL, adding 200. Mu.L of the mixed cells to a 96-well plate, standing at 37 ℃,5% CO 2 And incubating for 4-6h in the cell incubator.
(6) Wells of individual cells were recorded.
(7) When the wells of the individual cells in the 96-well plate were grown up, the medium was discarded, PBS was washed once, 100. Mu.L of 0.25% trypsin-EDTA was added, digestion was carried out at room temperature for about 2min, digestion was terminated by adding 2mL of DMEM/F12 (containing 10% serum + 25. Mu.M MSX), and the cells were blown off by a pipette. And transferring the cell sap to a 12-pore plate, taking the supernatant when the 12-pore plate is full, detecting whether the clone is positive by ELISA, and continuously performing expanded culture and freezing storage on the high-efficiency expressed positive clone.
Example 4: CHO-K1 cell strain domesticated into 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 (10 cm cell culture dish) were removed from the 37 ℃ incubator, the supernatant medium was discarded, the cells were washed once with pre-warmed 8mL PBS, and the PBS was discarded.
(3) Adding 0.25% of trypsin-EDTA per 10cm cell culture dish, digesting at room temperature for about 2min, observing under microscope that the cells shrink and become round and present as single cells.
(4) Digestion was stopped by adding 4mL of DMEM/F12 (containing 10% serum, 25. Mu.M MSX) and the cells were blown off with a pipette.
(5) The digested cells were transferred to a 15mL centrifuge tube and centrifuged at room temperature for 200g,5min.
(6) Cells were suspended in 100% DMEM/F12 (containing 10% serum, 25. Mu.M MSX) and counted.
(7) Dilute cells to 5 × 10 5 One cell/mL was inoculated into a 30mL culture medium in a 125mL shake flask. Placing the cell culture flask at 37 deg.C, 5% 2 Incubate 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 30min.
(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) Preparing: sterilizing the biological safety cabinet for 30min by ultraviolet; 100% DMEM/F12 (containing 10% serum, 25. Mu.M MSX), EX-CELL 302 in CO 2 The cell incubator was preheated to 37 ℃.
(12) The cells were removed from the 37 ℃ incubator, transferred to a 50mL centrifuge tube, and centrifuged at 200g for 5min at room temperature.
(13) DMEM/F12 (containing 10% serum, 25. Mu.M MSX) and EX-CELL 302 were mixed as 1:1 mixing, resuspending the cells, and counting.
(14) Diluting cellsTo 5X 10 5 One cell/mL was inoculated into a 30mL culture medium in a 125mL shake flask. The cell culture flask was left at 37 ℃ and 5% CO 2 Incubate 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 30min.
(16) Cell density and viability were counted every 24 h.
(17) The survival rate of the cells obtained after the second-generation culture is more than 95 percent; the cell survival rate obtained after three times of culture of the third generation to the sixth generation is more than 95 percent. After 7 weeks, the cells were seeded for 3 days and propagated for three generations with a density of 1X 10 6 Individual cells/mL with a cell viability of 95%, which cells are considered to have been adapted to suspension culture. The inoculation density is reduced to 3X 10 5 one/mL.
(18) After acclimation, both the 1D11 strain and the 20A5 strain meet the requirements, which indicates that both 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 CO 2 Taking out the shake flask cells by a constant temperature shaking table, and counting.
(3) The cells of the 1D11 and 20A5 strains obtained in example 4 were diluted to 2.5-3.5X 10 5 One cell/mL was inoculated into a 30mL culture medium in a 125mL shake flask. Placing the cell culture flask at 37 deg.C, 5% 2 Incubate overnight in a constant temperature shaker at 100 rpm/min.
(4) Counting the cell density and activity every 24h, measuring glucose, and adding the glucose to 4g/L when the blood sugar is lower than 2 g/L; 1mL of sample was taken daily and the supernatant was used to examine protein expression.
(5) Feeding (about day four): 70g/L CB5 was supplemented, and 10% of the basal medium was added.
(6) Beginning on day 5, CO was added 2 The incubator temperature was adjusted to 32 ℃.
(7) On day nine, 70g/L CB5 was supplemented and 10% of the basal medium was added.
(8) On the twelfth day, cells were harvested.
Example 6 construction and domestication of recombinant 293T cell line stably expressing CD2V-Fc protein
The present inventors also readily constructed stable cell lines expressing CD2V-Fc protein following the procedures of examples 1-5, and thus, a common engineered mammalian cell line is envisioned. 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 the present invention.
Example 7: protein purification
The cell culture medium of example 5 (about 100ml per batch) was collected, centrifuged at 4 ℃ for 30min at 8,000g, the supernatant was filtered through a 0.8 μm filter, and 80 μ L of the sample was added to 20 μ L of 5 XSDS-sample buffer for SDS-PAGE detection.
Column balancing: balancing 2-3 CV (column volume) with ultrapure water, and discharging ethanol preservation solution; then 5-10 CV are balanced with 1 XPBS.
Loading: 5mL of equilibrated ProteinA filler was combined with the cell culture supernatant roller bottle for 1h, flow Through (FT) was collected, and 80. Mu.L of the sample was added to 20. Mu.L of 5 XSDS-sample buffer 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.
And (3) elution: eluting the target protein with 0.1M glycine, pH 3.0buffer (adding appropriate amount of 1M Tris-HCl to the collection tube to neutralize the final eluate pH to 7.4), and collecting: 5 mL/tube; after sample mixing (Elutethregh-ET) 80. Mu.L of sample was added to 20. Mu.L of 5 XSDS-sample buffer for SDS-PAGE detection.
And (3) dialysis liquid exchange: the eluate containing the target protein was poured into a dialysis bag, dialyzed against 1XPBS at least 1,000 times, and 80. Mu.l of the sample was detected.
And (3) degerming and filtering: in a biosafety cabinet, a 0.22 μm low protein binding needle filter, or Nalgene filter with a 0.22 μm filter sterilized with a large volume of protein solution, was passed through and the filtered protein solution sample was stored in a freezer at-80 ℃. Protein concentration determination: determining the protein concentration by using a BCA method, wherein the protein concentration of the batch is respectively 2.5mg/ml and 2.8mg/ml, and the volume is about 40ml; by calculation (protein yield = protein concentration protein volume/volume of fermentation supernatant taken), the protein yields of both strains 1D11 and 20A5 were about 1-1.12g/L.
Example 8: nickel column protein purification
CD2V-Fc was prepared according to the method of examples 1 to 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 medium (about 100ml per batch) as in example 5 was collected, centrifuged at 4 ℃ for 30min at 8,000g, the supernatant was removed, filtered through a 0.8 μm filter, loaded, and 80 μ L of the sample was prepared and 20 μ L of 5 XSDS sample buffer was added for SDS-PAGE detection.
Column balancing: balancing 2-3 CV (column volume) with ultrapure water, and discharging ethanol preservation solution; then using BufferA (20 mM NaH) 2 PO 4 (pH 7.4), 500mM NaCl) was equilibrated at 2 to 3CV,4 to 7mL/min.
Loading: if one is used for the 5mL nickel pre-packed column, the sample is loaded at 1mL/min (the Flow rate is adjusted according to the volume of the pre-packed column, the retention time is 5 min), the Flow Through (FT) is collected, and 80. Mu.L of the sample is added to 20. Mu.L of 5 xSDS-sample buffer for SDS-PAGE detection.
Washing: by 4% of buffer B (20 mM NaH) 2 PO 4 (pH 7.4), 500mM NaCl,20mM imidazole) and a flow rate of 4mL/min, and protein which is not combined with the column and hybrid protein with weak combination ability are washed clean until OD280nm base line is stable.
And (3) elution: 50% of bufferb (20 mM NaH) 2 PO 4 (pH 7.4), 500mM NaCl,250mM imidazole) to baseline flush, 2mL/min, collect: 10 mL/tube; after sample mixing (Elutethregh-ET), 80. Mu.L of the sample was added to 20. Mu.L of 5 XSDS-sample buffer for SDS-PAGE detection.
Washing: 100% buffer B (20mM NaH2PO4 (pH 7.4), 500mM NaCl,500mM imidazole), 4mL/min, no collection, 2-3 column volumes washed until UV baseline was leveled. The ultrapure water is balanced for 2-3 CV. HisTrap excel columns can be stored with 2-3 CV equilibrium using 20% ethanol storage solution.
And (3) dialysis liquid change: the imidazole eluate containing the target protein was poured into a dialysis bag, dialyzed with 1XPBS for at least 1,000 times, and 80. Mu.l of the eluate was detected.
And (3) degerming and filtering: in a biosafety cabinet, a 0.22 μm low protein binding needle filter, or Nalgene filter with a 0.22 μm filter sterilized with a large volume of protein solution, was passed through and the filtered protein solution sample was stored in a freezer at-80 ℃.
Protein concentration determination: determining the protein concentration by using a BCA method, wherein the protein concentration of the batch is respectively 2.0mg/ml and 2.3mg/ml, and the volume is about 40ml; by calculation (protein yield = protein concentration protein volume/volume of fermentation supernatant taken), the protein yields of both strains 1D11 and 20A5 were about 0.8-0.92g/L.
Example 9: CD2V-Fc protein detection and stability verification
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, as shown in FIG. 3: from the figure, it can be calculated that the purity of SDS-PAGE of the purified CD2V-Fc protein 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 sample 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, diluting the CD2V-Fc protein by PBS (or normal saline) to a proper concentration to obtain an aqueous 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 following weight ratio of an antigen phase to the adjuvant of 1 and the volume ratio of 46;
emulsification: preheating the water phase and oil phase to 33 deg.C, slowly adding the water phase into the oil phase, stirring at 200-500rpm for 20-30min, standing at 20 deg.C for 1h, and standing at 4 deg.C 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 suction pipe, dripping the vaccine into cold water, observing (except for the 1 st drop), wherein the vaccine is dispersed in a cloud form, and judging the vaccine 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 separated out from the tube bottom is less than or equal to 0.25 mL;
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 one pig farm of Shaoxing) at 35 days of age (half of male and female) were randomly divided into 4 groups of 5 pigs each, and safety tests were conducted as follows.
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 for 2 weeks. Control group: each group was inoculated 1ml (vaccine in PBS) by intramuscular injection into the neck at 5 heads for 2 weeks.
During the experiment, the animal's spirit, clinical changes such as feeding, activity, drinking, inflammation change of injection site and excretion condition were observed every day, and 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 of dead pigs and no mental change are caused due to no inflammation phenomenon found at the injection part, so that inoculated animals do not suffer from any adverse reaction. The vaccine protein prepared by the invention has no obvious side effect even if injected at high dose (500 ug/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 with a concentration of CD2V-Fc protein (labeled 1 in the figure) of 2. Mu.g/well; the primary antibody is derived from serum of a pig immunized by CD2V-Fc, and the dilution ratio is 1; the secondary antibody is an HRP-labeled goat anti-pig IgG secondary antibody, and the dilution ratio is 1. 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 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 Hailong 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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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
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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
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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
225 230 235 240
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
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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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<211> 206
<212> PRT
<213> CD2V extracellular region protein amino acid sequence (PRT)
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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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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
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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
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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 of porcine Fc Protein (PRT)
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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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Gly Pro Ser Arg Glu Pro Gln Val Tyr Thr Leu Ser Pro Ser Ala Glu
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Glu Leu Ser Arg Ser Lys Val Ser Ile Thr Cys Leu Val Thr Gly Phe
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Tyr Pro Pro Asp Ile Asp Val Glu Trp Lys Ser Asn Gly Gln Pro Glu
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<213> nucleotide sequence (DNA) encoding CD2V-Fc
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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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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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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
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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
260 265 270
Asp Gly Val Glu Val His Thr Ala Gln Thr Arg Pro Lys Glu Glu Gln
275 280 285
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 (7)

1. A subunit fusion protein CD2V-Fc, wherein the subunit fusion protein CD2V-Fc contains an extracellular region of African swine fever virus surface envelope protein CD2V and an antibody Fc protein of a pig; 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, wherein the antibody Fc protein of the pig is the heavy chain constant region of IgG of the pig, the amino acid sequence is shown as SEQ ID No.3, and the amino acid sequence of the subunit fusion protein CD2V-Fc is shown as SEQ ID No. 1.
2. The subunit fusion protein CD2V-Fc of claim 1, wherein the subunit fusion protein CD2V-Fc further comprises a derivatized protein in which the amino acid sequence shown in SEQ ID NO1 includes the reversal of the order of CD2V and Fc and the placement of Fc at the amino terminus.
3. The subunit fusion protein CD2V-Fc of claim 2, wherein the subunit fusion protein CD2V-Fc further comprises one or more tag amino acids selected from 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.
4. The subunit fusion protein CD2V-Fc of claim 1, wherein the subunit fusion protein CD2V-Fc of African swine fever CD2V encodes a gene sequence as set forth in SEQ ID No. 4.
5. A method for producing the subunit fusion protein CD2V-Fc according to any one of claims 1 to 4, comprising the steps of:
1) Cloning a gene coded by a subunit fusion protein CD2V-Fc of the African swine fever CD2V shown as 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; the eukaryotic expression vector is pEE6.4, pEE12.4, pGL4.13, pcDNA3.1 and pcDNA3.3;
2) Transfecting recombinant plasmids containing genes coded by subunit fusion proteins of the African swine fever CD2V into CHO cells 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.
6. The method for preparing the African swine fever CD2V subunit fusion protein CD2V-Fc, according to the claim 5, wherein the expression system is mammalian cells, the mammalian cells are CHO cells, 293T cells, the CHO cells are DG44, DXB11, CHO-K1, CHO-S cells.
7. Use of a subunit fusion protein CD2V-Fc according to any one of claims 1 to 4 for the preparation of a vaccine for the diagnosis, prevention and treatment of African swine fever.
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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
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
CN114044805B (en) * 2020-08-26 2023-10-27 中国农业科学院兰州兽医研究所 Polypeptide for promoting pig organism to generate broad-spectrum immune response and application thereof
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
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
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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