Mammalian cell strain, method for preparing hog cholera virus soluble E2 recombinant antigen by using same and application thereof

Abstract

The invention discloses a mammalian cell strain, a method for preparing a hog cholera virus soluble E2 recombinant antigen by using the mammalian cell strain and application of the mammalian cell strain. The mammalian cell expression system with different cell generations can stably express a large amount of soluble CSFV-E2 recombinant protein, so that the production cost for producing the hog cholera E2 protein is effectively reduced, and further the mammalian cell expression system can be applied to production of the hog cholera E2 protein subunit vaccine.

Description

Technical Field

[0001] The present invention relates to a mammalian cell line, in particular to a mammalian cell line which stably and massively expresses soluble E2 recombinant antigen of classical swine fever virus, a method for producing soluble E2 recombinant antigen of classical swine fever virus and an application thereof. Background Art

[0002] Classical swine fever (CSF), also known as classical swine fever, is a highly contagious disease that infects pigs. Generally speaking, pigs have high fever, anorexia, diarrhea, neurological symptoms, systemic erythema, mass mortality, sow abortion or dead piglets. It is a highly contagious and highly pathogenic disease that infects pigs.

[0003] After being first discovered in 1903, classical swine fever has spread around the world, including Asia, Africa, Europe, Central and South America, and once an outbreak occurs, it often causes significant economic losses to the pig farming industry and even affects the global economy. Routine vaccination must be used to prevent and control the spread of classical swine fever in areas where classical swine fever is prevalent. If used properly, vaccination can effectively limit the spread of classical swine fever, prevent disease outbreaks, and establish protective immunity in uninfected pigs.

[0004] The pathogen of classical swine fever is classical swine fever virus (CSFV), which belongs to the genus Pestivirus of the family Flavivirdae. The CSFV genome is a single-stranded positive RNA with a total length of about 12.3kb, which is translated into 3898 amino acids to form a polyprotein, which is then processed by proteases into four structural proteins (C, Erns, E1 and E2) and eight non-structural viral proteins (Npro, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). Among them, E2, Erns and NS3 can induce antibody responses in the host.

[0005] There are two main types of commercial swine fever vaccines in common use: modified live vaccine (MLV) and swine fever virus envelope protein E2 unit vaccine. Although the MLV swine fever vaccine is cheap and effective, it is impossible to distinguish the immune response of pigs from MLV vaccination or natural infection in serum antibody testing. After pigs are vaccinated with MLV swine fever vaccine, the swine fever virus in the environment can continue to infect animals, and the immune response produced by animals after natural infection may still cause a certain degree of low virus content in the animal's body, resulting in the continued presence of swine fever virus in the environment and the host. In order to completely eradicate the source of infection of the virus, it is necessary to distinguish whether the reaction in the pigs is caused by vaccine immunization or natural infection in the pigs in the animal farms, and then carry out the eradication plan, which requires diagnostic reagents to distinguish these two types of immune responses.

[0006] Although commercially available insect cells can produce E2 recombinant antigens, their cost is high, so the development of low-cost E2 subunit vaccines is an urgent issue. In view of this, it is urgent to develop a low-cost method for producing E2 recombinant antigens to solve the various problems of the conventional technology. Summary of the invention

[0007] Therefore, one aspect of the present invention is to provide a mammalian cell line that stably expresses soluble E2 recombinant antigen of classical swine fever virus.

[0008] Another aspect of the present invention is to provide a method for producing a soluble recombinant E2 antigen of classical swine fever virus, which comprises culturing the above-mentioned mammalian cell line to increase the yield of the soluble E2 recombinant antigen.

[0009] Another aspect of the present invention is to provide a soluble recombinant E2 antigen of classical swine fever virus, which is produced by the above-mentioned mammalian cell line.

[0010] Another aspect of the present invention is to provide a subunit vaccine composition for pigs, which uses the above-mentioned soluble E2 recombinant antigen of classical swine fever virus as an active ingredient.

[0011] According to the above aspect of the present invention, a mammalian cell line stably expressing a soluble E2 recombinant antigen of classical swine fever virus is provided. This cell line is a Chinese hamster epithelial cell line (Chinese Hamster Ovary cell) CCC_E2_5E12 deposited with the German Culture Collection (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ; address: Inhoffen straβe 7B, 38124 Braunschweig, Germany) on May 17, 2023, with a deposit number of DSM ACC3376 and a classification name of Chinese hamster ovary cell.

[0012] In the above embodiment, the amino acid sequence of the aforementioned soluble E2 recombinant protein may be, for example, as shown in SEQ ID NO: 1.

[0013] In the above embodiments, the protein concentration of the aforementioned soluble E2 recombinant protein may be, for example, at least 800 mg / L cell culture fluid. In some examples, the protein concentration of the aforementioned soluble E2 recombinant protein may be, for example, at least 850 mg / L cell culture fluid. In other examples, the protein concentration of the aforementioned soluble E2 recombinant protein may be, for example, 850 mg / L cell culture fluid to 950 mg / L cell culture fluid.

[0014] In the above embodiment, the aforementioned E2 recombinant protein can be encoded by the nucleic acid sequence shown in SEQ ID NO:2, for example.

[0015] According to another aspect of the present invention, a method for producing a soluble E2 recombinant protein of classical swine fever virus is provided, wherein the method comprises culturing the mammalian cell line as described above to produce a soluble E2 recombinant antigen.

[0016] According to another aspect of the present invention, a soluble E2 recombinant antigen of classical swine fever virus is provided, which is produced by the mammalian cell line as mentioned above, wherein the amino acid sequence of the soluble E2 recombinant protein can be, for example, as shown in the sequence identification number (SEQ ID NO): 1.

[0017] According to another aspect of the present invention, a subunit vaccine composition for pigs is provided, which uses the above-mentioned soluble E2 recombinant antigen of classical swine fever virus as an active ingredient, and the effective number of administrations of this subunit vaccine composition for pigs to a subject can be, for example, once.

[0018] In the above embodiment, the effective dosage of the aforementioned E2 recombinant protein may be, for example, 25 μg / mL to 50 μg / mL.

[0019] The mammalian cell line of the present invention and the method for using the same for producing soluble E2 recombinant antigen of classical swine fever virus can greatly increase the yield of soluble E2 recombinant antigen of classical swine fever virus by utilizing mammalian cell expression systems of different cell generations, and can effectively reduce the production cost of soluble E2 recombinant antigen of classical swine fever virus, and then be applied to the production of classical swine fever E2 protein subunit vaccine.

[0020] It is to be understood that the foregoing general description and the following detailed description are merely exemplary, and are intended to provide further explanation of the invention as claimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] To make the above and other objects, features, advantages and embodiments of the present invention more clearly understood, the detailed description of the accompanying drawings is as follows:

[0022] Figure 1A and Figure 1B The colloidal images of SDS-PAGE of the transfected CHO cell culture fluid according to one embodiment of the present invention are shown respectively ( Figure 1A ) and immunoblotting ( Figure 1B ) color image.

[0023] FIG. 2A to FIG. 2B The colloidal images of SDS-PAGE of the E2 recombinant protein expressed by the CHO cell line CCC_E2_5E12 according to one embodiment of the present invention are shown respectively ( Figure 2A ) and immunoblotting ( Figure 2B ) color image.

[0024] FIG. 3A to FIG. 3B The colloidal images of SDS-PAGE of CHO cell lines CCC_E2_5E12 of different cell generations expressing E2 recombinant protein in cell fluid before purification according to one embodiment of the present invention are shown respectively ( Figure 3A ) and immunoblotting ( Figure 3B ) color image.

[0025] FIG. 4A to FIG. 4B The colloidal images of SDS-PAGE of the E2 recombinant protein expressed in the cell fluid of the CHO cell line CCC_E2_5E12 of different cell generations according to one embodiment of the present invention after purification are shown respectively ( Figure 4A ) and immunoblotting ( Figure 4B ) color image.

[0026] Figure 5 A bar graph showing the protein concentration of E2 recombinant protein after purification of cell fluids of CHO cell line CCC_E2_5E12 of different cell generations according to an embodiment of the present invention.

[0027] Wherein, the reference numerals are:

[0028] 101, 201, 203, 301: arrows. DETAILED DESCRIPTION

[0029] If the definition or use of a term in a reference is inconsistent or contrary to the definition of that term herein, the definition of that term herein shall apply rather than the definition of that term in the reference. Secondly, unless otherwise defined in the context, singular terms may include pluralities and plural terms may include the singular.

[0030] As mentioned above, the present invention provides a mammalian cell line and a method for using the same to produce a soluble E2 recombinant antigen of classical swine fever virus, which can utilize a mammalian cell expression system to mass produce a soluble E2 recombinant antigen of classical swine fever virus.

[0031] The terms "recombinant protein", "recombinant antigen", "protein", "peptide" and "exogenous polypeptide" as used herein are interchangeable and refer to polymers of amino acids, usually linked together by peptide bonds or disulfide bonds. "Peptide" may also be used for amino acid polymers in which one or more amino acid residues are naturally occurring amino acids and polymers thereof, or analogs or mimetics corresponding to naturally occurring amino acids. "Peptide" further includes modified amino acid polymers, for example, glycoproteins with carbohydrate residues, or phosphorylated peptides. Peptides, exogenous polypeptides and proteins may be produced by liquid phase synthesis, solid phase synthesis, or by genetic engineering, recombinant cells, prokaryotic expression systems, or eukaryotic expression systems. In one embodiment, the recombinant antigen referred to in the present invention is a soluble classical swine fever virus E2 (CSFV-E2) recombinant protein produced by a mammalian cell expression system.

[0032] As used herein, "amino acid" and "residue" are interchangeable and, when used in conjunction with a peptide, refer to naturally occurring and synthetic amino acids, amino acid analogs, amino acid mimetics, and non-naturally occurring amino acids that are chemically similar to the naturally occurring amino acids.

[0033] The strain of the "classic swine fever virus" referred to herein is not particularly limited, and may be, for example, the 2.1a subgroup of the classic swine fever virus. However, in other embodiments, the classic swine fever virus may also use other strains, depending on actual needs.

[0034] The "E2 recombinant protein" referred to herein may be, for example, a full-length E2 recombinant protein, such as the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence encoded by a nucleic acid sequence shown in SEQ ID NO: 2, wherein the amino acid sequence is, from N-terminus to C-terminus, a signal peptide (which facilitates secretion of the translated E2 protein into the culture medium), a sequence-modified E2 protein (HS2 strain, about 342 a.a.), a protease cleavage site (which facilitates removal of the histidine tag), and a histidine tag (His tag, which facilitates protein purification). The amino acid sequence shown in SEQ ID NO: 1 can be based on the gene sequence of the 2.1a gene subtype of the classical swine fever virus (Classical swine fever virus) published by Pan et al. (2005) (GenBank No.: AY526726.1), and the gene fragment of E2 (equivalent to nucleotides 1267-2292; the corresponding E2 amino acid fragment is the 423-764 amino acids of the complete sequence (GenBank No.: AAS20410.1)) is extracted and modified into a sequence suitable for production via a mammalian cell expression system. In some specific examples, in order to facilitate the construction of recombinant plasmids, restriction enzyme sites can be selectively added to both ends of the E2 recombinant gene sequence, wherein the type of restriction enzyme site is not particularly limited, and depends on the plasmid sequence to be constructed, and can be, for example, Bam HI and Not I restriction enzyme sites. In addition, in order to facilitate the subsequent purification of the protein, the C-terminus of the E2 recombinant protein can selectively add a human rhinovirus 3C protease (HRV 3C protease) cleavage site (about 8a.a., which is conducive to the removal of the histidine tag) and a histidine (His) tag [His tag; also known as polyhistidine], wherein the His tag may include but is not limited to 6 to 10 histidine residues. Therefore, the E2 recombinant protein obtained above is a soluble protein.

[0035] In some embodiments, the above-mentioned CSFV-E2 recombinant protein can be manufactured using conventional methods or the following methods. First, a protein expression step is performed on the prokaryotic transformant cell, wherein the prokaryotic transformant cell may include a first recombinant plasmid, which contains a recombinant gene with a nucleic acid sequence as shown in SEQID NO: 2 to express a CSFV-E2 recombinant protein with an amino acid sequence as shown in the sequence identification number (SEQ ID NO): 1. In order to subsequently purify the recombinant protein, a nucleic acid sequence for compiling a His tag may be selectively added to the 3' end of the above-mentioned recombinant gene. In some examples, the aforementioned nucleic acid sequence for compiling a His tag may be provided, for example, by a commercially available plasmid and designed to be connected to the 3' end of the above-mentioned recombinant gene. The nucleic acid sequence of the His tag is common knowledge in the technical field to which the present invention belongs and will not be described in detail here.

[0036] Next, the first recombinant plasmid of the aforementioned prokaryotic transformed cell is extracted, the E2 recombinant gene is cut out with a restriction enzyme, and then constructed into a second recombinant plasmid suitable for a mammalian cell expression system. The second recombinant plasmid is transfected into mammalian cells, and the E2 recombinant protein is expressed in large quantities and secreted into the cell culture medium. Then, the soluble E2 recombinant protein is recovered and purified.

[0037] In the above embodiments, the type of mammalian cells is not particularly limited, and may be, for example, a commercially available Chinese Hamster Ovary cell line (CHO cell). This cell line is a Chinese Hamster Ovary cell line CCC_E2_5E12 deposited with the German Microbiological Culture Collection Center (Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH, DSMZ; address: Inhoffen straβe 7B, 38124 Braunschweig, Germany) on May 17, 2023, with a deposit number of DSMACC3376, and is classified and named Chinese Hamster Ovary Cell.

[0038] In the above embodiment, after the E2 recombinant protein is recovered, the CSFV-E2 recombinant protein may be selectively subjected to a conventional column purification step to obtain a purified CSFV-E2 recombinant protein.

[0039] Generally speaking, cells of different cell generations of the above-mentioned mammalian cells can stably express and significantly increase the yield of soluble E2 recombinant antigen of classical swine fever virus, wherein the production of CSFV-E2 recombinant protein can obtain at least 800 mg (mg) of soluble CSFV-E2 recombinant protein, or at least 850 mg, or 850 mg to 950 mg of soluble CSFV-E2 recombinant protein per liter of cell culture medium. It is worth mentioning that the E2 recombinant protein produced by the mammalian cell expression system can be secreted into the cell culture medium, which can effectively increase the yield of E2 recombinant protein, and its yield is at least 80 to 100 times that of other expression systems (such as the expression system of baculovirus-infected insect cells). Secondly, the E2 recombinant protein produced by the mammalian cell expression system has a higher proportion of soluble protein, and at least 95% of the E2 recombinant protein is soluble protein, so a lower-cost process can be used to recover and purify the E2 protein, thereby significantly reducing the production cost of producing classical swine fever E2 recombinant protein. Furthermore, the proportion of soluble E2 recombinant protein produced by the mammalian cell expression system is relatively high, which can effectively improve the effectiveness of the vaccine produced. In some embodiments, a vaccine made by administering a single dose of soluble E2 recombinant protein can achieve 100% protection. In contrast, about 50% to 80% of the E2 recombinant protein produced by other expression systems (such as the baculovirus-infected insect cell expression system) is insoluble protein, and the yield of soluble protein is low, and it cannot be purified. Vaccines made with unpurified E2 recombinant protein are not only less effective, but also use a higher dose of antigen, and two doses (i.e., booster vaccination) must be administered to achieve 100% protection. In other words, the dosage of E2 recombinant protein produced by other expression systems (such as the baculovirus-infected insect cell expression system) is twice the dosage of soluble E2 recombinant protein produced by the mammalian cell expression system. Moreover, the E2 recombinant protein produced by other expression systems (such as the expression system of baculovirus-infected insect cells) cannot be purified and may contain allergens. After the vaccine prepared in this way is vaccinated in animals, some animals may cause side effects (such as allergic reactions or even death), thereby reducing the safety of the vaccine.

[0040] The above-mentioned soluble CSFV-E2 recombinant protein can form a bivalent or multivalent antigen with other antigen proteins, and only a single dose is required to provide effective immune protection. The "single dose" mentioned here means that the effective number of administrations of the pig subunit vaccine composition to the subject is one, without the need for booster immunity, and without side effects, which can effectively simplify the vaccination process.

[0041] In some embodiments, the effective dose of the CSFV-E2 recombinant protein in the above-mentioned subunit vaccine composition for pigs can be, for example, 2 mL per dose, each dose containing 25 μg / mL to 50 μg / mL. In some specific examples, the effective dose of the CSFV-E2 recombinant protein can be, for example, 25 μg / mL.

[0042] It is understood that the following specific recombinant protein sequences, specific formulations, specific dosages, specific detection methods, viewpoints, illustrations and embodiments are for illustration only and are not intended to be limiting conditions of the present invention. The main features of the present invention may be used in various embodiments without departing from the spirit and scope of the present invention. Therefore, those with common knowledge in the technical field to which the present invention belongs can easily determine the necessary technical features of the present case without departing from the spirit and scope of the present invention, and make various changes and modifications to the present invention to adapt to different uses and conditions.

[0043] Example: Preparation of Classical Swine Fever Virus E2 Recombinant Protein

[0044] 1. Construction of recombinant gene of classical swine fever virus E2

[0045] The amino acid sequence of the recombinant protein of classical swine fever virus E2 is shown in SEQ ID NO: 1. It is based on the gene sequence of the 2.1a gene subtype of classical swine fever virus (Classical swine fever virus) published by Pan et al. (2005) (GenBank No.: AY526726.1), and the gene fragment of E2 (equivalent to nucleotides 1267-2292; the corresponding E2 amino acid fragment is amino acids 423-764 of the complete sequence (GenBank No.: AAS20410.1)) was extracted and synthesized by Mingxin Technology Co., Ltd. and modified into a sequence suitable for expression via a mammalian expression system. Restriction sites with Bam HI and Not I were designed at both ends, a signal peptide (protein-tyrosine phosphatase, PTP; a total of 21 a.a.) was added to the N-terminus to facilitate the secretion of the translated E2 protein into the culture medium, and an HRV 3C protease cleavage site (a total of 8 a.a.) was added to the C-terminus to facilitate the removal of the histidine tag) and a histidine tag (His tag, a total of 8 a.a., which is conducive to protein purification), was cloned into the pcDNA3.4 expression vector and named PTP-E2, and its nucleic acid sequence is shown in SEQ ID NO:2.

[0046] 2. Screening of cell lines that stably express recombinant protein of classical swine fever virus E2

[0047] This example uses a commercially available mammalian cell expression system, such as the commercially available CHO cell expression system (ExpiCHO TMExpression system, product model A29129, Thermo Fisher Scientific), according to the operating manual of Thermo Company (Pub. No MAN0017764, Rev. 3.0), to produce E2 recombinant protein.

[0048] First, the PTP-E2 / pcDNA3.4 plasmid was cut into a linear vector using SspI restriction enzyme (BioLabs, R0132S) and then quenched with ExpiFectamine TM CHO transfection reagent (thermo, A29129) was transfected into ExpiCHO TM -S cells. ExpiCHO after transfection TM -S cells were first cultured in ExpiCHO containing 200 μg / mL G418 sulfate (G418sulfate, Youhe, G8168) TM Expression medium (ExpiCHO TM Expression Medium, Model A2910001, thermo). TM -S cells were 3×10 5 cells / mL, seeded with 30 mL of ExpiCHO TM 4 g / mL of glucose was added to the shake flask (125 mL) of expression medium on days 3, 5, and 7, and the cell culture medium was collected on days 0, 3, 5, 7, 10, 12, and 14 to analyze the expression level of the E2 recombinant protein.

[0049] The recombinant protein obtained in Section 2.1 was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and protein transfer by Western blot. The results are shown in Figure 2. Figure 1A (SDS-PAGE: E2), Figure 1B [Western blotting: E2, detected using anti-E2 antibody (product name: WH303, model: RAE0826, He Jun)] as shown.

[0050] See also Figure 1A and Figure 1B , which respectively show the colloidal images of SDS-PAGE of the transfected CHO cell culture fluid according to one embodiment of the present invention ( Figure 1A ) and immunoblotting ( Figure 1B ) color image.

[0051] Figure 1A The results showed that the E2 recombinant protein was successfully expressed in the supernatant. Figure 1A As indicated by the arrow 101 (E2 monomer, 46 kDa). Figure 1B The results showed that the recombinant protein expressed by the transfected CHO cells was confirmed to be the E2 recombinant protein (its sequence is shown in SEQ ID NO: 1).

[0052] Next, single cells were isolated by limiting dilution cloning, and then amplified to obtain a cell line that stably expressed the recombinant protein of classical swine fever virus E2.

[0053] Specifically, after the cells were serially diluted to a cell concentration of 1000 cells / mL, 0.1 mL of the cell solution was added to 39.9 mL of the expression culture medium, and added to a 96-well plate at a volume of about 200 μL per well, and cultured at 37°C and 8% carbon dioxide concentration. After culture for 17 days, the culture medium containing cells in each well was transferred to a 24-well plate, and 300 μL of the expression culture medium was added to each well, so that each well contained about 0.5 mL of the culture medium, and cultured at 37°C and 8% carbon dioxide concentration. Finally, a cell library of 235 cell clones was obtained. According to the protein production evaluation, the cell strain with the highest recombinant protein expression (i.e., a mammalian cell strain that stably expresses the soluble E2 recombinant antigen of classical swine fever virus) was selected and deposited in the German Culture Collection (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ; address: Inhoffen straβe7B, 38124 Braunschweig, Germany), named Chinese Hamster Ovary cell line (Chinese Hamster Ovary cell) CCC_E2_5E12, deposited on May 17, 2023, deposited with the number DSMACC3376, classified and named Chinese Hamster Ovary cell, and the cell line was confirmed to be viable on June 7, 2023.

[0054] 3. Expression and purification of E2 recombinant protein

[0055] Before transfection, the CHO cell line CCC_E2_5E12 (cell density reached 3×10 5 cells / mL), a total of 30 mL of stable culture medium (ExpiCHO TMStable Production AGT Medium, model A3711101, thermo) was cultured at 37°C and 8% carbon dioxide. On the 3rd, 5th and 7th days of culture, glucose was added to a final concentration of 4 g / L. On the 14th day of culture, the cells were separated by centrifugation at 2000g for 15 minutes and the supernatant (part of the culture medium) was taken out. After that, thiothionine (Sigma, CAS no. A4915) was added to the supernatant for precipitation to obtain a 20-40% precipitate. Then, the above precipitate was re-dissolved with 100 mL of buffer A [containing 50 mM tris (Tris) and 500 mM sodium chloride (NaCl)], and its pH value was pH 7.5. Next, the above-mentioned resolubilized material was filtered through a 0.45 μm filter membrane and then subjected to a column purification step. The culture medium was injected into a Ni-NTA column, and the E2 recombinant protein was washed out using a washing solution (containing 50 mM Tris, 500 mM sodium chloride and 250 mM imidazole) to obtain a high-purity E2 recombinant protein.

[0056] 4. Verification of Recombinant Protein Expression

[0057] The recombinant protein purified in Section 2.1 was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and protein transfer by Western blot. The results are shown in Figure 2. Figure 2A (SDS-PAGE: E2), Figure 2B [Western blot: E2, detected using anti-E2 antibody (product name: WH303, model: RAE0826, He Jun)] as shown.

[0058] The SDS-PAGE film containing the E2 recombinant protein was transferred to a polyvinylidene difluoride (PVDF) membrane (GE healthcare, Advanced), and reacted with 20 mL of blocking buffer [Tris Buffered Saline with Tween 20 (TBST) and 5% bovine serum albumin (BSA)] at room temperature (about 25°C) for 2 hours. Then, the blocking buffer was removed, and the PVDF membrane was reacted with 10 mL of anti-His tag antibody solution [antibody diluted 1:1000 and TBST buffer solution (1x PBS with 0.1% Tween-20)] at 4°C for 16 to 18 hours. Thereafter, the anti-His tag antibody solution was removed, and the PVDF membrane was rinsed three times with 10 mL of TBST buffer solution, each rinse for 20 minutes. Then, the PVDF membrane was reacted with anti-mouse antibody (anti-mouse antibody, dilution 1:10000) in TBST buffer solution at room temperature for 1 hour. After that, the TBST buffer solution containing the antibody was removed, and 10 mL of TBST buffer solution was added to wash the PVDF membrane three times, each time for 20 minutes. Then, the PVDF membrane was colored using a coloring agent (ECL stain kit, advanced), and the results are shown in Figure 2. Figure 2B shown.

[0059] See also FIG. 2A to FIG. 2B , which shows the SDS-PAGE colloidal image of the CSFV-E2 recombinant protein expressed by the CHO cell line CCC_E2_5E12 according to one embodiment of the present invention ( Figure 2A ) and immunoblotting ( Figure 2B ) color image. Figure 2A Lane 1 from left to right is a protein marker; Lane 2 is the purified CSFV2 E2 recombinant protein (E2 dimer; -β-ME) without β-mercaptoethanol (β-ME) treatment; Lane 3 is the purified CSFV2 E2 recombinant protein treated with β-ME, with a molecular weight of 46 kDa.

[0060] Figure 2A The results showed that the supernatant of CHO cell line CCC_E2_5E12 could indeed purify CSFV-E2 recombinant protein. Figure 2AIndicated by arrow 201 (E2 monomer, molecular weight 46 kDa; treated with β-mercaptoethanol, +β-ME) and arrow 203 (E2 dimer, molecular weight 92 kDa; not treated with β-ME, -β-ME). Figure 2B The results showed that the protein purified from the supernatant of CHO cell line CCC_E2_5E12 was confirmed to be CSFV E2 recombinant protein (its sequence is as follows

[0061] (shown in SEQ ID NO: 1).

[0062] In order to confirm whether the expression level of CSFV E2 recombinant protein in CHO cell line CCC_E2_5E12 of different cell generations is stable, the culture medium of the second, fourth, sixth, eighth and tenth generations of CHO cell line CCC_E2_5E12 was taken for SDS-PAGE gel electrophoresis and protein transfer immunoblotting analysis, and the results are shown as follows: Figure 3A and Figure 3B shown.

[0063] See also FIG. 3A to FIG. 3B , which respectively show the SDS-PAGE colloidal images of the CHO cell line CCC_E2_5E12 of different cell generations expressing E2 recombinant protein in the cell fluid before purification according to one embodiment of the present invention ( Figure 3A ) and immunoblotting ( Figure 3B ) color image.

[0064] Figure 3A and Figure 3B The results showed that the E2 recombinant protein could be stably expressed in the supernatant of the CHO cell line CCC_E2_5E12 at different cell generations before purification. Figure 3A and Figure 3B The color band signal pointed by arrow 301 is similar.

[0065] See also FIG. 4A to FIG. 4B , which respectively show the colloidal images of SDS-PAGE of the E2 recombinant protein expressed in the cell fluid of the CHO cell line CCC_E2_5E12 of different cell generations according to one embodiment of the present invention after purification ( Figure 4A ) and immunoblotting ( Figure 4B ) color image.

[0066] Figure 4A and Figure 4B The results showed that after the supernatant of CHO cell line CCC_E2_5E12 of different cell generations was purified, the E2 recombinant protein could indeed be stably expressed in the supernatant. Figure 4A and Figure 4BThe color band signal is close.

[0067] In addition, the commercially available electrophoresis colloid density determination method was used to detect the concentration of E2 recombinant protein purified from CHO cell line CCC_E2_5E12 of different cell generations. The results are as follows: Figure 5 shown.

[0068] See also Figure 5 , which shows a bar graph of the protein concentration of the purified E2 recombinant protein in the cell fluid of the CHO cell line CCC_E2_5E12 of different cell generations according to one embodiment of the present invention. Figure 5 The results showed that after purification of the supernatants of the CHO cell line CCC_E2_5E12 of different cell generations, there was no statistically significant difference in the expression levels of E2 recombinant protein in the CHO cell lines of different cell generations (T-test statistical analysis, P>0.05), among which the average protein concentration of E2 recombinant protein was at least 800 mg / L cell culture medium, preferably at least 850 mg / L cell culture medium, and more preferably 850 mg / L cell culture medium to 950 mg / L cell culture medium.

[0069] The above examples demonstrate that mammalian cell expression systems of different cell generations can stably express and significantly increase the yield of soluble E2 recombinant antigen of classical swine fever virus, effectively reduce the production cost of classical swine fever E2 protein, and further be applied to the production of classical swine fever E2 protein subunit vaccine.

[0070] In summary, the present invention uses specific amino acid sequences, specific cell lines, specific production methods or specific evaluation methods only to illustrate mammalian cell lines and methods for producing soluble E2 recombinant antigens of classical swine fever virus. However, those with common knowledge in the technical field to which the present invention belongs should understand that other amino acid sequences, other production methods or other evaluation methods can also be used for mammalian cell lines and methods for producing soluble E2 recombinant antigens of classical swine fever virus without departing from the spirit and scope of the present invention, and are not limited to the above.

[0071] According to the above embodiments, the mammalian cell line of the present invention and the method for using the same to produce soluble E2 recombinant antigen of classical swine fever virus have the advantage that the production of soluble E2 recombinant antigen of classical swine fever virus can be greatly improved by using mammalian cell expression systems of different cell generations, thereby effectively reducing the production cost of soluble E2 recombinant antigen of classical swine fever virus, and further being applied to the production of subunit vaccine compositions for pigs.

[0072] Although the present invention has been disclosed as above with several specific embodiments, various modifications, changes and substitutions may be made to the aforementioned disclosure, and it should be understood that in some cases, certain features of the embodiments of the present invention will be adopted but other features will not be used accordingly without departing from the spirit and scope of the present invention. Therefore, the spirit and scope of the claims of the present invention should not be limited to the above exemplary embodiments.

[0073]

Biological material storage

[0074] Domestic deposit information (please note the order of deposit institution, date and number)

[0075] none

[0076] Overseas deposit information (please note the order of deposit area, institution, date and number)

[0077] The Chinese Hamster Ovary cell line (Chinese Hamster Ovary cell) CCC_E2_5E12 was deposited with the German Type Culture Collection (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ; address: Inhoffen straβe 7B, 38124 Braunschweig, Germany) on May 17, 2023. The deposit number is DSMACC3376. It is classified and named Chinese Hamster Ovary cell, and the cell line was confirmed to be viable on June 7, 2023.

Claims

1. A mammalian cell line stably expressing a soluble E2 recombinant antigen of classical swine fever virus, characterized in that: The mammalian cell line that stably expresses the soluble E2 recombinant antigen of classical swine fever virus is the Chinese Hamster Ovary cell line CCC_E2_5E12, which was deposited with the German Culture Collection (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ) on May 17, 2023 and has the deposit number DSMACC3376.

2. The mammalian cell line stably expressing soluble E2 recombinant antigen of classical swine fever virus according to claim 1, characterized in that: The amino acid sequence of the soluble E2 recombinant protein is shown in SEQ ID NO:

1.

3. The mammalian cell line stably expressing soluble E2 recombinant antigen of classical swine fever virus according to claim 1, characterized in that: The protein concentration of the soluble E2 recombinant protein is at least 800 mg / L cell culture fluid.

4. The mammalian cell line stably expressing soluble E2 recombinant antigen of classical swine fever virus according to claim 1, characterized in that: The protein concentration of the soluble E2 recombinant protein is at least 850 mg / L cell culture fluid.

5. The mammalian cell line stably expressing soluble E2 recombinant antigen of classical swine fever virus according to claim 1, characterized in that: The protein concentration of the soluble E2 recombinant protein is 850 mg / L cell culture fluid to 950 mg / L cell culture fluid.

6. The mammalian cell line stably expressing soluble E2 recombinant antigen of classical swine fever virus according to claim 1, characterized in that: The E2 recombinant protein is encoded by a nucleic acid sequence as shown in SEQ ID NO:

2.

7. A method for producing a soluble E2 recombinant protein of classical swine fever virus, characterized in that: The method for producing the soluble E2 recombinant protein of classical swine fever virus comprises culturing the mammalian cell line as claimed in claim 1 to produce the soluble E2 recombinant antigen.

8. The method for producing soluble E2 recombinant protein of classical swine fever virus according to claim 7, characterized in that: The protein concentration of the soluble E2 recombinant protein is at least 800 mg / L cell culture fluid.

9. A soluble E2 recombinant antigen of classical swine fever virus, characterized in that: The soluble E2 recombinant antigen of classical swine fever virus is produced by the mammalian cell line according to claim 1, wherein an amino acid sequence of the soluble E2 recombinant protein is as shown in SEQ ID NO:

1.

10. The soluble E2 recombinant antigen of classical swine fever virus according to claim 9, characterized in that: The E2 recombinant protein is encoded by a nucleic acid sequence as shown in SEQ ID NO:

2.

11. A subunit vaccine composition for pigs, characterized in that: The subunit vaccine composition for pigs uses the soluble E2 recombinant antigen of classical swine fever virus as claimed in claim 9 as an active ingredient, and an effective number of administrations of the subunit vaccine composition for pigs to a subject is once.

12. The subunit vaccine composition for pigs according to claim 11, characterized in that: An effective dosage of the soluble E2 recombinant protein of classical swine fever virus is 25 μg / mL to 50 μg / mL.

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