A Highly Efficient Method for Purifying Porcine Epidemic Diarrhea Virus

Abstract

This invention provides a highly efficient method for purifying porcine epidemic diarrhea virus (PEDV), relating to the field of biology. The purification method includes the following steps: (1) adding concanavalin A and a precipitation promoter to the PEDV solution and mixing; (2) centrifuging and collecting the precipitate; (3) adding a dissociation solution to the precipitate and dissociating to obtain the purified virus solution. Using this method to purify PEDV, the impurity removal rate exceeds 90%, the recovery rate exceeds 90%, the viral load is large, and no high-value equipment is required.

Description

Technical Field

[0001] This invention relates to the field of biology, and more specifically to a highly efficient method for purifying porcine epidemic diarrhea virus. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) causing diarrhea in piglets is a significant infectious disease hindering the development of the pig industry. Epidemiological surveys show that the positive detection rate of PEDV samples is 62.1%–78.5%, the positive rate in pig farms is 71.4%–83.5%, and the mortality rate of infected suckling piglets is as high as 90%. Currently, this disease has become one of the most important swine diseases in the world, seriously affecting the healthy development of the pig industry and causing huge economic losses.

[0003] Currently, vaccination is the primary means of preventing and controlling porcine epidemic diarrhea (PEDV). Most PEDV vaccines are currently unpurified, and numerous impurities can cause severe side effects. Among existing purification methods such as centrifugation, membrane filtration, and chromatography, centrifugation and membrane filtration have very limited ability to remove contaminating proteins due to their low resolution. While chromatography achieves a higher rate of protein removal, the high cost of purification materials and equipment hinders its use in low-value-added veterinary vaccines. Therefore, developing novel, efficient, and low-cost PEDV antigen purification technologies is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a highly efficient method for purifying porcine epidemic diarrhea virus, which has high purification efficiency and low cost.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A highly efficient method for purifying porcine epidemic diarrhea virus includes the following steps:

[0007] (1) Add concanavalin A and precipitation promoter to the porcine epidemic diarrhea virus solution and mix them together;

[0008] (2) Centrifuge and collect the precipitate;

[0009] (3) Add dissociation solution to the precipitate, dissociate, and obtain purified virus solution.

[0010] In this invention, the final concentration of concanavalin A added to the porcine epidemic diarrhea virus solution is 1–2.4 μg / mL, and the viral load of the porcine epidemic diarrhea virus solution is 10. 6 -10 7.5 TCID 50 / mL.

[0011] In this invention, the precipitation formation promoter is a mixture of chondroitin sulfate a and dextran sulfate.

[0012] In this invention, the centrifugation speed in step (2) is 2000-8000 rpm.

[0013] In this invention, the final concentration of chondroitin sulfate a added to the porcine epidemic diarrhea virus solution in step (1) is 0.2-1.5 mg / mL, and the final concentration of dextran sulfate added to the porcine epidemic diarrhea virus solution is 0.2-1.5 mg / mL.

[0014] In this invention, the dissociation solution in step (3) contains one or more of mannose, methyl-α-D-mannopyranoside, and hexamannose-diN-acetylglucosamine.

[0015] In this invention, the dissociation solution also contains isopropanol and NaCl.

[0016] In this invention, the dissociation solution contains 150-450 mM methyl-α-D-mannopyranoside, 5-15 nM hexamannose-diN-acetylglucosamine, 10-250 mM NaCl, and isopropanol at a volume percentage of 1-6%.

[0017] In this invention, the solvent of the dissociation solution is a Tris buffer or PB buffer with a concentration of 0.005M-0.05M and a pH of 7.0-8.0.

[0018] In this invention, the shaking speed during the mixing process in step (1) is 100-500 rpm, and the mixing time is 1-5 hours; the dissociation time in step (3) is 10-60 minutes.

[0019] The applicant has identified a protein that can specifically recognize and directly precipitate PEDV virus particles: concanavalin A, which can efficiently purify PEDV virus particles. Purifying PEDV virus particles using concanavalin A has the following beneficial effects: (1) Impurity removal rate can reach 95%. Because the interaction between concanavalin A (Con A) and virus particles is highly specific, it can remove most impurities, including bovine serum albumin and cell debris, achieving a removal rate comparable to higher-cost chromatography techniques. (2) Recovery rate can meet the needs of large-scale industrial production. Using the method of this invention to purify PEDV, the PEDV virus particle recovery rate can reach approximately 95%. (3) High load capacity and no need for high-value equipment. After simple disruption and clarification of the viral fluid from cell proliferation, Con A and a highly efficient precipitation promoter are added, followed by stirring at room temperature. A low-speed centrifugation step yields a concentrated complex. Adding a highly efficient dissociation solution to the complex yields purified virus. The entire process is simple to operate, easy to scale up, has a larger loading capacity than chromatography methods, and does not require expensive purification equipment, thus significantly reducing production costs. Attached Figure Description

[0020] Figure 1 The graph shows the effect of Con A addition on purification efficiency. The horizontal axis represents the amount of Concanavalin A or phytohemagglutinin added, in μg, and the vertical axis represents the OD of the purified virus solution or dissociation solution. 450 The values ​​are: Con A-PEDV is the PEDV virus solution purified from Con A; Con A-Vero is the control group dissociation solution purified from Con A; PHA-PEDV is the PEDV virus solution purified from PHA; and PHA-Vero is the control group dissociation solution purified from PHA.

[0021] Figure 2 The OD values ​​of the purified PEDV virus solution corresponding to each precipitation promoter are as follows: 450 The values ​​represent the group on the x-axis and the purified virus solution's OD value on the y-axis. 450 value.

[0022] Figure 3 This is the result of optimizing the reaction time between PEDV cell culture medium and concanavalin A. The horizontal axis represents the reaction time in hours (h), and the vertical axis represents the OD of the purified virus solution. 450 value.

[0023] Figure 4 This represents the optimal centrifugal force optimization result, where the horizontal axis represents the centrifugal speed in rpm; and the vertical axis represents the OD (expansion / deceleration) value. 450 Value. ● represents the virus purification group, ○ represents the control group.

[0024] Figure 5 This is the optimal centrifugation time optimization result, where the horizontal axis represents centrifugation time in minutes; the vertical axis represents OD. 450 value.

[0025] Figure 6 This is an optimization of the dissociation fluid buffer system, where the horizontal axis represents the dissociation fluid group and the vertical axis represents OD. 450 value.

[0026] Figure 7 This is the standard curve of PEDV viral fluid, with OD on the horizontal axis. 450 The value is given on the ordinate, which is lg(PEDV titer). The standard curve is y = 0.4939x + 5.5878, where y is lg(PEDV titer) and x is OD. 450 value. Detailed Implementation

[0027] The present invention will be further illustrated by the following examples.

[0028] PEDV CV777 strain: published in Chen Jin, Huang Chunjuan, Yu Xiaoming, Hou Liting, Qiao Xuwen, Zheng Qisheng, Hou Jibo. Screening of PEDV subunit vaccine immune enhancers. Journal of North China Agricultural University. 2016, 31(2):205-210.

[0029] In this invention, room temperature refers to 20-30℃.

[0030] Example 1: Quantitative Detection of PEDV Virus Particles Using a Double Antibody Sandwich ELISA Method

[0031] The double-antibody sandwich ELISA method was used to quantitatively detect PEDV virus particles. The specific operation procedure is as follows: Sodium bicarbonate buffer (0.05 mol / mL, pH 9.6) was used as the coating solution. Anti-PEDV monoclonal antibody (purchased from Shandong Lvdou Biotechnology Co., Ltd.) was diluted with coating buffer to a concentration of 1 μg / mL and added to 96-well ELISA plates at 100 μL / well. The plates were incubated overnight at 4°C. The plates were washed three times with PBST solution (pH 7.2, 0.01 M PBS buffer containing 0.05% Tween 20). Each well was then blocked with 100 μL of PBST solution containing 1% (w / w) BSA. The plates were incubated at 37°C for 2 h, washed three times with PBST solution, and then 100 μL of the test sample was added to each well. The plates were incubated at 37°C for 1 h, washed three times with PBST solution, and then 100 μL of HRP-labeled anti-PEDV monoclonal antibody (purchased from Shandong Lvdou Biotechnology Co., Ltd.) was added to each well. The plates were incubated at 37°C for 1 h, washed with PBST solution, and then TMB chromogenic buffer was added. The reaction was incubated for 15 min, and the reaction was terminated with 2 M sulfuric acid solution. OD values ​​were measured using a microplate reader. 450 .

[0032] Example 2: Screening and Optimization of Agglutinin Usage

[0033] Preparation of PEDV cell culture medium: Vero cells were cultured until they formed a monolayer. The cell surface was washed twice with PBS, and then 30 mL of DMEM culture medium containing 2% (v / v) fetal bovine serum was added. The cells were seeded at a volume ratio of 1:100, and 300 μL of PEDV CV777 virus solution was added to the above cell culture medium. The cells were cultured at 37°C for 48 h under 5% CO2 conditions. When 90% of the cells showed cytopathic effects, the cell culture flasks were placed in a -20°C freezer and subjected to three freeze-thaw cycles. The cell suspension was collected and centrifuged at 6000 rpm for 10 min. The supernatant was collected as the PEDV cell culture medium, and its titer was determined to be 10. 6.9 TCID 50 / mL.

[0034] At a titer of 10 in 50 mL 6.9 TCID 50 Con A (concanavalin A, purchased from Sigma, C2010-250MG) was added to PEDV cell culture medium at concentrations of 0, 30, 60, 70, 80, 90, 100, and 120 μg, respectively. The mixture was shaken at 200 rpm for 2 hours at room temperature, centrifuged at 5000 rpm for 10 minutes, and the precipitate was collected. 30 mL of PBS (0.01 mol / L, pH 7.2) buffer containing 200 mM methyl-α-D-mannopyranoside was added to the precipitate, and the mixture was incubated at room temperature for 30 minutes to dissociate the virus, yielding purified PEDV virus solution. The PEDV virus solutions purified from different concentrations of Con A were labeled E1, E2, E3, E4, E5, E6, E7, and E8, respectively. Simultaneously, 50 mL of Vero cell lysis buffer was used instead of 50 mL of 10% titer PEDV virus solution. 6.9 TCID 50 The control group consisted of PEDV virus solution at a concentration of / mL, with other procedures following the same purification process as the PEDV virus solution. The control groups were treated with different concentrations of Con A, followed by dissociation to obtain dissociated solutions, labeled e1, e2, e3, e4, e5, e6, e7, and e8, respectively. See Table 1 for details.

[0035] Table 1. Numbering of virus solutions or dissociation solutions purified from Con A at different concentrations.

[0036]

[0037] E1-E8 and e1-e8 were detected using the double-antibody sandwich ELISA method described in Example 1, and OD was recorded. 450 Value. OD obtained from E1-E8 tests. 450The values ​​were 0.13, 0.178, 0.301, 0.831, 1.752, 2.882, 2.807, and 2.876, respectively, and the OD values ​​obtained from e1-e8 were... 450 The values ​​were 0.11, 0.13, 0.105, 0.1, 0.122, 0.106, 0.132, and 0.107, respectively. The OD values ​​of the Con A addition amount versus the purified virus solution were plotted. 450 The curve, the results are shown in Figure 1 .

[0038] In addition, following the same method used to purify PEDV viral fluid as described above (Con A), the study investigated the effect of using a 10-titer solution in 50 mL of fluid. 6.9 TCID 50 The effects of adding 0, 30, 60, 70, 80, 90, 100, and 120 μg of phytoagglutinin (PHA) to PEDV cell culture medium at a concentration of / mL on the purification of PEDV cell culture medium were investigated. A control group was also included, differing only in that PHA was used instead of Con A. The virus solutions or dissociation solutions purified by different concentrations of PHA are numbered as shown in Table 2.

[0039] Table 2. Numbering of virus solution or dissociation solution purified from PHA at different concentrations.

[0040]

[0041] OD obtained from F1-F8 tests 450 The values ​​were 0.13, 0.178, 0.185, 0.165, 0.752, 0.182, 0.207, and 0.176, respectively, and the OD values ​​obtained from f1-f8 were... 450 The values ​​were 0.09, 0.13, 0.112, 0.106, 0.131, 0.116, 0.102, and 0.097, respectively. A plot of PHA addition amount versus OD was then generated. 450 The curve, see Figure 1 .

[0042] OD obtained from F1-8 testing 450 The blank level indicates that PHA cannot bind to PEDV viral particles. When the amount of Con A added is less than 40 μg, OD... 450 The value increases slowly; when the amount of Con A added is 40–90 μg, the OD value increases slowly. 450 The value increases sharply; when the amount of ConA added is greater than 90 μg, the OD value increases dramatically. 450 Since the value remained at its highest level, Con A was chosen for the purification of PEDV, with an optimal addition concentration of 1.8 μg / mL.

[0043] Example 3: Screening of Precipitation Formation Promoters

[0044] At a titer of 10 in 50 mL 6.9 TCID 50 ConA was added to PEDV cell culture medium at a final concentration of 1.8 μg / mL. Then, five precipitation promoters listed in Table 3 were added (the concentrations of each substance in Table 3 refer to the final concentrations added to the PEDV cell culture medium). The mixture was shaken at 200 rpm for 2 hours at room temperature, centrifuged at 5000 rpm for 10 minutes, and the precipitate was collected. 35 mL of PBS buffer (0.01 mol / L, pH 7.2) containing 200 mM methyl-α-D-mannopyranoside was added to the precipitate, and the mixture was incubated at room temperature for 30 minutes to dissociate, yielding purified PEDV virus solution. The purified virus solutions corresponding to each precipitation promoter were numbered P1, P2, P3, P4, and P5, respectively. The OD values ​​of P1, P2, P3, P4, and P5 were detected using a double-antibody sandwich ELISA method (Example 1). 450 OD obtained from P1-5 detection 450 The values ​​were 2.079, 2.416, 1.835, 2.653, and 1.942, respectively. (See...) Figure 2 Therefore, the optimal precipitation formation promoters are chondroitin sulfate A and dextran sulfate.

[0045] Table 3. Numbers of purified PEDV virus fluids corresponding to different precipitation promoters.

[0046]

[0047] Example 4: Optimization of the reaction time between Con A and PEDV cell culture medium

[0048] At a titer of 10 in 50 mL 6.9 TCID 50 ConA, chondroitin sulfate a, and dextran sulfate were added to PEDV cell culture medium at a final concentration of 1.8 μg / mL, 0.3 mg / mL, and 0.7 mg / mL, respectively. The mixture was then shaken at 200 rpm at room temperature. The effect of the mixing time (0.5, 1, 1.5, 2, and 2.5 h) on the purification efficiency of ConA with PEDV cell culture medium was investigated. After shaking, the mixture was centrifuged at 4000 rpm for 10 min, and the precipitate was collected. 35 mL of PBS buffer (0.01 mol / L, pH 7.2) containing 200 mM methyl-α-D-mannopyranoside was added, and the mixture was dissociated at room temperature for 30 min to obtain the purified virus solution. The purified virus solutions collected after incubation of ConA with PEDV cell culture medium for 0.5, 1, 1.5, 2, and 2.5 h were labeled T1, T2, T3, T4, and T5, respectively. OD was detected using a double-antibody sandwich ELISA method (Example 1).450 OD obtained from T1-5 testing 450 The values ​​are 0.231, 0.673, 1.436, 2.658, and 2.679, respectively. (See...) Figure 3 Therefore, the optimal reaction time between ConA and PEDV cell culture medium is 2 hours.

[0049] Example 5: Optimization of Centrifugal Force and Centrifugation Time

[0050] 1. Optimization of centrifugal force

[0051] At a titer of 10 in 50 mL 6.9 TCID 50 ConA, chondroitin sulfate a, and dextran sulfate were added to 50 mL of PEDV cell culture medium at a final concentration of 1.8 μg / mL, designated as the virus purification group. ConA, at a final concentration of 1.8 μg / mL, was added to 50 mL of Vero cell lysate as the control group. The virus purification group and control group were mixed and incubated at 200 rpm for 2 h at room temperature. Then, the mixture was centrifuged at 1000, 2000, 4000, 6000, 8000, 10000, and 12000 rpm for 10 min, respectively, to investigate the effect of centrifugation speed on the purification results. After centrifugation, the precipitate was harvested, and 35 mL of PBS buffer (0.01 mol / L, pH 7.2) containing 200 mM methyl-α-D-mannopyranoside was added. The mixture was then dissociated at room temperature for 30 min to obtain the purified virus solution. The purified virus solutions harvested at different centrifugation speeds (1000, 2000, 4000, 6000, 8000, 10000, 12000 rpm) were labeled M1, M2, M3, M4, M5, M6, and M7, respectively. The dissociated solutions obtained from the control group at 1000, 2000, 4000, 6000, 8000, 10000, and 12000 rpm were labeled m1, m2, m3, m4, m5, m6, and m7, respectively. The OD values ​​of the purified virus solutions and dissociated solutions under each centrifugation condition were detected using a double-antibody sandwich ELISA method (Example 1). 450 OD obtained from M1-7 detection 450 The values ​​were 2.833, 2.945, 2.952, 2.815, 2.674, 1.915, and 0.943, respectively, and the OD values ​​obtained by m1-7 were... 450 The values ​​are 1.676, 1.819, 1.497, 1.026, 0.342, 0.229, and 0.121, respectively. (See...) Figure 4 When the centrifugation speed is high, the OD values ​​of the virus purification group and the control group are significantly higher. 450Both were relatively high, and non-specific impurities were also precipitated, affecting the impurity removal efficiency; when the centrifugation speed was low, the OD values ​​of the virus purification group and the control group were relatively high. 450 The OD values ​​were all low, indicating that the Con A-PEDV complex did not precipitate, affecting the recovery rate. When Con A reacted with the PEDV viral solution and centrifuged at 4000 rpm, the OD values ​​of the purified virus group were... 450 Higher, the OD corresponding to the control group was higher. 450 When maintaining a blank level, the optimal centrifugation speed is 4000 rpm.

[0052] 2. Optimization of centrifugation time

[0053] At a titer of 10 in 50 mL 6.9 TCID 50 ConA, chondroitin sulfate a, and dextran sulfate were added to a final concentration of 1.8 μg / mL to PEDV cell culture medium. The mixture was incubated at 200 rpm for 2 h at room temperature. The effect of centrifugation at 4000 rpm for 1, 2, 5, 10, 15, 20, 25, and 30 min on purification was investigated. After centrifugation, the precipitate was harvested, and 35 mL of PBS buffer (0.01 mol / L, pH 7.2) containing 200 mM methyl-α-D-mannopyranoside was added. The mixture was incubated at room temperature for 30 min to obtain the purified virus solution. The purified virus solutions obtained from centrifugation at 4000 rpm for 1, 2, 5, 10, 15, 20, 25, and 30 min were labeled N1, N2, N3, N4, N5, N6, N7, and N8, respectively. OD was detected using a double-antibody sandwich ELISA method. 450 OD obtained from N1-8 detection 450 The values ​​were 1.542, 1.807, 2.224, 2.685, 2.971, 2.968, 2.97, and 2.971, respectively. With increasing centrifugation time, the OD of the purified virus solution... 450 The OD of the purified virus solution gradually increased, and when the centrifugation time was greater than or equal to 15 min, the OD value increased. 450 The value remained relatively stable, indicating that the optimal centrifugation time was 15 minutes. Figure 5 ).

[0054] Example 6: Screening and Optimization of Dissociation Solutions

[0055] At a titer of 10 in 50 mL 6.9 TCID 50ConA (1.8 μg / mL), chondroitin sulfate (0.3 mg / mL), and dextran sulfate (0.7 mg / mL) were added to PEDV cell culture medium at a final concentration of 1.8 μg / mL. The mixture was incubated for 2 hours at 200 rpm on a shaker at room temperature. After centrifugation at 4000 rpm for 15 minutes, the precipitate was collected. 50 mL of dissociation solutions A, B, C, D, or E were added to investigate the effect of each solution on the purification effect. After adding the dissociation solution, the mixture was dissociated at room temperature for 30 minutes to obtain the purified virus solution.

[0056] Dissociation solution A: A 0.02M PB buffer solution with pH 7.5 containing 500mM mannose and 200mM NaCl. The 0.02M PB buffer solution with pH 7.5 is prepared as follows: 24.8mL of a 0.2M Na₂HPO₄ aqueous solution is mixed thoroughly with 15.2mL of a 0.2M NaH₂PO₄ aqueous solution, and then diluted 10 times with water.

[0057] Dissociation solution B: A 0.01M Tris buffer solution with pH 7.5 containing 500mM mannose and 200mM NaCl. The 0.01M Tris buffer solution with pH 7.5 is prepared as follows: 50mL of 0.1M Tris aqueous solution, 40.3mL of 0.1M hydrochloric acid, and 9.7mL of water are mixed thoroughly, and then diluted 5 times with water.

[0058] Dissociation solution C: pH 7.5, 0.01M Tris buffer (prepared as above) containing 200mM methyl-α-D-mannopyranoside and 50mM KCl.

[0059] Dissociation solution D: pH 7.5, 0.01M Tris buffer (prepared as above) containing 200mM methyl-α-D-mannopyranoside, 10nM Man6GlcNAc2 (hexamannose-diN-acetylglucosamine), 50mM NaCl and 2% isopropanol.

[0060] Dissociation solution E: pH 7.5, 0.02M PB buffer (prepared as above) containing 400mM methyl-α-D-mannopyranoside, 1nM Man9GlcNAc2 (nonamannose-diN-acetylglucosamine), 20mM NaCl and 5% isopropanol.

[0061] The OD of purified virus solution corresponding to each dissociation solution was detected using a double-antibody sandwich ELISA method (Example 1). 450 The OD value obtained by detecting the purified virus solution corresponding to the dissociation solution AE.450 The values ​​are 2.371, 2.470, 2.534, 2.636, and 2.576, respectively. (From...) Figure 6 It is evident that dissociation solution D exhibits the best dissociation effect.

[0062] Example 7: Construction of Standard Curve

[0063] 1. Construction of Standard Curve

[0064] Using the TCID50 (Cellular Half-Infection Dose) 50 The PEDV cell culture medium (Table 4) was accurately quantified as a standard, and the OD of the standard was detected using the double-antibody sandwich ELISA method described in Example 1. 450 Value, establish the titer and OD of the standard. 450 The standard curve of the linear regression relationship between the values ​​can be found in [the image / document]. Figure 7 The standard curve is y = 0.4939x + 5.5878, where y is lg(PEDV titer) and x is OD. 450 Value. Viral titer at 1.11 × 10⁻⁶. 4 ~3.2×10 7 TCID 50 When R is in the range of / mL 2 >0.99, CV<10%, indicating that the double-antibody sandwich ELISA method in Example 1 has good repeatability and high accuracy, and can stably and rapidly determine the PEDV content, which can be used to evaluate the virus recovery rate.

[0065] Table 4 Standard Product OD 450 Value and lgTCID 50 Correspondence

[0066] <![CDATA[OD 450 ]]> <![CDATA[lgTCID 50 ]]> 2.74 6.9 1.986 6.6 1.364 6.3 0.806 6.0 0.314 5.7

[0067] Example 8: Method and Effects of the Porcine Epidemic Diarrhea Virus Purification in This Invention

[0068] 1. The PEDV CV777 strain was purified using the virus purification method of this invention. The specific method is as follows:

[0069] At a titer of 10 in 50 mL 6.9 TCID 50To a final concentration of 1.8 μg / mL Con A, 0.3 mg / mL chondroitin sulfate a, and 0.7 mg / mL dextran sulfate in PEDV CV777 cell culture medium (i.e., the virus solution before purification), mix the mixture in a shaker at 200 rpm for 2 h at room temperature, centrifuge at 4000 rpm for 15 min, and collect the precipitate. Add 50 mL of dissociation buffer to the precipitate and dissociate it at room temperature for 30 min to obtain the purified virus solution. The dissociation buffer is a 0.01 M Tris buffer at pH 7.5, supplemented with 200 mM methyl-α-D-mannopyranoside, 10 nM Man9GlcNAc2, 50 mM NaCl, and 2% (v / v) isopropanol.

[0070] Determination of virus recovery rate using the purification method of this invention: The OD values ​​of the virus solution before and after purification were detected using the double-antibody sandwich ELISA method described in Example 1. 450 The value was substituted into the standard curve to obtain the TCID value of the virus solution before purification. 50 (denoted as T0) and the TCID of the purified virus solution 50 (Denotes it as T1). The virus recovery rate is calculated using the following formula: T0 is 10 6.9 / mL, T1 is 10 6.88 / mL, therefore, virus recovery rate = 10 6.88 / 10 6.9 ×100% = 95%.

[0071] The purification method of this invention was used to determine the removal rate of impurities and proteins: the total protein content in the virus solution before and after purification was determined using a BCA protein quantification kit. The removal rate was calculated using the following formula: The total protein content in the viral solution before purification is denoted as C1 (3.5 mg / mL), and the volume is V; the total protein content in the viral solution after purification is denoted as C2 (79.2 μg / mL), and the volume is V. The removal rate of impurity proteins = (1 - 79.2 / 3500) × 100%, which is higher than 95%.

[0072] 2. Purification of PEDV CV777 strain using control method 1

[0073] Purification of PEDV CV777 strain using control method 1 includes the following steps: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 6.9 TCID 50Add Con A to a final concentration of 800 μg / mL of PEDVCV777 cell culture medium (virus solution before purification), incubate at 25°C for 1 h, centrifuge at 10000 rpm for 25 min, and collect the precipitate. Add 5 mL of dissociation buffer F (pH 7.5, 0.01 M Tris buffer containing 200 mM methyl-α-D-mannopyranoside, 1 mM EDTA, and 100 mM NaCl) to the precipitate, and dissociate at 4°C for 15 min to obtain the purified virus.

[0074] Virus recovery rate determination: The OD of the virus solution before and after purification was detected using the double-antibody sandwich ELISA method described in Example 1. 450 The value was substituted into the standard curve to obtain the TCID value of the virus solution before purification. 50 (denoted as T0) and the TCID of the purified virus solution 50 (Denotes it as T2). The virus recovery rate is calculated using the following formula: T0 is 10 6.9 / mL, T2 is 10 6.87 / mL, therefore, virus recovery rate = 10 6.83 / 10 6.9 ×100% ÷ 10 = 9.3%.

[0075] Protein removal rate determination: The total protein content in the virus solution before and after purification was determined using the BCA protein quantification kit. The removal rate was calculated using the following formula: The total protein content in the virus solution before purification was denoted as C1 (3.5 mg / mL), and the volume was V (50 mL); the total protein content in the virus solution after purification was denoted as C3 (5.07 mg / mL), and the volume was V3 (5 mL). The removal rate of impurity proteins = [1 - (5070 × 5) ÷ (3500 × 50)] × 100%, which was 85.5%.

[0076] 3. Purification of PEDV CV777 strain using control method 2

[0077] Purification of PEDV CV777 strain using control method 2 includes the following steps: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 6.9 TCID 50 Add Con A to a final concentration of 800 μg / mL of PEDVCV777 cell culture medium (virus solution before purification), incubate at 25°C for 2 h, centrifuge at 4000 rpm for 25 min, and collect the precipitate. Add 50 mL of dissociation buffer F (pH 7.5, 0.01 M Tris buffer containing 200 mM methyl-α-D-mannopyranoside, 1 mM EDTA, and 100 mM NaCl) to the precipitate, and dissociate at room temperature for 30 min to obtain the purified virus.

[0078] Virus recovery rate determination: The OD of the virus solution before and after purification was detected using the double-antibody sandwich ELISA method described in Example 1. 450 The value was substituted into the standard curve to obtain the TCID value of the virus solution before purification. 50 (denoted as T0) and the TCID of the purified virus solution 50 (Denotes it as T3). The virus recovery rate is calculated using the following formula: T0 is 10 6.9 / mL, T3 is 10 5.98 / mL, therefore, virus recovery rate = 10 5.98 / 10 6.9 ×100% = 12%.

[0079] Protein removal rate determination: The total protein content in the virus solution before and after purification was determined using the BCA protein quantification kit. The removal rate was calculated using the following formula: The total protein content in the viral solution before purification is denoted as C1 (3.5 mg / mL), and the total protein content in the viral solution after purification is denoted as C4 (0.63 mg / mL), with a volume of V. The removal rate of impurity proteins is calculated as (1 - 630 / 3500) × 100%, which is 82%.

Claims

1. A highly efficient porcine epidemic diarrhea virus purification method, characterized by It comprises the following steps: (1) adding Concanavalin A and a precipitate formation accelerator into the porcine epidemic diarrhea virus liquid and mixing; (2) centrifuging and taking the precipitate; (3) adding a dissociation solution into the precipitate to dissociate and obtain the purified virus liquid; The precipitate formation accelerator is a mixture of chondroitin sulfate a and dextran sulfate; in step (1), the final concentration of chondroitin sulfate a added into the porcine epidemic diarrhea virus liquid is 0.2-1.5 mg / mL, and the final concentration of dextran sulfate added into the porcine epidemic diarrhea virus liquid is 0.2-1.5 mg / mL; The dissociation solution contains 150-450 mM methyl-α-D-mannopyranoside, 5-15 nM hexamannose-di-N-acetylglucosamine, 10-250 mM NaCl and 1-6% isopropyl alcohol in volume percentage.

2. The purification method of claim 1, wherein The final concentration of Concanavalin A added to the porcine epidemic diarrhea virus liquid was 1-2.4 μg / mL, and the virus content of the porcine epidemic diarrhea virus liquid was 10 6 -10 7.5 TCID 50 / mL.

3. The purification method of claim 2, wherein The centrifugation speed in step (2) is 2000-8000 rpm.

4. The purification method of claim 3, wherein The solvent of the dissociation solution is Tris buffer or PB buffer with a concentration of 0.005 M-0.05 M and a pH of 7.0-8.

0.

5. The purification method of claim 4, wherein The shaking speed during the mixing in step (1) is 100-500 rpm, and the mixing time is 1-5 hours; the dissociation time in step (3) is 10-60 minutes.

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