Subunit fusion protein RBD-Fc on surface of porcine coronavirus D and preparation method and application of subunit fusion protein RBD-Fc

Abstract

The invention provides a subunit fusion protein RBD-Fc on the surface of a porcine D-type coronavirus and a preparation method and application thereof, the preparation method comprises the following steps: 1) cloning a gene sequence as shown in SEQ ID NO.5 into a eukaryotic expression vector to obtain a recombinant plasmid containing a fusion protein RBD-Fc coding gene; (2) transfecting a recombinant plasmid containing the fusion protein RBD-Fc coding gene into an expression cell; 3) culturing, screening and domesticating the expression cells in the step 2) to obtain a highly expressed cell strain; and 4) fermenting and culturing the cell strain in the step 3), and purifying to obtain the subunit fusion protein RBD-Fc. According to the present invention, the RBD-Fc can be subjected to mass soluble expression, the protein is stable, the problems that the porcine D-coronavirus surface S protein cannot be subjected to large-scale expression and the like in the prior art are solved, the preparation method is simple, and the cost is low.

Description

Technical Field

[0001] The present invention belongs to the technical field of expression and purification of recombinant proteins of biological products, and relates to a subunit fusion protein RBD-Fc on the surface of swine delta coronavirus, and a preparation method and application thereof. Background Art

[0002] Porcine deltacoronavirus, also known as PDCoV (Porcine Deltacoronavirus), was first named by Woo et al. in Hong Kong in 2012. Woo et al. performed RdRp gene detection on 7,140 nucleic acid samples collected from wild birds and mammals, and after comparing the genetic detection, they proposed that it was a deltacoronavirus.

[0003] Like PEDV and TGEV, PDcoV can cause severe diarrhea or vomiting in piglets, and further lead to dehydration and death. Domestic epidemiological surveys have found that the disease is co-infected with the PEDV virus, with a co-infection rate of up to 60%, posing a potential huge risk to the pig industry.

[0004] Like other coronaviruses, PDCoV is a single-stranded, positive-sense RNA virus with a spike-shaped, crown-like structure. It is approximately 25.4 kb in length and, from the 5' to the 3' end, consists of the following: the 5' untranslated region (5'-UTR), open reading frames 1a / 1b (ORF 1a / 1b), spike protein S, small envelope membrane protein, membrane protein, nonstructural protein 6 (NS6), nucleocapsid protein, accessory protein 7 (NS7), and the 3' untranslated region (3'-UTR). The spike protein S plays a crucial role in binding to cell receptors, mediating viral invasion and infection. It determines the virus's tissue tropism and is the primary antigen that induces neutralizing antibodies. Therefore, the S protein is the primary target antigen in coronavirus vaccine design. The receptor binding domain (RBD) is the primary target for inducing neutralizing antibodies. The present inventors analyzed the amino acid sequences of the S protein of PDCoV prevalent in China reported by NCBI from 2018 to 2022 and found that the (300-419aa) of the recently isolated strains remained essentially unchanged, and the RBD sequence of the existing inactivated vaccine (CHN-HN-2014 strain) and the recently reported strains were essentially identical. This can be used to prepare recombinant subunit vaccines.

[0005] Chinese patent application number 202210947100.X, entitled "A porcine delta coronavirus genetically engineered subunit vaccine and its application", provides a method for producing RBD protein. The RBD protein with correct structural folding is expressed and prepared using an insect baculovirus system, but the yield is low and the cost of large-scale industrial production is high, making it impossible to produce and apply. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a fusion protein RBD-Fc containing the surface S protein of swine delta coronavirus that can be produced industrially on a large scale and is safe and stable, as well as a preparation method and application thereof;

[0007] In order to solve this problem, the inventors fully analyzed and studied the currently available data on delta coronavirus. The inventors first optimized the RBD protein codons and then designed the expression. It was found that before and after optimization, the yield was still very low and did not increase significantly.

[0008] Furthermore, in order to make the target protein RBD easier to express, the inventors proposed adding a porcine antibody heavy chain constant region sequence, namely the Fc fragment, to the C-terminus of RBD in eukaryotic cells to promote the secretion and stable expression of the RBD protein, and to enable the recombinant protein to be secreted and expressed in the form of a dimer.

[0009] Ultimately, the inventors developed a method for stably and efficiently expressing RBD protein antigens and its construction and expression in a CHO system. The resulting monoclonal cell line, capable of secreting and expressing the subunit fusion protein RBD-Fc, exhibits high expression yields, allowing for the production of large quantities of pure RBD-Fc fusion protein through a single-step affinity chromatography step.

[0010] According to one aspect of the present invention, the present invention provides a subunit fusion protein RBD-Fc on the surface of porcine delta coronavirus, wherein the subunit fusion protein RBD-Fc contains the extracellular region and cell receptor binding site sequence of the porcine delta coronavirus surface envelope protein S protein and a pig antibody heavy chain constant region sequence Fc protein sequence; wherein the amino acid sequence of the extracellular region RBD of the porcine delta coronavirus surface envelope protein S is shown in SEQ ID NO.3; the Fc protein is an antibody heavy chain constant region sequence that can form a dimer, and its sequence is shown in SEQ ID NO.2.

[0011] According to the technical solution of the present invention, preferably, the amino acid sequence of the subunit fusion protein RBD-Fc is as shown in SEQ ID NO.4.

[0012] According to the technical solution of the present invention, preferably, the subunit fusion protein RBD-Fc also includes a derived protein having immunogenicity after substitution, deletion or addition of one or several amino acids in the amino acid sequence in SEQ ID NO.4.

[0013] According to the technical solution of the present invention, preferably, a detection or purification tag is connected to the amino terminus or carboxyl terminus of the amino acid sequence shown in SEQ ID NO.4, wherein the tag is selected from one of poly-Arg, poly-His, flag, c-myc, and HA.

[0014] According to the technical solution of the present invention, preferably, the subunit fusion protein RBD-Fc includes a protein derived by reversing the order of the RBD protein and the porcine antibody heavy chain constant region Fc in the amino acid sequence in SEQ ID NO.4, such as placing the porcine antibody heavy chain constant region Fc at the amino terminus.

[0015] According to the technical solution of the present invention, preferably, the swine delta coronavirus S protein is the amino acid sequence 300K-419V of the coronavirus extracellular region, including a derived protein that is substituted, deleted or added with one or several amino acids in the amino acid sequence in SEQ ID NO.2 and has immunogenicity.

[0016] According to the technical solution of the present invention, preferably, the expression system of the swine delta coronavirus subunit fusion protein includes but is not limited to mammalian cells.

[0017] According to the technical solution of the present invention, preferably, the mammalian cells are CHO cells.

[0018] According to the technical solution of the present invention, preferably, the gene sequence encoded by the subunit fusion protein RBD-Fc is as shown in SEQ ID NO.5.

[0019] According to another aspect of the present invention, a method for preparing a subunit fusion protein RBD-Fc is provided, comprising the following steps: 1) cloning the gene sequence encoding the amino acid sequence of SEQ ID NO.1 after codon optimization as shown in SEQ ID NO.5 into a eukaryotic expression vector to obtain a recombinant plasmid containing a gene encoding the fusion protein RBD-Fc; 2) transfecting the recombinant plasmid into expression cells; 3) culturing, screening, and acclimating the expression cells described in step 2) to obtain a highly expressing cell strain; and 4) fermenting and culturing the cell strain described in step 3), and obtaining the subunit fusion protein RBD-Fc after purification.

[0020] In the technical solution of the present invention, preferably, in step 1), the eukaryotic expression vector may be pEE6.4, pEE12.4, pGL4.13, pcDNA3.1, pcDNA3.3. Preferably, the eukaryotic expression vector is pEE12.4.

[0021] In the technical solution of the present invention, preferably, in step 2), the expression cell is a mammalian cell, and the mammalian cell is a CHO cell. The CHO cell can be a DG44, DXB11, CHO-K1, or CHO-S cell strain. Preferably, the CHO cell is a CHO-K1 cell.

[0022] To facilitate purification of the subunit RBD-Fc protein, those skilled in the art can attach a tag as shown in Table 1 to the amino terminus or carboxyl terminus of the amino acid sequence shown in SEQ ID NO. 4 through conventional techniques. In this example, Poly-His is specifically used as an example and is attached to the carboxyl terminus of the amino acid sequence shown in SEQ ID NO. 4.

[0023] Table 1 Tags and their amino acid sequences

[0024]

[0025] The present invention constructs and screens a CHO cell line that stably and efficiently secretes and expresses the subunit fusion protein RBD-Fc of the swine delta coronavirus S protein in suspension. The cell line has a high yield (yield of up to 1.5 g / L), is easy to purify, and only requires one-step affinity chromatography to achieve a purity of over 90% of the target protein, far exceeding the needs of subunit vaccines and diagnostic reagents), and is easy to produce on a large scale. Therefore, the problem of the S protein being unable to be expressed on a large scale is solved, and production costs are reduced. In addition, because the CHO cell line used for production is highly controllable during culture, easy to control quality, and the protein produced is stable between batches, it has high biosafety (no viruses, no risk of virus shedding). BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Represents the pEE12.4-OPTI-RBD-Fc plasmid map.

[0027] Figure 2 Indicates the double enzyme digestion identification results of pEE12.4-OPTI-RBD-Fc: M is DNA Marker: DL15000Marker; 1, 2, 3, 4 are the double enzyme digestion electrophoresis results of pEE12.4-OPTI-RBD-Fc EcoR I / Spe I, with band sizes of 7197 bp and 2689 bp, respectively. DETAILED DESCRIPTION

[0028] The present invention will be further described below with reference to the accompanying drawings and embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention.

[0029] The strains, plasmids and reagents used in the examples of the present invention are all commercially available products.

[0030] The sources of the reagents and drugs of the present invention are listed as follows:

[0031] CHO-K1 cells were obtained from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences and the Cell Bank of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences;

[0032] Cell culture medium and serum were purchased from Gibco, USA;

[0033] The eukaryotic expression vector pEE12.4 was purchased from Shanghai Linyuan Biotechnology Co., Ltd.;

[0034] Lipofectamine LTX was purchased from Thermo Fisher, USA;

[0035] Methionine sulfoximine (L-methionine sulfoximine, MSX) was purchased from Sigma;

[0036] BCA protein quantification kit was purchased from Thermo Fisher, USA;

[0037] Example 1: S protein expression and design

[0038] 1.1. Selection of swine deltacoronavirus S protein

[0039] The surface envelope protein S protein and the receptor-binding region RBD of swine delta coronavirus are important antigens that induce the body to produce protective neutralizing antibodies. Researchers have attempted to express the RBD protein using various systems, but none have been able to express and purify the protein on a large scale. This may be due to its instability when expressed alone. To address this important technical problem, the present invention introduces a porcine heavy chain constant region Fc sequence at the carboxyl terminus of the RBD protein to ensure correct structural folding and protein stability of the RBD protein. At the same time, to improve expression and facilitate screening in CHO cells, the inventors codon-optimized the encoded RBD-Fc fusion protein.

[0040] 1.2. Codon Optimization of the S Protein of Swine Delta Coronavirus

[0041] Our laboratory analyzed the amino acid sequences of the S protein of PDCoV reported in China by NCBI from 2018 to 2022. We found that the recently isolated strain (300-419aa) remained essentially unchanged, and the RBD sequence of the existing inactivated vaccine (CHN-HN-2014 strain) and the recently reported strain were essentially identical. The inventors used the sequence published in GenBank (GenBank: UDM84933.1) as a template and selected the RBD at 300K-419V. The inventors performed codon optimization and found that secretory protein expression in CHO cells was not optimal. To further enhance the stability of secretory expression of the RBD protein, the inventors added a porcine Fc sequence to the carboxyl terminus of the RBD protein. The amino acid sequence is shown in SEQ ID NO. 3. Finally, the inventors optimized the amino acid codons encoding the RBD-Fc fusion protein. The codon-optimized gene sequence is shown in SEQ ID NO. 5. Synthesis of this sequence was commissioned to Nanjing GenScript Biotechnology Co., Ltd.

[0042] Example 2: Construction of pEE12.4-OPTI-RBD-Fc recombinant plasmid

[0043] 2.1. PCR amplification of target fragment OPTI-RBD-Fc

[0044] 2.1.1 PCR reaction

[0045] (1) Primer design and synthesis

[0046] Upstream primer: 5'-ACGAAGCTTGCCGCCACCATGAC-3'

[0047] Downstream primer: 5'-TGAATTCTTAATGGTGATGG-3'

[0048] (2) The sample loading system is 50 μL, as shown in the following table:

[0049]

[0050]

[0051] PCR amplification procedure:

[0052]

[0053] 2.1.2. Gel recovery of PCR products

[0054] (1) Label the sample collection EP tube, adsorption column, and collection tube;

[0055] (2) Weigh the marked empty EP tube and record the value;

[0056] (3) Carefully cut the single target DNA band from the agarose gel using a scalpel and place it into a clean 1.5 mL centrifuge tube;

[0057] (4) Add 600 μL of PC buffer to the 1.5 mL centrifuge tube in step (3) and place in a 50°C water bath for about 5 min, gently turning the centrifuge tube upside down to ensure that the gel is fully dissolved;

[0058] (5) Column equilibration: Add 500 μL of equilibration solution BL to the adsorption column CB2 (the adsorption column is pre-placed in the collection tube), centrifuge at 12,000 rpm / min for 1 min, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube;

[0059] (6) Add the solution obtained in step (5) to the adsorption column CB2, let it stand for 2 minutes, centrifuge at 10,000 rpm / min for 30 seconds, discard the waste liquid in the collection tube, and then place the adsorption column CB2 into the collection tube;

[0060] (7) Add 600 μL of PW buffer to the adsorption column, let it stand for 3 min, centrifuge at 10,000 rpm / min for 30 s, discard the waste liquid in the collection tube, and place the adsorption column CB2 in the collection tube;

[0061] (8) Repeat step (7);

[0062] (9) Centrifuge the empty adsorption column at 12,000 rpm / min for 2 min to remove as much rinse solution as possible. Place the adsorption column at room temperature for 10 min to dry thoroughly.

[0063] (10) Place the adsorption column CB2 in the collection tube, add 50 μL of Elution Buffer (preheated at 65°C) dropwise to the middle of the adsorption membrane, let it stand for 3 min, and centrifuge at 12,000 rpm / min for 2 min;

[0064] (11) Remove the centrifuge tube from step (10) from the centrifuge, discard the adsorption column CB2 in the middle, cover the centrifuge tube, and retain the DNA sample in the centrifuge tube;

[0065] (12) Store the DNA sample in step 11 at 4°C and prepare agarose gel electrophoresis to identify the DNA fragments recovered from the gel.

[0066] 2.2. Double enzyme digestion reaction of PCR products and vectors

[0067] (1) Label the 1.5 mL EP tubes to be used, add the sample and mix well according to the following table: 50 μL reaction system

[0068]

[0069] (2) Place the 1.5 mL EP tube in step (1) in a constant temperature water bath at the optimal temperature for the corresponding enzyme and incubate for 2-3 hours.

[0070] Gel recovery of double enzyme digestion products: Take out the double enzyme digestion system and perform agarose gel electrophoresis to recover the DNA fragments therein. The method is the same as the gel recovery of PCR products in 1.2.1.

[0071] 2.3 Ligation reaction

[0072] (1) Prepare several clean 1.5 mL EP tubes, label them, and place them in an EP tube rack for use.

[0073] (2) Add the sample to a 1.5 mL EP tube and mix thoroughly according to the table below.

[0074]

[0075] (3) After adding the sample according to the table in step (2), place each 10 μl reaction system in a 16°C low-temperature cooling liquid circulation machine and water bath for 10-16 hours;

[0076] (4) Take out the EP tube from step (3) and place it in a 65°C water bath for 15 minutes;

[0077] (5) Take out the EP tube from step (4) and store it at 4°C.

[0078] 2.4 Conversion reaction

[0079] (1) Quickly add 10 μL of ligation reaction solution to 100 μL of competent cells, pipette to mix, and incubate on ice for 30 min;

[0080] (2) Remove the sample tube and place it in a 42°C water bath for 100 seconds, then immediately place it in an ice bath for 2 minutes;

[0081] (3) Remove the sample tube and add 600 μL of liquid LB medium to the sample tube in a clean bench. Then place the sample tube in a 37°C constant temperature shaker at 220 rpm / min for 1 h.

[0082] (4) Plate coating: Take out the sample tube in step (3), centrifuge at 8,000 rpm / min for 2 min at room temperature, remove 600 μL of supernatant, and resuspend the bacteria at the bottom of the tube with the remaining supernatant. Place the resuspended bacterial solution in the center of the corresponding transformation plate and spread the bacterial solution in the center of the transformation plate evenly with a bacterial spreader.

[0083] (5) Place the transformation plate prepared in step (4) upright in a biochemical constant temperature incubator and incubate at 37°C for 1 hour. Then, invert the transformation plate and incubate for 15 hours.

[0084] (6) Observe the transformation results.

[0085] 2.5 Plasmid extraction and double enzyme digestion identification

[0086] 2.5.1 Plasmid Extraction

[0087] (1) Use a 10 μL pipette tip to pick a single colony from the transformation plate and transfer it to 5 mL of LB liquid medium containing ampicillin resistance. Incubate the culture at 37°C, 220 rpm / min, and shake overnight.

[0088] (2) Transfer the bacterial suspension to a 1.5 mL EP tube and centrifuge at room temperature, 12,000 rpm / min, for 2 min. Discard the supernatant.

[0089] (3) Add 250 μL of plasmid extraction reagent P1 buffer to the EP tube in step (2) to thoroughly suspend the bacteria;

[0090] (4) Add 250 μL P2 buffer to the solution in step (3), immediately gently invert the centrifuge tube 5-10 times to mix, and let it stand at room temperature for 2-4 minutes;

[0091] (5) Add 350 μL P3 buffer to the solution in step (4) and immediately mix by gently inverting the tube 5-10 times; let it stand at room temperature for 2-4 minutes;

[0092] (6) Centrifuge the solution from step (5) at room temperature, 14,000 rpm / min, for 10 min;

[0093] (7) Transfer the supernatant solution from step (6) to the center of the adsorption column and centrifuge at room temperature, 12,000 rpm / min, for 30 s, and discard the liquid in the collection tube;

[0094] (8) Add 500 μL of buffer DW1 to the center of the adsorption column, centrifuge at room temperature, 12,000 rpm / min, 30 s, and discard the liquid in the collection tube;

[0095] (9) Add 500 μL of wash solution to the center of the adsorption column and centrifuge at room temperature, 12,000 rpm / min, for 30 s. Discard the liquid in the collection tube and repeat once.

[0096] (10) Empty adsorption column, centrifuge at room temperature, 12,000 rpm, 2 min.

[0097] (11) Place the adsorption column in a clean 1.5 mL centrifuge tube. Add 30 μL of Eliution Buffer to the center of the adsorption membrane. Let it stand at room temperature for 5 min. Centrifuge at room temperature at 12,000 rpm for 2 min. Save the DNA solution in the tube.

[0098] 2.5.2 Double enzyme digestion identification

[0099] (1) Mark the 1.5mL EP tubes to be used and add samples according to the following table: 20μL reaction system

[0100]

[0101] (2) Place the 20 μL reaction system in the EP tube in step (1) in a 37°C constant temperature water bath for 2 hours.

[0102] (3) The double enzyme digestion system sample in step (2) was subjected to agarose gel electrophoresis to check whether the inserted fragment size was correct; the experimental results are shown in Figure 2 : Enzyme digestion confirmed that the construction was correct.

[0103] (4) Select the clone with the correct insert and send it to a sequencing company for sequencing.

[0104] Example 3: Transfection of CHO-K1 cells with pEE12.4-OPTI-RBD-Fc recombinant plasmid and establishment of monoclonal screening

[0105] 3.1 CHO-K1 cell transfection

[0106] (1) Preparation: Sterilize in a biosafety cabinet with UV for 30 min; preheat DMEM / F12 (containing 10% serum and 1% double antibody), DMEM / F12, and PBS to 37°C in a 37°C water bath.

[0107] (2) Remove cells (10 cm cell culture dish) from the 37°C incubator, discard the supernatant culture medium, wash the cells once with 8 mL of pre-warmed PBS, and discard the PBS.

[0108] (3) Add 1-2 mL of 0.25% trypsin-EDTA to each 10 cm cell culture dish and digest at room temperature for about 2 minutes. Observe under a microscope to see that the cells are wrinkled and rounded and appear as single cells.

[0109] (4) Add 4 mL of DMEM / F12 (containing 10% serum and 1% double antibody) to terminate the digestion reaction, and use a pipette to blow away the cells.

[0110] (5) Transfer the digested cells to a 15 mL centrifuge tube and centrifuge at room temperature, 200 g, for 5 min.

[0111] (6) Resuspend the cells in DMEM / F12 (containing 10% serum and 1% double antibody) and count.

[0112] (7) Dilute cells to 2×10 5 2 mL of the mixed cells were added to a six-well plate, and the six-well plate was placed in a 37°C, 5% CO2 cell culture incubator and incubated overnight.

[0113] (8) Take out the cell culture dish prepared in step (7) and observe the cell status: when the cell confluence reaches 80%-90%, transfection can be started. Before transfection, replace the culture medium with antibiotic-free and serum-free DMEM / F12, 2 mL / well.

[0114] (9) Plasmid dilution: Dilute the plasmid with OPTI-MEM. Add 2.5 μg of plasmid to 125 μL OPTI-MEM, then add 2.5 μL plus, mix well, and let stand at room temperature for 5 min.

[0115] (10) Dilute Lipofectamine LTX: Add 9 μL Lipofectamine LTX to 125 μL OPTI-MEM, then add 2.5 μL plus, mix gently, and let stand at room temperature for 5 minutes.

[0116] (11) Gently mix the mixtures from step (10) and step (11). Let stand at room temperature for 5 minutes, then add dropwise to a six-well plate and distribute evenly.

[0117] (12) Place the six-well plate in a 37°C, 5% CO2 cell culture incubator and culture for 4-6 hours.

[0118] (13) Medium change: discard the supernatant culture medium, add 2 mL of DMEM / F12 (containing 10% serum and 1% double antibody), and place the six-well plate in a cell culture incubator at 37°C and 5% CO2 for culture.

[0119] 3.2. Pressurized screening

[0120] Start pressurization 24 hours after transfection: remove the six-well plate cells from the 37°C incubator, discard the supernatant culture medium, add 2mL DMEM / F12 (containing 10% serum + 25μM MSX), and pressurize for 7 days. Observe the cells in the middle and change the medium frequently for dead cells.

[0121] 3.3 Monoclonal Screening

[0122] (1) When the negative control cell stock is about 10-20%, about 7 days later, monoclonal screening begins.

[0123] (2) Remove the six-well plate, discard the culture medium, wash once with PBS, then add 300 μL of 0.25% trypsin-EDTA, digest at room temperature for about 2 minutes, add 2 mL of DMEM / F12 (containing 10% serum + 25 μM MSX) to terminate the digestion reaction, and blow the cells apart with a pipette.

[0124] (3) Transfer the digested cells to a 15 mL centrifuge tube and centrifuge at room temperature, 200 g, for 5 min.

[0125] (4) Resuspend the cells in DMEM / F12 (containing 10% serum + 25 μM MSX) and count.

[0126] (5) Plating: Dilute cells to 5 cells / mL, take 200 μL of the mixed cells and add them to a 96-well plate, and place it in a 37°C, 5% CO2 cell culture incubator for 4-6 hours.

[0127] (6) Recording of single cell wells.

[0128] (7) When a single cell has grown in a 96-well plate, discard the culture medium, wash once with PBS, add 100 μL of 0.25% trypsin-EDTA, and digest for about 2 minutes at room temperature. Add 2 mL of DMEM / F12 (containing 10% serum + 25 μM MSX) to terminate the digestion reaction, and use a pipette to disperse the cells. Transfer the cell solution to a 12-well plate. When the 12-well plate is full, take the supernatant and test whether the clone is positive by ELISA. The positive clones with high expression continue to be expanded and frozen.

[0129] Example 4: CHO-K1 cell line acclimated to suspension culture

[0130] (1) Preparation: Sterilize in a biosafety cabinet with UV for 30 min; preheat DMEM / F12 (containing 10% serum and 25 μM MSX) to 37°C in a 37°C water bath.

[0131] (2) Remove cells (10 cm cell culture dish) from the 37°C incubator, discard the supernatant culture medium, wash the cells once with 8 mL of pre-warmed PBS, and discard the PBS.

[0132] (3) Add 1-2 mL of 0.25% trypsin-EDTA to each 10 cm cell culture dish and digest at room temperature for about 2 minutes. Observe under a microscope to see that the cells are wrinkled and rounded and appear as single cells.

[0133] (4) Add 4 mL of DMEM / F12 (containing 10% serum and 25 μM MSX) to terminate the digestion reaction, and disperse the cells with a pipette.

[0134] (5) Transfer the digested cells to a 15 mL centrifuge tube and centrifuge at room temperature, 200 g, for 5 min.

[0135] (6) The cells were suspended in 100% DMEM / F12 (containing 10% serum and 25 μM MSX) and counted.

[0136] (7) Dilute cells to 5×10 5 Inoculate 30 mL of culture medium in a 125 mL shake flask with 100 cells / mL. Place the cell culture flask in a 37°C, 5% CO2 cell incubator on an orbital shaker at 120 rpm / min and incubate overnight.

[0137] (8) The biosafety cabinet surface should be wiped and disinfected with 75% alcohol and then irradiated with ultraviolet light for 30 minutes.

[0138] (9) Count the cell density and viability every 24 hours.

[0139] (10) After the first generation of cells has been cultured once, the second generation of cells is cultured when the cell survival rate reaches 94-97%.

[0140] (11) Preparation: UV sterilize in a biosafety cabinet for 30 min; 100% DMEM / F12 (containing 10% serum, 25 μM MSX) and EX-CELL 302 were placed in a CO2 cell culture incubator and preheated to 37°C.

[0141] (12) Remove the cells from the 37°C incubator and transfer them to a 50 mL centrifuge tube. Centrifuge at 200 g for 5 min at room temperature.

[0142] (13) DMEM / F12 (containing 10% serum, 25 μM MSX) and EX-CELL 302 were mixed at a ratio of 1:1, the cells were resuspended, and the cells were counted.

[0143] (14) Dilute cells to 5×10 5 Inoculate 30 mL of culture medium in a 125 mL shake flask with 100 cells / mL. Place the cell culture flask in a 37°C, 5% CO2 cell culture incubator on an orbital shaker at 120 rpm / min and incubate overnight.

[0144] (15) The biosafety cabinet surface should be wiped and disinfected with 75% alcohol and then irradiated with ultraviolet light for 30 minutes.

[0145] (16) Count the cell density and viability every 24 h.

[0146] (17) The cell survival rate after two cultures of the second generation was greater than 95%; the cell survival rate after three cultures of the third to sixth generations was greater than 95%. After 7 weeks, the cells were propagated for three generations 3 days after inoculation, and the density reached 1×10 6cells / mL, and the cell viability reaches 95%, the cells are considered to have adapted to suspension culture. The seeding density is reduced to 3×10 5 pieces / mL.

[0147] (18) After domestication, the 3D10, 9C11, and 2D8 strains all met the requirements, indicating that the 3D10, 9C11, and 2D8 strains were successfully domesticated.

[0148] Example 5: Cell shake flask fermentation

[0149] (1) Preparation of subculture medium: 60% CD-CHO + 40% Ex-cell 302 were placed in a 37°C water bath and preheated to 37°C.

[0150] (2) Remove the cells from the CO2 constant temperature shaker and count them.

[0151] (3) Dilute the 3D10 cells obtained in Example 4 to 2.5-3.5×10 5 Inoculate 30 mL of culture medium in a 125 mL shake flask with 100 cells / mL. Incubate the cell culture flask in a 37°C, 5% CO2 incubator at 100 rpm / min overnight.

[0152] (4) Count cell density and viability every 24 h, measure glucose, and add glucose to 4 g / L when blood glucose is lower than 2 g / L. Take 1 mL of sample every day, and use the supernatant to detect protein expression.

[0153] (5) Feed (about the fourth day): Supplement with 70 g / L CB5, adding 10% of the basal medium.

[0154] (6) Starting from the 5th day, adjust the CO2 incubator temperature to 32℃.

[0155] (7) On the ninth day, 70 g / L CB5 was added, which was 10% of the basal medium.

[0156] (8) On the thirteenth day, the cell supernatant was harvested.

[0157] Example 6: Construction and acclimation of a recombinant CHO-S cell line stably expressing RBD-Fc protein

[0158] Following the procedures of Examples 1-5, the present inventors also readily constructed stable cell lines expressing RBD-Fc proteins. Therefore, common engineered mammalian cell lines can be foreseen. This method can readily be used to construct stable cell lines expressing recombinant RBD-Fc, thereby enabling large-scale production of the protein. Therefore, this approach falls within the scope of protection of the present invention.

[0159] Example 7: Protein purification

[0160] The cell culture medium of Example 5 (about 100 ml for each batch) was collected and centrifuged at 8,000 g for 30 min at 4°C. The supernatant was collected and filtered through a 0.8 μm filter. The sample was loaded, and 80 μL of the sample was reserved and added to 20 μL of 5× SDS-sample buffer for SDS-PAGE detection.

[0161] Column equilibration: Equilibrate with ultrapure water for 2-3 CV (column volume), drain out the ethanol storage solution; then equilibrate with equilibration buffer for 5-10 CV.

[0162] Sample loading: Connect a 5 ml GE Excel prepacked column to an AKTA column at 1 ml / min, collect the flow through (FT), take 80 μL of sample and add 20 μL of 5× SDS-sample buffer for SDS-PAGE detection.

[0163] Wash impurities: Elute impurities with elution buffer containing 20mM imidazole and 50mM imidazole, respectively, and collect: 5mL / tube; after collecting and mixing the samples, take 80μL of the sample and add 20μL of 5×SDS-sample buffer for SDS-PAGE detection.

[0164] Elution: Elute the impurities with 500 mM imidazole elution buffer, collection: 5 mL / tube; after collecting and mixing the samples (Elutethrough-ET), take 80 μL of sample and add 20 μL of 5×SDS-sample buffer for SDS-PAGE detection.

[0165] Dialysis solution exchange: Pour the eluate containing the target protein into the dialysis bag, dialyze at least 1,000 times with 1× PBS, and take 80μl sample for detection.

[0166] Sterile Filtration: In a biosafety cabinet, filter the protein through a 0.22μm low-protein binding syringe filter, or filter large amounts of protein solution through a sterile 0.22μm Nalgene filter. Store the filtered protein sample at -80°C. Purified protein was determined to be over 90% pure by SDS-PAGE, as shown in the figure (Figure 1 shows SDS-PAGE analysis of purified subunit fusion protein RBD-Fc: 1 is the marker, 2 is the subunit fusion protein (RBD-Fc in the loading buffer containing β-mercaptoethanol as a reducing agent), and 3 is the RBD-Fc subunit fusion protein (RBD-Fc in the loading buffer without β-mercaptoethanol as a reducing agent). This figure will be added later). Using a loading buffer without β-mercaptoethanol as a reducing agent, the results indicate that RBD-Fc exists as a dimer with a molecular weight approximately twice that of RBD-Fc in the loading buffer containing β-mercaptoethanol as a reducing agent. Protein concentration determination: The protein concentration was determined by the BCA method. The protein concentration of this batch was 3 mg / ml and the volume was approximately 50 ml. After calculation (protein yield = protein concentration * protein volume / fermentation supernatant volume), the protein yield was approximately 1500 mg / L.

[0167] Example 8: Western-blot identification and stability verification of RBD-Fc protein

[0168] 8.1. SDS-PAGE detection

[0169] The protein purified in Example 7 was subjected to western-blot detection, the transfer time was 1 h, the antibody used was swine D coronavirus positive serum (donated by Guangdong Provincial Animal Health Institute), the dilution ratio was 1:1000, the incubation time was 1 h, and the secondary antibody was HRP-labeled sheep anti-swine polyclonal antibody (purchased from Quanshijin Bio). The results are shown in the figure (the figure shows the Western ern-blot detection results after purification of RBD-Fc protein: 1 is Marker, 2 is RBD-Fc fusion protein containing β-mercaptoethanol reducing agent, 3 is RBD-Fc subunit fusion protein without β-mercaptoethanol reducing agent, 3 is Marker. This figure will be supplemented later.): As can be seen from the results in the figure, the purified RBD-Fc protein can effectively bind to the positive antibody.

[0170] 8.2 Stability Verification

[0171] The protein (3 mg / ml) purified in Example 7 was diluted to 1 mg / ml and divided into 16 portions, each 0.5 ml; 8 portions were placed in a 4°C refrigerator, and samples were taken at 1 week, 2 weeks, 3 weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks, and 20 weeks, for 8 consecutive times; 8 portions were placed in a -20°C refrigerator, and samples were taken at 1 week, 2 weeks, 3 weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks, and 20 weeks, for 8 consecutive times; the protein concentration was detected by BCA after each sampling, and the results are shown in the following table:

[0172]

[0173] Judging from the changes in BCA protein concentration, the protein remained basically stable during the two groups of experiments.

[0174] Example 9: Preparation of RBD-Fc protein subunit vaccine

[0175] 9.1 Vaccine Preparation

[0176] Preparation of aqueous phase: Based on the RBD-Fc protein content in the vaccine, dilute the RBD-Fc protein to an appropriate concentration using PBS (or normal saline) to prepare the aqueous phase;

[0177] Oil phase preparation: Based on the total amount of vaccine to be prepared, measure an appropriate amount of ISA 201VG adjuvant at a weight ratio of 1:1 between antigen phase and adjuvant and a volume ratio of 46:54;

[0178] Emulsification: Preheat both the water phase and the oil phase to 33°C, slowly add the water phase to the oil phase, stir at 200-500 rpm for 20-30 minutes, let stand at 20°C for 1 hour, and then place at 4°C overnight;

[0179] Packaging and storage: Pack as needed and store at 4℃ for later use after passing inspection.

[0180] 9.2 Vaccine Testing

[0181] Physical properties: Observe the appearance by visual inspection (whether it is a milky white emulsion);

[0182] Use a clean pipette to draw a small amount of vaccine and drop it into cold water. Observe (except the first drop). The vaccine should diffuse in a cloud-like manner, indicating that it is a water-in-oil-in-water dosage form.

[0183] Add 10 ml of the vaccine to a centrifuge tube and centrifuge at 3000 r / min for 15 min. If the amount of water precipitated at the bottom of the tube is ≤ 0.5 mL, it is considered stable.

[0184] Use a viscometer to test the viscosity of the vaccine, which should be between 20-50cp to be considered qualified.

[0185] 9.3. Animal Experiments of RBD-Fc Protein Subunit Vaccine

[0186] 10.3.1 Immunity Test

[0187] The vaccine (100 μg / dose) was prepared according to the method in 10.1 above. Six healthy New Zealand white rabbits were randomly divided into two groups of three. Each rabbit was immunized with 2 ml intramuscularly for a total of two immunizations, with a booster 21 days after the first immunization. Blood was collected before immunization, 21 days after immunization, and 7 days after the second immunization. Serum was isolated and assayed for antibody levels by ELISA. As shown in the table below, antibody levels reached a high of 1:102,400 21 days after the first immunization and 1:819,600 7 days after the second immunization.

[0188]

[0189] 10.3.2. ELISA Antibody Titer Detection

[0190] (1) Coating: Dilute the purified tS protein prepared in the inventor's laboratory to 2 μg / ml with coating solution (50 mM carbonate buffer, pH 9.5), add 100 μl to each well of the ELISA plate, seal with sealing film, and place in a refrigerator at 4°C overnight;

[0191] (2) Washing: After taking the ELISA plate out of the refrigerator, place it in a plate washer and wash it using PBST;

[0192] (3) Blocking: Add 200 μl of blocking solution (5% skim milk) to each well, seal with sealing film, and incubate at 37°C for 2 h;

[0193] (4) Sample preparation: Dilute the serum appropriately with blocking solution according to known information and required dosage;

[0194] (5) Washing: Same as (2);

[0195] (6) Sample addition: Add diluted serum and use blocking solution as a negative control, incubate at 37°C for 1 h;

[0196] (7) Washing: Same as (2);

[0197] (8) Add secondary antibody: add 100 μl of appropriately diluted HRP-labeled secondary antibody to each well and incubate at 37°C for 0.5 h;

[0198] (9) Washing: Same as (2);

[0199] (10) Color development: Add 100 μl of TMB color development solution to each well in the dark and incubate at 37°C for 10 min.

[0200] (11) Termination: Add 50 μl of stop solution (2 M H2SO4) to each well to terminate the reaction;

[0201] (12) Detection: Measure the OD value of the sample at a wavelength of 450 nm and analyze the data;

[0202] (13) Result analysis: The criteria for judging antibody positivity are: P / N ≥ 2.1, OD450 ≥ 0.1.

[0203] The present invention is illustrated by the above examples, however, it should be understood that the present invention is not limited to the specific examples and embodiments described herein. These specific examples and embodiments are included herein to assist those skilled in the art in practicing the present invention. Further improvements and modifications will readily occur to those skilled in the art without departing from the spirit and scope of the present invention, and the present invention is therefore limited only by the content and scope of the appended claims, which are intended to cover all alternatives and equivalents included within the spirit and scope of the present invention as defined by the appended claims.

[0204]

[0205]

[0206]

Claims

1. A subunit fusion protein RBD-Fc on the surface of swine delta coronavirus, characterized in that: The subunit fusion protein RBD-Fc contains the extracellular region and cell receptor binding site (RBD) sequence of the porcine delta coronavirus surface envelope protein S protein and a pig Fc protein sequence; wherein, the amino acid sequence of the extracellular region RBD of the porcine delta coronavirus surface envelope protein S is shown in SEQ ID NO.3; the Fc protein is a porcine antibody heavy chain constant region protein sequence that can form a dimer, and its sequence is shown in SEQ ID NO.

2.

2. The subunit fusion protein RBD-Fc according to claim 1, characterized in that The amino acid sequence of the subunit fusion protein RBD-Fc is shown in SEQ ID NO.

4.

3. The subunit fusion protein RBD-Fc according to claim 1 or 2, characterized in that Also included are derivative proteins having immunogenicity after substitution, deletion or addition of one or several amino acids in the amino acid sequence shown in SEQ ID NO.

4.

4. The subunit fusion protein RBD-Fc according to claim 3, characterized in that A detection or purification tag is connected to the amino terminus or carboxyl terminus of the amino acid sequence shown in SEQ ID NO. 4, wherein the tag is selected from one of poly-Arg, poly-His, flag, c-myc, and HA.

5. The subunit fusion protein RBD-Fc according to claim 4, characterized in that The tag is poly-His, and the amino acid sequence of the subunit fusion protein RBD-Fc is shown in SEQ ID NO.

1.

6. The subunit fusion protein RBD-Fc according to claim 5, characterized in that The gene sequence encoding the subunit fusion protein RBD-Fc is shown in SEQ ID NO.

5.

7. A method for preparing the subunit fusion protein RBD-Fc according to any one of claims 1 to 6, characterized in that: The preparation method comprises the following steps: 1) Optimizing the codons encoding the amino acid sequence of SEQ ID NO. 1 to obtain the gene sequence shown in SEQ ID NO. 5, and cloning the gene sequence of SEQ ID NO. 5 into a eukaryotic expression vector to obtain a recombinant plasmid containing the gene encoding the fusion protein RBD-Fc; 2) transfecting the recombinant plasmid into expression cells; 3) obtaining a highly expressing cell line by culturing, screening, and acclimating the expressing cells described in step 2); and 4) Fermenting and culturing the cell line described in step 3), and obtaining the subunit fusion protein RBD-Fc after purification.

8. The preparation method according to claim 7, characterized in that In step 1), the eukaryotic expression vector is pEE6.4, pEE12.4, pGL4.13, pcDNA3.1, pcDNA3.3, or pEE12.

4.

9. The preparation method according to claim 7, characterized in that In step 2), the expression cells are mammalian cells, the mammalian cells are CHO cells, and the CHO cells are CHO-K1 cells.

10. Use of any subunit fusion protein RBD-Fc according to claim 1 in the preparation of a vaccine for diagnosis and prevention of swine delta coronavirus.

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