Purification method of porcine epidemic diarrhea virus s protein, product and application thereof

Abstract

The application provides a porcine epidemic diarrhea virus S protein purification method, a product and an application thereof. The purification method comprises the following steps: sequentially subjecting a virus protein sample to deep filtration, liquid replacement and composite chromatography, and finally collecting a flow-through liquid to obtain purified porcine epidemic diarrhea virus S protein; and the filler of the composite chromatography comprises any one of Capto Core 400, Seplife Suncore 400 or VDX Core A. The purification method maximizes the purification efficiency and yield of the porcine epidemic diarrhea virus S protein, has a simple process flow, reduces the purification cost, and has an outstanding large-scale application prospect.

Description

Technical Field

[0001] This invention belongs to the field of protein purification technology, specifically relating to a method for purifying porcine epidemic diarrhea virus S protein, its products, and applications. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes severe watery diarrhea, vomiting, dehydration, and high mortality in piglets. It belongs to the genus Alphacoronavirus in the family Coronaviridae. The spike protein (S protein) of PEDV is the core functional protein for viral invasion of host cells, and it is also the main target of neutralizing antibodies and a key antigen in vaccine design. The molecular weight of the S protein monomer is 170-200 kDa, and it can be expressed and secreted extracellularly as a trimer in CHO cell expression systems.

[0003] CN108822191A discloses a porcine epidemic diarrhea virus S protein. The S protein with a complete structure and modified sugar was obtained through the CHO expression system. The protein was purified by nickel column purification, which showed excellent immunogenicity. However, it was found that in the process of large-scale production, the nickel column purification process was cumbersome, time-consuming, costly, and inefficient, which affected the quality of the protein and large-scale industrial production.

[0004] Therefore, developing an efficient method for purifying the S protein of porcine epidemic diarrhea virus is of great significance. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a purification method for porcine epidemic diarrhea virus (PEDV) S protein, along with its products and applications. The purification method of the present invention maximizes the purification efficiency and yield of PEDV S protein, has a simple process flow, reduces purification costs, and has outstanding prospects for large-scale application.

[0006] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for purifying the S protein of porcine epidemic diarrhea virus, the purification method comprising: The viral protein sample was subjected to deep filtration, liquid exchange and complex chromatography in sequence, and finally the flow-through was collected to obtain purified porcine epidemic diarrhea virus S protein. The packing material for the composite chromatography includes any one of Capto Core 400, Seplife Suncore 400, or VDX ​​Core A.

[0007] The purification method of this invention maximizes the purification efficiency and yield of porcine epidemic diarrhea virus S protein. The process is simple, reduces purification costs, and has outstanding prospects for large-scale application.

[0008] When a specific composite chromatography packing is used for purification, target substances in the sample with a molecular weight greater than the cutoff (greater than 20 nm) cannot enter the packing and appear at the flow-through position; impurities with a molecular weight smaller than the cutoff (such as host cell proteins, host cell DNA, endotoxins, etc.) can enter the packing and be fixed by the ligand adsorption column inside the packing. They are then washed off during the CIP stage of chromatography, thereby achieving the separation of the target substance and impurities.

[0009] Preferably, the viral protein sample is a cell culture medium containing porcine epidemic diarrhea virus S protein.

[0010] Preferably, the pore size of the deep filter is 0.1~30 μm, for example, it can be 0.1 μm, 0.2 μm, 0.45 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, etc.

[0011] Deep filtration can trap cell debris, colloidal particles, lipid droplets, flocculants, etc. in the sample, resulting in a clear and transparent liquid free of suspended matter, which is beneficial for subsequent purification.

[0012] Preferably, the fluid replacement includes replacing the liquid medium of the viral protein sample with a phosphate buffer containing sodium chloride.

[0013] Buffer replacement can remove excess inorganic salts from the original sample solution, standardize ionic strength, replace the solution medium with the equilibrium buffer for downstream composite chromatography, eliminate process fluctuations caused by differences in the buffer system, stabilize the system pH and osmotic pressure, and maintain the native conformation and antigenic activity of PEDVS protein.

[0014] Preferably, the concentration of sodium chloride is 0.1~0.2 M, for example, it can be 0.1 M, 0.11 M, 0.12 M, 0.13 M, 0.14 M, 0.15 M, 0.16 M, 0.17 M, 0.18 M, 0.19 M or 0.2 M, etc.

[0015] Preferably, the concentration of the phosphate buffer solution is 18~22 mM, for example, it can be 18 mM, 18.5 mM, 19 mM, 19.5 mM, 20 mM, 20.5 mM, 21 mM, 21.5 mM or 22 mM, etc.

[0016] Preferably, the pH of the phosphate buffer solution is 7.3 to 7.8, for example, it can be 7.3, 7.35, 7.4, 7.45, 7.5, 7.55, 7.6, 7.65, 7.7, 7.75 or 7.8.

[0017] Preferably, the liquid exchange method includes gel filtration chromatography and / or hollow fiber ultrafiltration.

[0018] Preferably, the liquid exchange method is gel filtration chromatography.

[0019] Gel filtration chromatography is preferred for liquid exchange, resulting in clearer samples, a more stable purification system, and higher yield and purity after purification.

[0020] Preferably, the packing material for the gel filtration chromatography is Bestdex G25.

[0021] Preferably, the equilibrium flow rate in the gel filtration chromatography is 60~90 cm / h, for example, it can be 60 cm / h, 65 cm / h, 70 cm / h, 75 cm / h, 80 cm / h, 85 cm / h or 90 cm / h, etc.

[0022] Preferably, the sample loading flow rate in the gel filtration chromatography is 60~90 cm / h, for example, it can be 60 cm / h, 65 cm / h, 70 cm / h, 75 cm / h, 80 cm / h, 85 cm / h or 90 cm / h, etc.

[0023] Preferably, the sample loading volume in the gel filtration chromatography does not exceed 1 / 4 of the column volume, for example, it can be 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9 or 1 / 10, etc.

[0024] Preferably, the packing material for the composite chromatography is Capto Core 400.

[0025] Capto Core 400 is the preferred packing material for composite chromatography, as it offers better purification results.

[0026] Preferably, the equilibrium flow rate in the composite chromatography is 90~150 cm / h, for example, it can be 90 cm / h, 95 cm / h, 100 cm / h, 105 cm / h, 110 cm / h, 115 cm / h, 120 cm / h, 125 cm / h, 130 cm / h, 135 cm / h, 140 cm / h, 145 cm / h or 150 cm / h, etc.

[0027] Preferably, the flow rate for loading the sample in the composite chromatography is 60~90 cm / h, for example, it can be 60 cm / h, 65 cm / h, 70 cm / h, 75 cm / h, 80 cm / h, 85 cm / h or 90 cm / h, etc.

[0028] Preferably, the sample loading volume in the composite chromatography does not exceed 10 times the column volume, for example, it can be 10 times, 9 times, 8 times, 7 times, 6 times, 5 times, 4 times, 3 times, 2 times or 1 times, etc.

[0029] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0030] Secondly, the present invention provides porcine epidemic diarrhea virus S protein obtained by the purification method described in the first aspect.

[0031] Thirdly, the present invention provides the purification method described in the first aspect or the use of the porcine epidemic diarrhea virus S protein as described in the second aspect in the preparation of porcine epidemic diarrhea virus vaccines.

[0032] The vaccine prepared using the porcine epidemic diarrhea virus S protein purified in this invention has advantages over existing porcine epidemic diarrhea vaccines, including high safety, fewer side effects in pigs, good uniformity, and good immunization effect.

[0033] Compared with the prior art, the present invention has the following beneficial effects: (1) The composite packing material was used to purify porcine epidemic diarrhea virus S protein. The single processing volume was large and the purification effect was good. Compared with affinity nickel column chromatography, molecular sieve chromatography and other methods, the purification efficiency and protein yield were greatly improved.

[0034] (2) Composite packing belongs to core-shell type composite packing, which combines the high loading capacity of large pore medium with the high resolution advantage of small particle packing. It is an important supplement and upgrade of traditional agarose-based packing. It has the advantages of less damage to porcine epidemic diarrhea virus S protein, good removal of impurities, high protein recovery rate and large single processing capacity, making it very suitable for large-scale application.

[0035] (3) The purified porcine epidemic diarrhea virus S protein has the advantages of high safety, controllable quality, good batch-to-batch consistency, relatively good stability and flexible adjuvant compatibility, and can finely regulate the type of immune response. Attached Figure Description

[0036] Figure 1 This is an SDS-PAGE image of the purified sample from Example 1. Detailed Implementation

[0037] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0038] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0039] Referring to CN108822191B, cell culture medium containing porcine epidemic diarrhea virus S protein was obtained using the CHO cell expression system. The culture medium was centrifuged at 8000 g for 30 min at 4°C, and the supernatant was collected as the virus protein sample for purification in the following specific embodiments.

[0040] The filter selected is the COBB MDS840PC-TMP, with a filtration accuracy of 0.1~30 μm.

[0041] Bestdex G25 was purchased from Bogelon, Capto Core 400 and Capto Core 700 were purchased from Sitopan, Seplife Suncore 400 and Q Large Scale HP were purchased from Lanxiao Technology, and VDX Core A was purchased from Weidu Bio.

[0042] Example 1 This embodiment provides a method for purifying the S protein of porcine epidemic diarrhea virus, the purification method comprising: (1) Deep filtration: Pass the viral protein sample through a filter and collect the filtrate 1.

[0043] (2) Buffer exchange: Add Bestdex G25 to the chromatography column and equilibrate with equilibration buffer (20mM PBS + 0.15M NaCl, pH 7.5) at a flow rate of 75 cm / h. Then, set the sample loading volume to 1 / 4 column volume, and slowly pass the filtrate 1 through the chromatography column at a flow rate of 70 cm / h. Collect the flow-through liquid 1, which is the protein solution after solution replacement. Collect from the beginning of the UV absorption peak rise until the UV absorption peak declines and then stop collecting. Wash the chromatography column with 4 column volumes of 0.5M NaOH, then rinse with 4 column volumes of pure water until the conductivity is leveled off, and finally store with 0.01M NaOH.

[0044] (3) Composite chromatography: Capto Core 400 composite packing material was added to the chromatography column and equilibrated with equilibration buffer (20mM PBS + 0.15M NaCl, pH 7.5) at a flow rate of 120 cm / h. Then, the sample loading volume was set to 10 column volumes, and the flow-through buffer 1 was slowly passed through the chromatography column at a flow rate of 80 cm / h. The flow-through buffer 2 was collected, which is the purified porcine epidemic diarrhea virus S protein. Collection was stopped when the UV absorption peak started to rise and when the UV absorption peak dropped to a plateau. The chromatography column was washed with washing buffer (20mM PBS + 1M NaCl, pH 7.5) to remove non-specifically bound proteins, followed by rinsing with 2 column volumes of pure water, then 4 column volumes of 1M NaOH + 30 vol% isopropanol, and then rinsing with 4 column volumes of pure water until the conductivity leveled off. Finally, it was stored with 0.01M NaOH.

[0045] Filtrate 1, flow-through 1, and flow-through 2 were subjected to SDS-PAGE electrophoresis analysis. The results are as follows: Figure 1 As shown.

[0046] Example 2 This embodiment provides a method for purifying the S protein of porcine epidemic diarrhea virus, the purification method comprising: (1) Deep filtration: Pass the viral protein sample through a filter and collect the filtrate 1.

[0047] (2) Buffer exchange: Add Bestdex G25 to the chromatography column and equilibrate with equilibration buffer (22mM PBS + 0.1M NaCl, pH 7.8) at a flow rate of 60 cm / h. Then, set the sample loading volume to 1 / 5 column volume, and slowly pass the filtrate 1 through the chromatography column at a flow rate of 60 cm / h. Collect the flow-through liquid 1, which is the protein solution after solution replacement. Collect from the beginning of the UV absorption peak rise until the UV absorption peak declines and then stop collecting. Wash the chromatography column with 5 column volumes of 0.5M NaOH, then rinse with 5 column volumes of pure water until the conductivity is leveled off, and finally store with 0.01M NaOH.

[0048] (3) Composite chromatography: Capto Core 400 composite packing material was added to the chromatography column and equilibrated with equilibration buffer (22 mM PBS + 0.1 M NaCl, pH 7.8) at a flow rate of 90 cm / h. Then, the sample loading volume was set to 9 column volumes. Flow-through buffer 1 was slowly passed through the chromatography column at a flow rate of 60 cm / h, and flow-through buffer 2 was collected, which is the purified porcine epidemic diarrhea virus S protein. Collection was stopped when the UV absorption peak started to rise and when the UV absorption peak declined and leveled off. The chromatography column was washed with washing buffer (20 mM PBS + 1 M NaCl, pH 7.5) to remove non-specifically bound proteins, followed by rinsing with 2 column volumes of pure water, then 3 column volumes of 1 M NaOH + 30 vol% isopropanol, and then rinsing with 3 column volumes of pure water until the conductivity leveled off. Finally, it was stored with 0.01 M NaOH.

[0049] Example 3 This embodiment provides a method for purifying the S protein of porcine epidemic diarrhea virus, the purification method comprising: (1) Deep filtration: Pass the viral protein sample through a filter and collect the filtrate 1.

[0050] (2) Buffer exchange: Add Bestdex G25 to the chromatography column and equilibrate with equilibration buffer (18mM PBS + 0.2M NaCl, pH 7.3) at a flow rate of 90 cm / h. Then, set the sample loading volume to 1 / 6 column volume, and slowly pass the filtrate 1 through the chromatography column at a flow rate of 90 cm / h. Collect the flow-through liquid 1, which is the protein solution after solution replacement. Collect from the beginning of the UV absorption peak rise until the UV absorption peak declines and then stop collecting. Wash the chromatography column with 3 column volumes of 0.5M NaOH, then rinse with 3 column volumes of pure water until the conductivity is leveled off, and finally store with 0.01M NaOH.

[0051] (3) Composite chromatography: Capto Core 400 composite packing material was added to the chromatography column and equilibrated with equilibration buffer (18mMPBS + 0.2M NaCl, pH 7.3) at a flow rate of 150 cm / h. Then, the sample loading volume was set to 8 column volumes. Flow-through buffer 1 was slowly passed through the chromatography column at a flow rate of 90 cm / h, and flow-through buffer 2 was collected, which is the purified porcine epidemic diarrhea virus S protein. Collection was stopped when the UV absorption peak started to rise and when the UV absorption peak declined and leveled off. The chromatography column was washed with washing buffer (20mMPBS + 1M NaCl, pH 7.5) to remove non-specifically bound proteins, followed by rinsing with 3 column volumes of pure water, then 5 column volumes of 1M NaOH + 30 vol% isopropanol, and then rinsing with 5 column volumes of pure water until the conductivity leveled off. Finally, it was stored with 0.01M NaOH.

[0052] Example 4 This embodiment provides a method for purifying the S protein of porcine epidemic diarrhea virus. The only difference between this method and Example 1 is that the composite packing material Capto Core 400 is replaced with the composite packing material Seplife Suncore 400, while all other steps remain unchanged.

[0053] Example 5 This embodiment provides a method for purifying the S protein of porcine epidemic diarrhea virus. The only difference between this method and Example 1 is that the composite packing material Capto Core 400 is replaced with the composite packing material VDX Core A, while all other steps remain unchanged.

[0054] Example 6 This embodiment provides a method for purifying the S protein of porcine epidemic diarrhea virus. The only difference between this method and that of Example 1 is that the liquid exchange method in step (2) is different, and hollow fiber ultrafiltration is used for liquid exchange.

[0055] Set the shear rate to 6000 s. -1The transmembrane pressure range was 13 psi. Filtrate 1 was replaced with a 30 kDa hollow fiber ultrafiltration column to replace the liquid medium with equilibration buffer (20 mM PBS + 0.15 M NaCl, pH 7.5), and flow-through solution 1 was collected.

[0056] Comparative Example 1 This comparative example provides a method for purifying the S protein of porcine epidemic diarrhea virus. The only difference between this method and Example 1 is that the composite packing material Capto Core 400 is replaced with the composite packing material Capto Core 700, while all other steps remain unchanged.

[0057] Comparative Example 2 This comparative example provides a method for purifying the S protein of porcine epidemic diarrhea virus. The only difference between this method and Example 1 is that the chromatography method in step (3) is different. Anion exchange chromatography is performed, and the composite packing material Capto Core 400 is replaced with the anion exchange packing material Q Large Scale HP. All other steps remain unchanged.

[0058] Test case SDS-PAGE electrophoresis analysis was performed on the flow-through solution 2 from Examples 1-6 and Comparative Examples 1-2. The yield and purity of the target protein are shown in Table 1.

[0059] As shown in Examples 1-5 and Comparative Examples 1-2, the present invention uses specific composite chromatography packing materials for purification, which achieves superior yield and purity compared to other chromatography methods and packing materials. As shown in Example 6, the liquid exchange method affects the stability of the purification system, thereby affecting the yield and purity of the target protein.

[0060] Table 1 This invention illustrates a method for purifying porcine epidemic diarrhea virus S protein, its products, and applications through the above embodiments. However, this invention is not limited to the above embodiments, meaning that it does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

[0061] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0062] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for purifying the S protein of porcine epidemic diarrhea virus, characterized in that, The purification method includes: The viral protein sample was subjected to deep filtration, liquid exchange and complex chromatography in sequence, and finally the flow-through was collected to obtain purified porcine epidemic diarrhea virus S protein. The packing material for the composite chromatography includes any one of Capto Core 400, Seplife Suncore 400, or VDX ​​Core A.

2. The purification method according to claim 1, characterized in that, The viral protein sample was a cell culture medium containing the S protein of porcine epidemic diarrhea virus; Preferably, the pore size of the deep filter is 0.1~30 μm.

3. The purification method according to claim 1 or 2, characterized in that, The fluid replacement involves replacing the liquid medium of the viral protein sample with a phosphate buffer containing sodium chloride. Preferably, the concentration of the sodium chloride is 0.1~0.2 M; Preferably, the concentration of the phosphate buffer solution is 18-22 mM; Preferably, the pH of the phosphate buffer solution is 7.3 to 7.

8.

4. The purification method according to any one of claims 1 to 3, characterized in that, The liquid exchange methods include gel filtration chromatography and / or hollow fiber ultrafiltration.

5. The purification method according to any one of claims 1 to 4, characterized in that, The liquid replacement method is gel filtration chromatography; Preferably, the packing material for the gel filtration chromatography is Bestdex G25; Preferably, the equilibrium flow rate in the gel filtration chromatography is 60~90 cm / h; Preferably, the sample loading flow rate in the gel filtration chromatography is 60~90 cm / h; Preferably, the sample loading volume in the gel filtration chromatography does not exceed 1 / 4 of the column volume.

6. The purification method according to any one of claims 1 to 5, characterized in that, The packing material for the composite chromatography is Capto Core 400.

7. The purification method according to any one of claims 1 to 6, characterized in that, The equilibrium flow rate in the composite chromatography is 90~150 cm / h; Preferably, the sample loading flow rate in the composite chromatography is 60~90 cm / h.

8. The purification method according to any one of claims 1 to 7, characterized in that, In the composite chromatography, the sample loading amount does not exceed 10 times the column volume.

9. The porcine epidemic diarrhea virus S protein obtained by the purification method according to any one of claims 1 to 8.

10. The purification method according to any one of claims 1 to 8 or the use of the porcine epidemic diarrhea virus S protein according to claim 9 in the preparation of porcine epidemic diarrhea virus vaccine.

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