Purification method of S subunit protein of porcine epidemic diarrhea virus

Abstract

The invention provides a method for purifying S subunit protein of porcine epidemic diarrhea virus, which is characterized by comprising the following steps of: 1) replacing a cultured supernatant with a buffer solution A to remove a culture medium and obtain a crude pure solution; and (2) carrying out anion exchange chromatography on the obtained crude pure liquid, and collecting flow-through liquid. According to the purification method, the anion exchange chromatography is combined after liquid exchange, so that the purity of the protein is improved, DNA residues are reduced, and meanwhile, the stability of the protein is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary biological products and relates to a method for purifying the S subunit protein of porcine epidemic diarrhea virus. Background Art

[0002] Porcine Epidemic Diarrhea (PED) is an intestinal infectious disease caused by Porcine Epidemic Diarrhea Virus (PEDV) with vomiting, diarrhea and dehydration as the main symptoms. The disease is susceptible to pigs of all ages, especially nursing piglets under 7 days old, with a mortality rate of up to 50%-90% after infection. In recent years, the incidence and mortality of the disease in my country have been on the rise, causing significant economic losses to the pig farming industry.

[0003] PEDV belongs to the Coronavirus genus of the Coronavirus family, group 1, and has a typical coronavirus morphology. PEDV particles are polymorphic, tend to be spherical, and are wrapped with a capsule on the periphery. The capsule is also covered with radial fibrils, with an average diameter of about 130nm. The S protein is a 20nm club-shaped glycoprotein that extends out of the viral particle envelope, with a molecular weight of about 180-220kDa and is composed of about 1383 amino acids. The protein is rich in cysteine ​​and contains 29 potential N-glycosylation sites, but the N-glycosylation sites cannot be cleaved by proteases after the virion matures, greatly reducing the cell fusion and infectivity of the virus, which is one of the important reasons for the difficulty in artificial cell culture of PEDV. Based on the similarity of the conserved sequences of PEDV and other coronavirus S proteins, the PEDV S protein is divided into two domains, S1 (1-789aa) and S2 (790-1383aa), of which S1 is located on the surface of the virus and its main function is to recognize receptors and bind to host cell receptors, and mediate the production of neutralizing antibodies. S2 is mainly responsible for the fusion of the viral envelope and the host cell membrane, and for the introduction of viral RNA into the host cell, thereby causing cell infection. In addition, the PEDV S protein is also an immunogenic protein that induces the host's humoral immune response. Therefore, the PEDV S protein is currently a candidate protein for the development of genetically engineered subunit vaccines.

[0004] CN201810310540.8 discloses a porcine epidemic diarrhea virus S protein. A sugar-modified S protein with a complete structure is obtained through a CHO expression system. The protein is purified by nickel column purification, and has very good immunogenicity. However, it was found that in the large-scale production process, the nickel column purification of the protein is cumbersome, time-consuming, costly, and not efficient enough, which affects the quality of the protein and large-scale industrial production. Summary of the invention

[0005] In order to solve the above technical problems of the prior art, the present invention provides a method for purifying the S subunit protein of porcine epidemic diarrhea virus, and the purification method comprises the following steps:

[0006] 1) replacing the supernatant after cell culture with buffer A to remove the culture medium and obtain a crude pure solution; wherein the buffer A is a phosphate buffer, Tris buffer, Hepes buffer or PBS containing a neutral salt; and

[0007] 2) The crude pure solution obtained in step 1) is subjected to anion exchange chromatography, and the flow-through liquid is collected.

[0008] Here, the crude pure solution is a buffer A solution of S protein containing impurities. The neutral salt is preferably NaCl. Alkali metal or alkaline earth metal soluble neutral salts are also acceptable.

[0009] In a preferred technical solution of the present invention, the S subunit protein is a rod-shaped glycoprotein on the envelope of porcine epidemic diarrhea virus expressed by a CHO expression system.

[0010] In a preferred technical solution of the present invention, the S subunit protein is a rod-shaped glycoprotein on the envelope of porcine epidemic diarrhea virus expressed by mammalian cells such as HEK293, BHK, VERO, SP2 / 0 and insect baculovirus expression system.

[0011] In the preferred technical scheme of the present invention, the amino acid sequence of the S subunit protein is preferably as shown in SEQ ID NO.1; the porcine epidemic diarrhea virus S subunit protein can be a soluble S subunit protein when expressed in mammalian cells and insect baculovirus expression systems, and its amino acid sequence can be different from SEQ ID NO.1. The cell culture fluid obtained by culturing it is removed from the cell debris to obtain the cell supernatant, which is also the cultured supernatant described in the present invention.

[0012] In a preferred technical solution of the present invention, in step 1), the liquid replacement is performed using a hollow fiber ultrafiltration column, a membrane package, a dialysis or a desalting column.

[0013] In a preferred technical solution of the present invention, the molecular weight cutoff of the hollow fiber ultrafiltration column or membrane package is 30 kDa.

[0014] In a preferred technical solution of the present invention, the hollow fiber ultrafiltration column or membrane is coated with liquid at least seven times.

[0015] In the preferred technical solution of the present invention, in step 1), the liquid is concentrated before changing, and the concentration multiple is 2 to 5 times. That is, the concentrated cell supernatant is 2 to 5 times more concentrated than the fermented supernatant.

[0016] In the preferred technical solution of the present invention, in step 2), the filler of the filler column of the anion exchange chromatography is a strong anion exchange filler or a weak anion exchange filler; wherein the strong anion exchange filler is a filler with -CH2-N + -(CH3)3 ligand filler, the weak anion exchange filler is a filler with -CH2-CH2-N + -(CH2-CH3)2 or -CH2-CHOH-CH2-N + -(CH2-CH3)2 ligand filler.

[0017] In a preferred technical scheme of the present invention, the strong anion exchange filler is Mini Q, Mono Q, SOURCE15Q, RESOURCE Q, SOURCE 30Q, Q Sepharose High Performance, Q Sepharose Fast Flow, Q Sepharose XL, Q Sepharose Big Beads, Capto Q, Capto Q ImpRes, or Qlarge scaleHP; the weak anion exchange filler is DEAE Sepharose Fast Flow, ANX Sepharose 4Fast Flow, or Capto DEAE.

[0018] In a preferred technical solution of the present invention, the strong anion exchange filler is Qlarge scale HP or RESOURCE Q.

[0019] In a preferred technical solution of the present invention, the NaCl concentration in the buffer A is 300 mM.

[0020] In a preferred technical solution of the present invention, in steps 1) and 2), the buffer A is a PBS solution containing a NaCl solution, with a pH of 7.4.

[0021] In a preferred technical solution of the present invention, in step 2), the anion exchange chromatography includes column equilibration, sample loading, washing, and column washing.

[0022] In the preferred technical solution of the present invention, more preferably, in step 2), the column equilibration comprises: taking anion exchange filler and loading it into an empty chromatography column; equilibration with cooled water for injection, discharging concentrated ethanol storage solution, and then equilibration with buffer A, and then equilibration with buffer B, and finally equilibration with buffer A. Here, the difference between buffer B and buffer A is only that the concentration of neutral salt in buffer A is 300mM, and the concentration of neutral salt in buffer B is not less than 1000mM.

[0023] In the preferred technical solution of the present invention, more preferably, in step 2), the loading comprises: purifying the crude pure liquid after the liquid replacement treatment in step 1) through an anion exchange filler, and collecting the flow-through liquid. Here, the crude pure liquid is an S protein solution in buffer A containing impurities.

[0024] In the preferred technical scheme of the present invention, more preferably, in step 2), the washing comprises: washing the column with buffer A until no blue color appears in the Coomassie Brilliant Blue G250 test, collecting the washing liquid, and combining it with the flow-through liquid as the purified protein liquid.

[0025] In the preferred technical scheme of the present invention, more preferably, in step 2), the column cleaning comprises: first washing with buffer B, then washing with water, then washing with 0.5M sodium hydroxide, and finally washing with water until the pH is detected to be neutral, and storing with 20% ethanol.

[0026] Among them, the flushing step is only for improving the yield of protein, and the column cleaning is for the reuse of fillers and reducing costs.

[0027] Compared with the prior art, the purification method of the present invention improves the efficiency of large-scale purification by combining anion exchange chromatography after concentration and liquid exchange, has a simple and controllable process, and reduces DNA residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 AKTA profiles to explore salt concentration for anion exchange chromatography;

[0029] Figure 2 This is an SDS-PAGE image of anion exchange chromatography to explore salt concentration;

[0030] Figure 3 Agarose gel image showing salt concentration in anion exchange chromatography;

[0031] Figure 4 is the SDS-PAGE image of anion exchange chromatography, where M is maker, 1 is supernatant, 2 is flow-through, 3 is equilibrium solution, and 4 is eluent;

[0032] Figure 5 It is an agarose gel image of anion exchange chromatography, where M is maker, 1 is supernatant, 2 is flow-through, 3 is equilibrium solution, and 4 is eluent;

[0033] Figure 6 It is the molecular sieve diagram of the supernatant before anion exchange chromatography;

[0034] Figure 7 It is the molecular sieve diagram of the flow-through liquid after anion exchange chromatography;

[0035] Figure 8The SDS-PAGE images of purification by anion exchange chromatography with different buffers, where M is maker, 1 is phosphate buffer, 2 is Tris buffer, and 3 is Hepes buffer;

[0036] Fig. 9 This is the SDS-PAGE image of the protein after nickel column purification;

[0037] Fig.10 This is the molecular sieve diagram of the protein after nickel column purification;

[0038] Fig.11 The nickel column purified S protein was stored at 2-8°C, where M is the marker, 1 is the S protein, and A, B, C, D, E, and F are stored for 0 months, 1 month, 3 months, 6 months, 9 months, and 12 months;

[0039] Fig.12 The purified S protein was stored at 2-8°C after the hollow fiber solution was replaced, where M is the marker, 1 is the S protein, and A, B, C, D, E, and F are stored for 0 months, 1 month, 3 months, 6 months, 9 months, and 12 months;

[0040] Fig.13 This is the situation of hollow fiber liquid replacement combined with anion-purified S protein stored at 2-8°C, where M is Marker, 1 is S protein, and A, B, C, D, E, and F represent storage for 0 month, 1 month, 3 months, 6 months, 9 months, and 12 months. DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention, but are not intended to limit the present invention.

[0042] The strains, plasmids and reagents used in the examples of the present invention are all commercially available products.

[0043] According to the applicant's prior application CN201810310540.8 or other documents, mammalian cells or insect baculovirus are used for culture to obtain cell culture fluid, and cell debris is removed to obtain cell supernatant.

[0044] The solution preparations in the examples are shown in Tables 1 and 2 below.

[0045] Table 1

[0046] Buffer A Buffer Neutral salt pH Buffer A1 <![CDATA[20mM NaH2PO4]]> 300mM NaCl 7.4 Buffer A2 20mM Tris 300mM NaCl 7.4 Buffer A3 20mM Hepes 300mM NaCl 7.4 Buffer A4 1× PBS 300mM NaCl 7.4

[0047] Table 2

[0048] Buffer B Buffer Neutral salt pH Buffer B1 <![CDATA[20mM NaH2PO4]]> 1000mM NaCl 7.4 Buffer B2 20mM Tris 1000mM NaCl 7.4 Buffer B3 20mM Hepes 1000mM NaCl 7.4 Buffer B4 1× PBS 1000mM NaCl 7.4

[0049] Here: the difference between buffer B (including buffer B1, buffer B2, buffer B3, buffer B4) and buffer A (including buffer A1, buffer A2, buffer A3, buffer A4) is only that the concentration of neutral salt in buffer A is 300mM, and the concentration of neutral salt in buffer B is not less than 1000mM.

[0050] Example 1: Determination of salt concentration in ion exchange chromatography

[0051] Prepare the solution according to Table 3.

[0052] The cell supernatant was dialyzed into Buffer 1 and used as the sample.

[0053] According to Table 4, the sample was loaded, balanced, and eluted by gradient method. The filler was RESOURCE Q 1ml pre-packed column. The samples were collected for agarose gel detection of DNA and SDS-PAGE detection of protein to determine the neutral salt concentration.

[0054] Table 3

[0055] Solution name <![CDATA[NaH2PO4]]> NaCl pH Buffer 1 20mM - 7.4 Buffer 2 20mM 1M 7.4

[0056] Table 4

[0057] step volume <![CDATA[NaH2PO4]]> NaCl pH Remark Loading 20ml - - - 0.2m / min balance 4 / 5 / 6 tubes 20mM 0 7.4 1ml / min 0-50％ B 7 / 8 / 9 / 10 tube 20mM 0-0.5M 7.4 0.5ml / min, 20CV 50％ B 11 tubes 20mM 0.5M 7.4 0.5ml / min 100％ B 12 / 13 tube 20mM 1M 7.4 1ml / min

[0058] From Tables 3 and 4, and Figure 1 , Figure 2 and Figure 3 Result analysis: A small amount of target protein hangs on the column, and the target protein is eluted before DNA. DNA is eluted when eluting with 347mM NaCl in tube 9, that is, the concentration of neutral salt should not be higher than 347mM, so the concentration of neutral salt in buffer A is determined to be 300mM. The concentration of neutral salt in buffer B is not less than 1000mM, and the purpose is to wash the column for reuse. Here: the difference between buffer B and buffer A is only that the concentration of neutral salt in buffer A is 300mM, and the concentration of neutral salt in buffer B is not less than 1000mM.

[0059] Example 2: Hollow fiber concentration and liquid replacement

[0060] The collected cell supernatant was concentrated using a 30 kDa hollow fiber ultrafiltration column at a shear rate of 6000 s -1, the transmembrane pressure range is 13-14.5psi, and it can be appropriately concentrated 2-5 times. Then, the concentrated cell supernatant is replaced with a 30kDa hollow fiber ultrafiltration column, wherein buffer A4 is added to the concentrated solution at the same flow rate as the permeate end, and the same volume of liquid is replaced. When the permeate end volume is at least 7 times the concentrated sample volume, the liquid replacement is stopped and the protein solution is collected.

[0061] Example 3: Membrane Pack Concentration and Fluid Replacement

[0062] The collected cell supernatant was concentrated and replaced with a 30kDa membrane package, wherein the pressure at the inlet end was adjusted to 20-30psi, and the pressure at the reflux end was not greater than 10psi, and the supernatant was concentrated 2-5 times. Then, the concentrated cell supernatant was replaced with a 30kDa membrane package, wherein buffer A4 was added to the concentrated solution at the same flow rate as the permeate end, and the volume was replaced. When the permeate end volume was 7 times the volume of the concentrated sample, the replacement was stopped and the protein solution was collected.

[0063] Example 4: Anion Exchange Chromatography

[0064] Sample processing: The concentrated protein solution was filtered through a 0.45-0.8 μm filter membrane and used as the crude pure solution.

[0065] Column equilibration: Take anion exchange filler (filler is Q Large Scale HP) and load it into an empty chromatography column. Equilibrate with cooled water for injection for 2 to 3 column volumes (CV), drain the concentrated ethanol storage solution, and then equilibrate with buffer A4 (1×PBS (300mmol / LNaCl) for 2 to 3CV, then equilibrate with buffer B4 (1×PBS (1000mmol / L NaCl)) for 2 to 3CV, and finally equilibrate with buffer A4 for 2 to 3CV.

[0066] Sampling: The crude pure liquid after sample treatment is purified through anion exchange filler, and the flow-through liquid is collected. Please refer to Table 5. Here, the crude pure liquid is a buffer A4 solution of S protein containing impurities.

[0067] Washing: Wash the column with buffer A4 until no blue color appears in the Coomassie Brilliant Blue G250 test. Collect the washing solution and combine it with the flow-through solution as the purified protein solution.

[0068] Column cleaning: first wash with buffer B4 for 3CV, then wash with water for 2CV, then wash with 0.5M sodium hydroxide for 2CV, finally wash with water until the pH is neutral, and store with 20% ethanol.

[0069] Detection: The supernatant, flow-through, flushing fluid and eluate of the anion exchange chromatography were subjected to SDS-PAGE to detect protein purity and agarose electrophoresis to detect DNA residue.

[0070] The experimental results can be found in Figure 4 , Figure 5 , Figure 6 , Figure 7 and Table 5.

[0071] Table 5: Q Large Scale HP 20ml (gravity column)

[0072] step Volume(ml) Solution Loading 100 1×PBS (300 mM NaCl) balance 15 1×PBS (300 mM NaCl) 100％B 50 1×PBS (1000mM NaCl)

[0073] from Figure 1 It can be seen that the purity of the target protein is increased from 80% to 90% by anion exchange chromatography.

[0074] from Figure 2 It can be seen that most of the DNA was attached to the column by anion exchange chromatography and eluted with buffer B4.

[0075] from Figure 3 and Figure 4 It can be seen that by comparing the molecular sieve before and after anion exchange chromatography, it was found that the target protein did not polymerize and the 8ml peak was significantly removed.

[0076] Example 5: Detection of exogenous DNA residues

[0077] DNA residue detection was performed using a CHO DNA residue detection kit (PCR-fluorescent probe method). The results are shown in Table 6. After anion exchange chromatography, there was almost no DNA residue.

[0078] Table 6

[0079]

[0080]

[0081] Example 6: Purification with different buffers

[0082] Buffer A with different buffer components was prepared and anion exchange chromatography was performed according to the method of Example 4. The experimental results are shown in Figure 8 and Table 7. The results showed that the protein purity of anion exchange chromatography using phosphate buffer, Tris buffer and Hepes buffer was above 90%, and there was almost no residual DNA.

[0083] Table 7

[0084]

[0085] Example 7: Purification of S protein using other anion exchange fillers

[0086] Mini Q, Mono Q, SOURCE 15Q, SOURCE 30Q, Q Sepharose HighPerformance, Q Sepharose Fast Flow, Q Sepharose XL, Q Sepharose Big Beads, CaptoQ, Capto Q ImpRes, DEAE Sepharose Fast Flow, ANX Sepharose 4Fast Flow and CaptoDEAE were used to purify S protein using the same method as described in Example 4. The results are shown in Table 8, and all of them were able to improve protein purity and remove DNA.

[0087] Table 8

[0088]

[0089]

[0090] Example 8: Comparative Example Nickel Column Purification

[0091] (1) Column equilibration: Use ultrapure water to equilibrate for 2-3CV (column volume), drain the ethanol storage solution; then use Buffer C (50mM NaH2PO4, 500mM NaCl, pH 7.4) to equilibrate for 2-3CV. (2) Loading: Load the supernatant with a peristaltic pump, set the appropriate flow rate according to the volume of the nickel column, and flow through and collect the flow-through liquid.

[0092] (3) Equilibration: Equilibrate with 10 CV of Buffer C.

[0093] (4) Elution: Elute the target protein with Buffer D (50 mM NaH2PO4, 500 mM NaCl, 400 mM imidazole, pH 7.4).

[0094] (5) Dialysis: The eluted target protein was dialyzed into buffer A4 (1×PBS (300 mM NaCl)).

[0095] (6) Detection: Take the dialyzed protein sample for SDS-PAGE, DNA residue and molecular sieve detection.

[0096] Table 8

[0097]

[0098] from Fig. 9 , Fig.10As shown in , and Table 8, the SDS-PAGE purity of the protein after nickel column purification was above 90%, but the molecular sieve results showed that the protein aggregated and there was a lot of DNA residue.

[0099] Example 9: Effects of different purification methods on protein stability

[0100] The proteins obtained by nickel column purification, hollow fiber liquid exchange, and hollow fiber liquid exchange combined with anion purification were stored at 2-8°C and analyzed by SDS-PAGE at 0, 1, 3, 6, 9, and 12 months. The results are shown in Fig.11 , Fig.12 , Fig.13 .

[0101] from Fig.11 , Fig.12 , Fig.13 It can be seen that different purification methods have a certain influence on the stability of the protein. The proteins obtained by nickel column purification and hollow fiber liquid exchange combined with anion purification are relatively stable, while the protein obtained by hollow fiber liquid exchange is degraded. Therefore, the purification method of the present invention improves the stability of the protein by combining anion exchange chromatography after liquid exchange compared with hollow fiber liquid exchange, and the protein does not aggregate, and the process is relatively simple and controllable.

[0102] The present invention is illustrated by the above examples, however, it should be understood that the present invention is not limited to the specific embodiments and embodiments described herein. The purpose of including these specific embodiments and embodiments here is to help those skilled in the art to practice the present invention. Any person skilled in the art will readily make further improvements and perfections without departing from the spirit and scope of the present invention, and therefore the present invention is only limited by the content and scope of the claims of the present invention, which is intended to cover all alternatives and equivalents included in the spirit and scope of the present invention as defined by the appended claims.

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[0106]

Claims

1. A method for purifying the S subunit protein of porcine epidemic diarrhea virus, characterized in that: The purification method comprises the following steps: 1) replacing the cultured supernatant with buffer A to remove the culture medium and obtain a crude pure solution; wherein the buffer A is a phosphate buffer, Tris buffer, Hepes buffer or PBS containing a neutral salt, and the neutral salt is NaCl; and 2) The crude pure solution obtained in step 1) is subjected to anion exchange chromatography, and the flow-through liquid is collected.

2. The purification method according to claim 1, characterized in that The amino acid sequence of the S subunit protein is shown in SEQ ID NO.

1.

3. The purification method according to claim 1, characterized in that In step 1), the liquid is replaced by a hollow fiber ultrafiltration column, a membrane package, a dialysis or a desalting column.

4. The purification method according to claim 3, characterized in that The molecular weight cut-off of the hollow fiber ultrafiltration column or membrane package is 30 kDa.

5. The purification method according to claim 4, characterized in that The hollow fiber ultrafiltration column or membrane has a liquid exchange multiple of at least 7 times.

6. The purification method according to claim 1, characterized in that In step 1), the solution is concentrated before changing the solution, and the concentration multiple is 2 to 5 times.

7. The purification method according to claim 1, characterized in that In step 2), the filler of the filler column of the anion exchange chromatography is a strong anion exchange filler or a weak anion exchange filler; wherein the strong anion exchange filler is a filler with -CH2-N + -(CH3)3 ligand filler, the weak anion exchange filler is a filler with -CH2-CH2-N + -(CH2-CH3)2 or -CH2-CHOH-CH2-N + -(CH2-CH3)2 ligand filler.

8. The purification method according to claim 7, characterized in that The strong anion exchange filler is Mini Q, Mono Q, SOURCE 15Q, RESOURCE Q, SOURCE 30Q, Q Sepharose High Performance, Q Sepharose Fast Flow, Q Sepharose XL, Q Sepharose Big Beads, Capto Q, Capto QImpRes, or Qlarge scale HP; the weak anion exchange filler is DEAE Sepharose Fast Flow, ANX Sepharose 4 Fast Flow, or Capto DEAE.

9. The purification method according to claim 8, characterized in that The strong anion exchange filler is Qlargescale HP or RESOURCE Q.

10. The purification method according to claim 1, characterized in that: The NaCl concentration in the buffer A was 300 mM.

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