Method for purifying envelope virus

Abstract

The invention relates to the technical field of biology, and particularly provides a purification method of an envelope virus. According to the purification method, through the steps of virus particle release, virus clarification and virus purification, the virus purity is improved to the maximum extent, the virus recovery rate is increased, the envelope virus recovery rate is 83% or above, the impurity protein removal rate is 92% or above, and the virus activity is not obviously lost.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and specifically provides a method for purifying enveloped viruses. Background Art

[0002] The envelope is a lipid bilayer membrane composed of double-layer lipids, polysaccharides and proteins wrapped around the virus capsid. Its outer surface has spike-shaped glycoproteins that can bind to receptors and enter cells. Viruses with envelope structures are collectively referred to as enveloped viruses. In addition to the lipid bilayer membrane, there are generally matrix proteins responsible for virus assembly and nucleoproteins responsible for storing tissue nucleic acids. Because the lipid bilayer membrane is involved in the assembly of the virus, enveloped viruses are almost toxic and morphologically incomplete and deformed. Envelope viruses are mostly animal viruses and can be called the most threatening pathogens. Many viruses in nature belong to enveloped viruses, such as influenza virus, rabies virus, dengue virus, new coronavirus, porcine respiratory and reproductive syndrome virus, African swine fever virus, and Newcastle disease virus. These viruses not only seriously threaten the health of humans and animals, but also affect the development of the global economy and society.

[0003] There is usually no specific medicine for infectious diseases. Vaccine immunization is an effective means of preventing and controlling infectious diseases, including inactivated vaccines, attenuated live vaccines and subunit vaccines. Inactivated vaccines are vaccines prepared by inactivating strong strains or attenuated virus beads that retain immunogenicity after large-scale cultivation. Attenuated live vaccines are vaccines prepared by reducing the pathogenicity of natural strong strains to weak strains with good immunogenicity through methods such as biological continuous passage. The focus of these commercial vaccines is to improve the purity of antigens and ensure the titer of the virus to achieve good immune effects and safety. The virus fluid propagated by cells is affected by impurities such as culture medium components, cell metabolites and cell fragments, which can easily cause side effects such as allergies and fever in the body after immunization, so the virus needs to be purified.

[0004] The traditional method of removing solid impurities is to use density gradient ultracentrifugation, which requires expensive ultracentrifuges and is very costly; excessive centrifugal force and high osmotic pressure of gradient solutions can easily damage the integrity of enveloped viruses, causing the envelope to fall off, which in turn causes a decrease in immunogenicity. Centrifugation is the most commonly used clarification process, and its limitation is that it can remove large particles and cell debris in the culture medium but cannot remove soluble impurities.

[0005] Although column chromatography can obtain relatively pure viral antigens, the heterologous impurities present in the cultured viral antigens directly affect the yield and purity of the viral antigens.

[0006] Therefore, it is of great significance to develop a virus purification method with a simple purification technology route, little damage to the enveloped virus structure, high yield and high purity.

[0007] In view of this, this application is hereby filed. Summary of the invention

[0008] The purpose of the present application is to provide a method for purifying enveloped viruses to improve the purity and yield of enveloped viruses while reducing costs.

[0009] In order to achieve the above purpose, this application adopts the following technical solutions:

[0010] A method for purifying enveloped viruses comprises the following steps:

[0011] (1) Virus particle release: The culture medium is repeatedly frozen and thawed to lyse the cells and release the virus particles;

[0012] (2) Virus clarification: remove insoluble impurities by centrifugation and deep filtration;

[0013] (3) Virus purification: Purify the virus solution using anion exchange chromatography. Further, the enveloped virus includes porcine reproductive and respiratory syndrome virus, pseudorabies virus or feline herpes virus.

[0014] Furthermore, the turbidity of the supernatant after centrifugation is 100-200 NTU.

[0015] Furthermore, the centrifugal rotation speed is 5000-8000 rpm.

[0016] Furthermore, the turbidity of the virus solution after deep filtration is 30 to 80 NTU.

[0017] Furthermore, the pore size of the filter membrane used for the deep filtration is 0.45 to 1.2 μm.

[0018] Furthermore, the filler used in the anion exchange chromatography is selected from Fractogel EMD TMAE, FractogelDEAE, Q Sepharose Fast Flow, DEAE Sepharose Fast Flow, DEAE-SHV2, HPQ-30 strong anion exchange medium, HPQ-60 strong anion exchange medium, UniGel-30Q, UniGel-30DEAE, Q Beads 6FF or HiSelect Q 6FF.

[0019] Compared with the prior art, the technical effects of this application are:

[0020] The enveloped virus purification method provided in the present application maximizes the purity of the virus, increases the virus recovery rate, and has good versatility. The method can improve the virus recovery rate (up to 83% or more) and purity (the removal rate of foreign proteins is above 92%), without obvious loss of virus activity and has a wide range of applications, which can meet the requirements of virus research on virus purity and structural integrity and can achieve production amplification. DETAILED DESCRIPTION

[0021] The technical solution of the present application will be described clearly and completely below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.

[0022] The purification method provided in the present application has a wide range of applications. For example, common enveloped viruses such as porcine reproductive and respiratory syndrome virus, pseudorabies virus or feline herpes virus can be efficiently purified and have strong versatility.

[0023] The present application provides a method for purifying enveloped viruses, comprising the following steps:

[0024] (1) Virus particle release: The culture medium is repeatedly frozen and thawed to lyse the cells and release the virus particles.

[0025] After the virus is amplified by cell culture, the virus particles can be released by repeated freezing and thawing of the cells, which is beneficial for subsequent purification. In some embodiments, most cells can be lysed by repeated freezing and thawing about three times.

[0026] (2) Virus clarification: Remove insoluble impurities by centrifugation and deep filtration.

[0027] In order to remove insoluble impurities in the culture medium of cell disruption, large particles and most of the cell fragments can be removed by centrifugation, and then insoluble impurities such as cell fragments can be removed by deep filtration.

[0028] In some embodiments, the turbidity in the supernatant after centrifugation is 100-200 NTU. Preferably, a centrifuge is used. The speed of the centrifuge can be 5000-8000 rpm, for example, but not limited to 5000 rpm, 5500 rpm, 6000 rpm, 6500 rpm, 7000 rpm, 7500 rpm or 8000 rpm. The centrifugation time can be adjusted according to the speed, such as centrifugation for 30 minutes at a speed of 5000 rpm / min, and sampling is performed during the centrifugation to monitor the turbidity.

[0029] In some embodiments, the turbidity of the virus solution after deep filtration is 30 to 80 NTU. Preferably, a deep filter is used, and the pore size of the filter membrane is 0.45 to 1.2 μm, for example, but not limited to 0.45 μm, 0.8 μm or 1 μm. Different sizes of deep filtration filters can be selected according to the volume of the culture solution. For example, in different embodiments, a single layer of 23 cm is required to filter 500 mL of culture solution. 2 Filter, sampling is performed during the deep filtration process to monitor turbidity.

[0030] Conventional filtration is surface filtration, and particles accumulate quickly, which is suitable for coarse filtration with low precision. Different from conventional filtration, deep filtration is suitable for filtration with high precision requirements according to the pore size and particle interception area.

[0031] (3) Virus purification: The clarified virus solution was purified using anion exchange chromatography.

[0032] In some embodiments, the filler used in the anion exchange chromatography is selected from Fractogel EMD TMAE, Fractogel DEAE, Q Sepharose Fast Flow, DEAE Sepharose Fast Flow, DEAE-SHV2, HPQ-30 strong anion exchange medium, HPQ-60 strong anion exchange medium, UniGel-30Q, UniGel-30DEAE, Q Beads6FF or HiSelect Q 6FF.

[0033] The anion exchange chromatography method is as follows:

[0034] 1) Pre-packed chromatography column: Close the liquid outlet of the chromatography column, add the filler into the required chromatography column, let it stand for 0.5 h, open the liquid outlet to drain the excess liquid, and connect the column to the chromatography system.

[0035] 2) Column balance: Place each pipette of the chromatograph into the corresponding prepared solution, start the chromatograph, expel the bubbles, rinse with sterile deionized water for 5CV, 1mol / L NaOH for 5CV, and buffer A for 10CV.

[0036] Buffer A: 20 mmol / L PB, pH 7.0.

[0037] Buffer B: 20mmol / L PB, 1mol / L NaCl, pH 7.0.

[0038] 1mol / L NaOH.

[0039] 20% ethanol.

[0040] All the above solutions must be filtered through a 0.45μm filter membrane before use.

[0041] 3) Sample loading and equilibration: After sample loading, rinse with buffer A for 5CV.

[0042] 4) Elution: Use a linear salt concentration gradient for 20CV, such as 10% buffer B, 20% buffer B, 30% buffer B, 40% buffer B, 50% buffer B, 60% buffer B, etc. Collect samples according to the elution peak for detection.

[0043] 5) Chromatographic column treatment: flush with buffer B for 5CV, reverse flush with 1mol / L NaOH for 5CV, and finally flush with 20% ethanol for 2CV to preserve the chromatography column to avoid microbial growth.

[0044] The detection method of the purified virus liquid is as follows:

[0045] The turbidity, virus titer and protein concentration of virus samples after centrifugation, filtration, solution exchange and chromatography were tested to analyze the effectiveness of the purification process.

[0046] The method for detecting the virus titer is as follows:

[0047] TCID 50 The virus titer of the sample was detected by the method and the virus recovery rate was calculated:

[0048] The method for determining protein content is:

[0049] The total protein content of the virus solution was determined using a protein concentration determination kit, and the removal rate of impurity proteins was calculated:

[0050]

[0051] The present invention removes impurities in the cell culture medium through centrifugation and filtration processes, and purifies the virus by anion chromatography. The virus recovery rate is above 83%, the impurity protein removal rate is above 92%, and there is no obvious loss of virus activity.

[0052] The present application is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0053] Example 1 Purification of porcine reproductive and respiratory syndrome virus

[0054] A method for purifying porcine reproductive and respiratory syndrome virus comprises the following steps:

[0055] 1) The virus solution of porcine reproductive and respiratory syndrome virus NVDC-JXA1 strain cultured in Marc-145 cells was repeatedly frozen and thawed three times to lyse the cells and release the virus particles.

[0056] 2) Use a high-speed centrifuge to pre-treat the culture medium in 1)

[0057] ① Set the speed to 5000 rpm / min and centrifuge for 30 minutes.

[0058] ②Collect the supernatant.

[0059] ③ Detect sample turbidity and TCID 50 and total protein content.

[0060] 3) Deep filtration:

[0061] ① The pore size of the filter membrane is 0.45μm and the size of a single layer is 23cm 2 , connect the deep filter device according to the instructions, the filtration flow rate is 10L / min / m 2 , when the pressure reaches 1 bar, stop filtering.

[0062] ② Collect samples to test turbidity and TCID 50 .

[0063] 4) Virus purification using membrane ultrafiltration, molecular sieve and ion exchange chromatography

[0064] 4.1) Virus purification using membrane ultrafiltration system

[0065] ①Membrane package is 300kD, 50cm 2 , the quantity is 1 piece, the clarified liquid is replaced with 10 times volume of 20mmol / L PB, pH7.0 solution, and finally the virus liquid is concentrated 10 times.

[0066] ② Collect concentrated samples and test TCID 50 .

[0067] 4.2) Purification of viruses by molecular sieve

[0068] ① Molecular sieve balance: Place each pipette of the chromatograph into the corresponding prepared solution, start the chromatograph, expel the bubbles, and rinse with 20mmol / L PBS, pH7.0 at a flow rate of 1ml / min for 120min until the baseline is stable.

[0069] ② Loading and sample collection: Load 5 ml of the sample at a flow rate of 1 ml / min. The UV280 peak appears around 65 min, and 10 ml of the sample is collected based on the elution peak.

[0070] ③ Molecular sieve treatment: flush with 20% ethanol at a flow rate of 1 ml / min for 120-140 min until the baseline is stable and then store the molecular sieve.

[0071] ④ Collect purified samples and test TCID 50 and total protein content.

[0072] 4.3) Purify the virus by anion exchange chromatography.

[0073] ① Pre-packed chromatography column: Close the liquid outlet of the chromatography column, mix the filler and add 100 mL of filler into the XK 26 / 40 chromatography column. After standing for 0.5 h, open the liquid outlet to drain the excess liquid and connect the column to the chromatography system.

[0074] ② Balance the column: Place each pipette of the chromatograph into the corresponding prepared solution, start the chromatograph, expel the bubbles, rinse with sterile deionized water at a flow rate of 90cm / h for 5CV, 1mol / L NaOH for 5CV, and buffer A for 10CV until the baseline is stable. The solutions include 20mmol / L PB, pH7.0 (buffer A), 20mmol / L PB, 1mol / L NaCl, pH7.0 (buffer B), 1mol / L NaOH, and 20% ethanol, all of which need to be filtered through a 0.45μm filter membrane before use.

[0075] ③ Sample loading and equilibration: Load the sample at a flow rate of 90 cm / h for 5 CV, and then rinse with buffer A for 5 CV until the baseline is stable.

[0076] ④ Washing: Use 20% buffer B to wash for 5CV until the baseline is stable, then use 60% buffer B to elute and collect the elution peak.

[0077] ⑤ Column treatment: flush with buffer B at a flow rate of 90 cm / h for 5CV, reverse flush with 1 mol / L NaOH for 5CV, and finally flush with 20% ethanol for 2CV to preserve the column to avoid microbial reproduction.

[0078] ⑥ TCID analysis of purified virus 50 and protein content detection.

[0079] 5) Experimental results

[0080] ① Turbidity detection: During the experiment, it was found that some cell fragments were still floating in the supernatant of the culture medium after centrifugation and could not be removed. The turbidity of the samples before and after centrifugation and after deep filtration of the culture medium was tested. The experimental results are shown in Table 1. The turbidity of the samples was significantly reduced after deep filtration and could be used for purification.

[0081] Table 1 Sample turbidity detection

[0082] ② Virus titer detection: TCID was performed on samples after centrifugation, deep filtration, ultrafiltration purification, molecular sieve purification, and chromatography purification. 50 The experimental results are shown in Table 2. No obvious loss was observed in the deep filtration stage. Compared with the three purification methods, molecular sieve could not achieve sample concentration, and the virus titer was low after purification. The virus recovery rate after ion exchange chromatography purification was higher than that after ultrafiltration purification. Therefore, anion chromatography method was preferred for virus purification.

[0083] Table 2 Sample titer detection and virus recovery rate

[0084] ③ Protein content detection: The total protein content of the samples after pretreatment, deep filtration, ultrafiltration purification, molecular sieve purification and chromatography purification was detected respectively. The test results are shown in Table 3. The total protein content in the pretreated porcine respiratory and reproductive syndrome virus fluid was 2.8 mg / ml, and the total protein content after deep filtration was 2.4 mg / ml. The total protein content after ultrafiltration purification was 5.1 mg / ml, and the impurity protein removal rate was 82%; the total protein content after molecular sieve purification was 0.3 mg / ml, and the impurity protein removal rate was 79%; the total protein content after chromatography purification was 0.8 mg / ml, and the impurity protein removal rate was 94%. Therefore, the anion chromatography method is preferred for virus purification.

[0085] Table 3 Total protein concentration of samples and removal rate of impurity proteins after purification

[0086] In summary, the recovery rate of porcine respiratory and reproductive syndrome virus purified by the present application was 83%, the removal rate of foreign proteins was 94%, and there was no obvious loss of virus activity.

[0087] Example 2 Preparation of porcine reproductive and respiratory syndrome virus vaccine composition

[0088] The concentrated porcine reproductive and respiratory syndrome virus unpurified antigen and purified antigen prepared in Example 1 were inactivated and mixed with a water-in-oil-in-water emulsion adjuvant (for example, 206 adjuvant from SEPPIC, France) at a volume ratio of 50:50, and stirred at 120 rpm for 15 minutes at 30° C. The specific ratios are shown in Table 4.

[0089] Table 4 Porcine reproductive and respiratory syndrome virus vaccine composition ratio Components Vaccine 1 Vaccine 2 <![CDATA[Unpurified antigen (TCID 50 / ml)]]> <![CDATA[10 6.0 ]]> - <![CDATA[Purified antigen (TCID 50 / ml)]]> - <![CDATA[10 6.0 ]]> Adjuvant content (V / V%) 50 50

[0090] Example 3 Immunogenicity Test of Porcine Reproductive and Respiratory Syndrome Virus Vaccine Composition

[0091] Fifteen PRRSV antigen-antibody negative piglets at 42 days of age were randomly divided into three groups, 5 piglets per group. Group 1 was inoculated with vaccine 1, and group 2 was inoculated with vaccine 2, with an immunization dose of 2 ml per piglet; group 3 was inoculated with PBS 2 ml per piglet.

[0092] The challenge was carried out 28 days after immunization with porcine reproductive and respiratory syndrome virus NVDC-JXA1 strain, and the challenge dose was 10 5.5 TCID 50 / head, clinical symptoms observed are shown in Table 5.

[0093] Table 5 Results of immunogenicity test of porcine reproductive and respiratory syndrome virus vaccine composition

[0094] The results showed that when piglets were immunized with the porcine reproductive and respiratory syndrome virus vaccine composition (vaccine 2) purified and prepared by the method of the present application and challenged with the NVDC-JXA1 strain, the virus infection was blocked (clinical symptoms appeared), providing 100% (5 / 5) protection for the piglets; when piglets were immunized with the porcine reproductive and respiratory syndrome virus vaccine composition (vaccine 1) that was not purified and challenged with the NVDC-JXA1 strain, the virus infection could not be completely blocked (clinical symptoms appeared), and 80% (4 / 5) limited protection could be provided for the piglets; all the challenged control piglets showed clinical symptoms after challenge, and some pigs died.

[0095] The above shows that the porcine reproductive and respiratory syndrome virus vaccine composition prepared by the antigen purified by the method of the present application has a good immune effect and can provide 100% protection; it shows that the purification method of the present application does not damage the virus structure, has a high yield and high purity, can meet the requirements of virus research on virus purity and structural integrity and can achieve production scale-up, which is of great significance.

[0096] Example 4 Safety Test of Porcine Reproductive and Respiratory Syndrome Virus Vaccine Composition

[0097] Ten litters of 28-day-old PRRSV antigen-antibody negative piglets were randomly divided into two groups, 5 litters / group. Group 4 was vaccinated with vaccine 1, and group 5 was vaccinated with vaccine 2, with an immunization dose of 4 ml / head. The animals were observed for 21 days after immunization to observe the reactions of the immunized animals, and some piglets were dissected to observe the absorption of the vaccine at the injection site. The results are shown in Table 6.

[0098] Table 6 Safety test of porcine reproductive and respiratory syndrome virus vaccine composition

[0099] The results showed that overdose immunization of piglets with the porcine reproductive and respiratory syndrome virus vaccine composition (vaccine 2) purified and prepared by the method of the present application had no adverse reactions and was safe; overdose immunization of piglets with the unpurified porcine reproductive and respiratory syndrome virus vaccine composition (vaccine 1) resulted in about 32% adverse reactions.

[0100] The above shows that the porcine reproductive and respiratory syndrome virus vaccine composition prepared by the antigen purified by the method of the present application has good safety. The porcine reproductive and respiratory syndrome virus vaccine composition prepared without purification cannot achieve the effect brought by the purification method of the present application in terms of safety.

[0101] Example 5 Purification of Pseudorabies Virus

[0102] A method for purifying porcine pseudorabies virus comprises the following steps:

[0103] 1) The virus solution of the gE-deficient strain of the porcine pseudorabies virus HN1201 strain cultured in PK-15 cells was repeatedly frozen and thawed three times to lyse the cells and release the virus particles.

[0104] 2) Use a centrifuge to pretreat the culture medium in 1)

[0105] ① Centrifuge at 8000 rpm / min for 20 minutes.

[0106] ②Collect the supernatant.

[0107] ③ Detect sample turbidity and TCID 50 and total protein content.

[0108] 3) Deep filtration:

[0109] ① The pore size of the filter membrane is 0.45μm and the size of a single layer is 23cm 2 , connect the deep filter device according to the instructions, the filtration flow rate is 10L / min / m 2 , when the pressure reaches 1 bar, stop filtering.

[0110] ② Collect samples to test turbidity and TCID 50 .

[0111] 4) The virus was purified by membrane ultrafiltration, molecular sieve (Sepharose 4FF) and ion exchange chromatography.

[0112] 4.1) Virus purification by membrane ultrafiltration system

[0113] ①Membrane package is 300kD, 50cm 2 , the quantity is 1 piece, the clarified liquid is replaced with 10 times volume of 20mmol / L PB, pH7.0 solution, and finally the virus liquid is concentrated 4 times.

[0114] ② Collect concentrated samples and test TCID 50 .

[0115] 4.2) Purification of viruses by molecular sieve

[0116] ① Molecular sieve balance: Place each pipette of the chromatograph into the corresponding prepared solution, start the chromatograph, expel the bubbles, and rinse with 20mmol / L PBS, pH7.0 at a flow rate of 1ml / min for 120min until the baseline is stable.

[0117] ② Loading and sample collection: Load 5 ml of the sample at a flow rate of 1 ml / min. The UV280 peak appears around 75 minutes, and the sample is collected according to the elution peak.

[0118] ③ Molecular sieve treatment: flush with 20% ethanol at a flow rate of 1 ml / min for 120-140 min until the baseline is stable and then store the molecular sieve.

[0119] ④ Collect purified samples and test TCID 50 and total protein content.

[0120] 4.3) Purify the virus by anion exchange chromatography.

[0121] ① Pre-packed chromatography column: Close the liquid outlet of the chromatography column, mix the filler and add 100 mL of filler into the XK 26 / 40 chromatography column. After standing for 0.5 h, open the liquid outlet to drain the excess liquid and connect the column to the chromatography system.

[0122] ② Balance the column: Place each pipette of the chromatograph into the corresponding prepared solution, start the chromatograph, expel the bubbles, rinse with sterile deionized water at a flow rate of 90cm / h for 5CV, 1mol / L NaOH for 5CV, and buffer A for 10CV until the baseline is stable. The solutions include 20mmol / L PBS, pH7.0 (buffer A), 20mmol / L PBS, 1mol / L NaCl, pH7.0 (buffer B), 1mol / L NaOH, and 20% ethanol, all of which need to be filtered through a 0.45μm filter membrane before use.

[0123] ③ Sample loading and equilibration: Load the sample at a flow rate of 90 cm / h for 10 CV, and then rinse with buffer A for 5 CV until the baseline is stable.

[0124] ④ Washing: Use 5% buffer B to wash for 5CV until the baseline is stable, then use 50% buffer B to elute and collect the elution peak.

[0125] ⑤ Column treatment: flush with buffer B at a flow rate of 90 cm / h for 5CV, reverse flush with 1 mol / L NaOH for 5CV, and finally flush with 20% ethanol for 2CV to preserve the column to avoid microbial reproduction.

[0126] ⑥ TCID analysis of purified virus 50 and protein content detection

[0127] 5) Experimental results

[0128] ① Turbidity detection: The turbidity of the samples before and after centrifugation and after deep filtration of the culture medium was detected. The experimental results are shown in Table 7. The turbidity of the samples was significantly reduced after deep filtration and can be used for purification.

[0129] Table 7 Sample turbidity detection

[0130] ② Virus titer detection: TCID was performed on samples after centrifugation, deep filtration, ultrafiltration purification, molecular sieve purification, and chromatography purification. 50 The experimental results are shown in Table 8. There is no loss in the deep filtration stage. Compared with the three purification methods, the TCID 50 The virus recovery rate is higher than that of the sample after molecular sieve purification, but the virus recovery rate is best after chromatography purification, so the anion chromatography method is preferred for virus purification.

[0131] Table 8 Sample titer detection sample Volume (mL) <![CDATA[TCID 50 ]]> Virus recovery rate Centrifugation 2000 7.61 / Depth Filtration 2000 7.61 / Ultrafiltration purification 100 8.12 81％ Molecular sieve purification 15 7.0 74％ Anion exchange chromatography purification 100 8.59 95％

[0132] ③ Protein content detection: The total protein concentration of the samples after pretreatment, deep filtration, molecular sieve purification and anion chromatography purification was detected respectively, and the removal rate of impurity proteins after purification was calculated. The test results are shown in Table 9. The total protein content in the pretreated pseudorabies virus liquid was 3.5 mg / ml, and the total protein content after deep filtration was 3.2 mg / ml. The total protein content after ultrafiltration purification was 3.5 mg / ml, and the impurity protein removal rate was 75%; the total protein content after molecular sieve purification was 0.3 mg / ml, and the impurity protein removal rate was 74%; the total protein content after anion chromatography purification was 1.25 mg / ml, and the impurity protein removal rate was 96%. The anion chromatography purification method is the best.

[0133] Table 9 Sample protein content detection

[0134] In summary, the recovery rate of the porcine pseudorabies virus purified by the present application was 95%, the removal rate of foreign proteins was 96%, and there was no obvious loss of virus activity.

[0135] Example 6 Preparation of porcine pseudorabies virus vaccine composition

[0136] The purified concentrated antigen of pseudorabies virus prepared in Example 5 and the antigen without any purification treatment were added with 10% (v / v) formaldehyde solution to make the final concentration of formaldehyde 0.2% (v / v), inactivated at 37°C for 18 hours, stirred once every 4 hours, each stirring for 10 minutes, and after complete inactivation, mixed with 206 adjuvant (product of SEPPIC, France) at a volume ratio of 54:46, and stirred at 120 rpm for 15 minutes at 30°C. The specific ratios are shown in Table 10.

[0137] Table 10: Porcine pseudorabies virus vaccine composition ratio

[0138] Example 7 Immunogenicity test of porcine pseudorabies virus vaccine composition

[0139] Twelve PRV antigen-antibody negative piglets at 21 days of age were randomly divided into 3 groups, 4 piglets per group. Group 6 was inoculated with vaccine 3, and group 7 was inoculated with vaccine 4, with an immunization dose of 2 ml per piglet; group 8 was inoculated with PBS 2 ml per piglet.

[0140] The challenge dose was 2×10 8.0 TCID 50 / head, clinical symptoms and mortality are shown in Table 11.

[0141] Table 11 Results of immunogenicity test of porcine pseudorabies virus vaccine composition

[0142] The results showed that after the pseudorabies virus gE deleted strain vaccine purified and prepared by the method of the present application was used to immunize piglets, the virus infection could be blocked (clinical symptoms appeared), and 100% (0 / 4) protection could be provided to the piglets; after the pseudorabies virus gE deleted strain vaccine was not purified and prepared, although the virus infection could not be blocked (clinical symptoms appeared), it could provide 100% (0 / 4) protection to the piglets; all the control piglets died 4 days after the infection.

[0143] Therefore, the purified vaccine provided in the present application has better protective power than the unpurified antigen vaccine and shows good immune protection.

[0144] Example 8 Purification of Feline Herpes Virus

[0145] A method for purifying feline herpes virus comprises the following steps:

[0146] 1) The feline herpesvirus type 1 strain 64 virus liquid cultured in F81 cells (feline herpesvirus type 1 strain 64 (Felineherpesvirus type 1, Strain 64) is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: V202321, a deposit address of Wuhan University, Wuhan, China, and a deposit date of March 21, 2023.) was repeatedly frozen and thawed three times to lyse the cells and release the virus particles.

[0147] 2) Use a high-speed centrifuge to pre-treat the culture medium in 1)

[0148] ① Set the speed to 8000 rpm / min and centrifuge for 10 minutes.

[0149] ②Collect the supernatant.

[0150] ③ Detect sample turbidity and TCID 50 and total protein content.

[0151] 3) Deep filtration:

[0152] ① The pore size of the filter membrane is 0.45μm and the size of a single layer is 23cm 2 , connect the deep filter device according to the instructions, the filtration flow rate is 10L / min / m 2 , when the pressure reaches 1 bar, stop filtering.

[0153] ② Collect samples to test turbidity and TCID 50 .

[0154] 4) Virus purification using membrane ultrafiltration, molecular sieve and ion exchange chromatography

[0155] 4.1) Virus purification by membrane ultrafiltration system

[0156] ①Membrane package is 300kD, 50cm 2 , the quantity is 1 piece, and the clarified liquid is concentrated 3 times.

[0157] ② Replace the clarified liquid with 10-fold volume of 20mmol / L PB, pH 7.0 solution, and finally concentrate the virus 2-fold.

[0158] ③ Collect concentrated samples and test TCID 50 .

[0159] 4.2) Purification of viruses by molecular sieve

[0160] ① Molecular sieve balance: Place each pipette of the chromatograph into the corresponding prepared solution, start the chromatograph, expel the bubbles, and rinse with 20mmol / L PBS, pH7.0 at a flow rate of 1ml / min for 120min until the baseline is stable.

[0161] ② Loading and sample collection: Load 5 ml of the sample at a flow rate of 1 ml / min. The UV280 peak appears around 65 minutes, and the sample is collected according to the elution peak.

[0162] ③ Molecular sieve treatment: flush with 20% ethanol at a flow rate of 1 ml / min for 120-140 min until the baseline is stable and then store the molecular sieve.

[0163] ④ Collect purified samples and test TCID 50 and total protein content.

[0164] 4.3) Purify the virus by anion exchange chromatography.

[0165] ① Pre-packed chromatography column: Close the liquid outlet of the chromatography column, mix the filler and add 100 mL of filler into the XK 26 / 40 chromatography column. After standing for 0.5 h, open the liquid outlet to drain the excess liquid and connect the column to the chromatography system.

[0166] ② Balance the column: Place each pipette of the chromatograph into the corresponding prepared solution, start the chromatograph, expel the bubbles, rinse with sterile deionized water at a flow rate of 90cm / h for 5CV, 1mol / L NaOH for 5CV, and buffer A for 10CV until the baseline is stable. The solutions include 20mmol / L PB, pH7.0 (buffer A), 20mmol / L PB, 1mol / L NaCl, pH7.0 (buffer B), 1mol / L NaOH, and 20% ethanol, all of which need to be filtered through a 0.45μm filter membrane before use.

[0167] ③ Sample loading and equilibration: Load the sample at a flow rate of 90 cm / h for 4 CV, collect the flow-through, and then rinse with buffer A for 5 CV until the baseline is stable.

[0168] ④ Washing: Use 20% buffer B to wash for 5CV until the baseline is stable, then use 60% buffer B to elute and collect the elution peak.

[0169] ⑤ Column treatment: flush with buffer B at a flow rate of 90 cm / h for 5CV, reverse flush with 1 mol / L NaOH for 5CV, and finally flush with 20% ethanol for 2CV to preserve the column to avoid microbial reproduction.

[0170] ⑥ TCID analysis of purified virus 50 and protein content detection.

[0171] 5) Experimental results

[0172] ① Turbidity detection: During the experiment, it was found that some cell fragments were still floating in the supernatant of the culture medium after centrifugation and could not be removed. The turbidity of the samples before and after centrifugation and after deep filtration of the culture medium was tested. The experimental results are shown in Table 12. The turbidity of the samples was significantly reduced after deep filtration and could be used for purification.

[0173] Table 12 Sample turbidity detection

[0174] ② Virus titer detection: TCID was performed on samples after centrifugation, deep filtration, ultrafiltration purification, molecular sieve purification, and chromatography purification. 50 The experimental results are shown in Table 13. No significant loss of virus was observed during the pretreatment and deep filtration stages. Compared with the three purification methods, molecular sieve cannot achieve sample concentration, and the virus titer after purification is low; the virus recovery rate after ion exchange chromatography purification is higher than that after ultrafiltration purification, so anion chromatography is preferred for virus purification.

[0175] Table 13 Sample titer detection

[0176] ③ Protein content detection: The total protein content of the samples after pretreatment, deep filtration, ultrafiltration purification, molecular sieve purification and chromatography purification was detected respectively, and the removal rate of impurity proteins after purification was calculated. The test results are shown in Table 14. The total protein content in the pretreated feline herpes virus liquid was 3.2 mg / ml, and the total protein content after deep filtration was 3.0 mg / ml. The total protein content after ultrafiltration purification was 3.5 mg / ml, and the impurity protein removal rate was 45%; the total protein content after molecular sieve purification was 0.4 mg / ml, and the impurity protein removal rate was 80%; the total protein content after anion chromatography purification was 1.0 mg / ml, and the impurity protein removal rate was 92%. Therefore, the impurity protein removal effect is best with the anion chromatography purification method.

[0177] Table 14 Sample protein content detection

[0178] In summary, the recovery rate of the feline herpes virus purified by the present application was 87%, the removal rate of foreign proteins was 92%, and there was no obvious loss of virus activity.

[0179] Note that the above are only preferred embodiments of the present application and the technical principles used. Those skilled in the art will understand that the present application is not limited to the specific embodiments described herein, and that various obvious changes, readjustments and substitutions can be made by those skilled in the art without departing from the protection scope of the present application. Therefore, although the present application is described in more detail through the above embodiments, the present application is not limited to the above embodiments, and may also include more other equivalent embodiments without departing from the technical concept of the present application, all of which belong to the protection scope of the present application.

Claims

1. A method for purifying enveloped viruses, characterized in that: The following steps are involved: (1) Virus particle release: The culture medium is repeatedly frozen and thawed to lyse the cells and release the virus particles; (2) Virus clarification: remove insoluble impurities by centrifugation and deep filtration; (3) Virus purification: The virus solution was purified using anion exchange chromatography.

2. The purification method according to claim 1, characterized in that The enveloped virus includes porcine reproductive and respiratory syndrome virus, pseudorabies virus or feline herpes virus.

3. The purification method according to claim 1, characterized in that The turbidity of the supernatant after centrifugation was 100-200 NTU.

4. The purification method according to claim 3, characterized in that The centrifugal rotation speed is 5000-8000 rpm.

5. The purification method according to claim 1, characterized in that The turbidity of the virus solution after deep filtration is 30-80 NTU.

6. The purification method according to claim 5, characterized in that The pore size of the filter membrane used for the deep filtration is 0.45-1.2 μm.

7. The purification method according to claim 1, characterized in that The filler used in the anion exchange chromatography is selected from Fractogel EMD TMAE, Fractogel DEAE, Q Sepharose Fast Flow, DEAE Sepharose Fast Flow, DEAE-SHV2, HPQ-30 strong anion exchange medium, HPQ-60 strong anion exchange medium, UniGel-30Q, UniGel-30DEAE, Q Beads 6FF or HiSelect Q 6FF.

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