CN111763661B - Virus purification method, vaccine composition prepared by virus purification method and application of vaccine composition - Google Patents

Virus purification method, vaccine composition prepared by virus purification method and application of vaccine composition Download PDF

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CN111763661B
CN111763661B CN201910262281.0A CN201910262281A CN111763661B CN 111763661 B CN111763661 B CN 111763661B CN 201910262281 A CN201910262281 A CN 201910262281A CN 111763661 B CN111763661 B CN 111763661B
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virus
antigen
nuclease
vaccine composition
vaccine
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CN111763661A (en
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田克恭
侯瑞卿
郜玮
张许科
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Pulaike Biological Engineering Co Ltd
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Abstract

The invention provides a purification method of a virus antigen, which comprises the following steps: removing macromolecular impurities in the proliferated virus liquid by medium-speed centrifugation, treating the virus liquid with nuclease to remove the macromolecular impurities, adding EDTA to inhibit the nuclease, and performing purification and separation by gel chromatography to obtain a purified virus antigen. The antigens produced by the process of the invention are in the same TCID 50 The titer of the generated neutralizing antibody is higher than that of the purified inactivated antigen during content, and the neutralizing antibody has good immunogenicity.

Description

Virus purification method, vaccine composition prepared by virus purification method and application of vaccine composition
Technical Field
The invention relates to the field of virus purification and the field of prepared virus-containing pharmaceutical preparations, in particular to a virus purification method and a vaccine composition prepared by the virus purification method.
Background
Purification of viral antigens is a key step in research and production. In order to remove impurities in the culture process, a good virus antigen purification process should be able to effectively remove various foreign proteins, nucleic acids and other impurities brought in the culture process, and have a good antigen recovery rate.
The traditional virus vaccine purification adopts a density gradient ultracentrifugation process, the process needs an ultracentrifuge with high price, and the cost for preparing the vaccine is high; excessive centrifugal force and high permeability of gradient solution are easy to damage the activity and integrity of virus, and the sample amount of one-time processing is limited, which brings difficulty to the industrialization of vaccine.
Although the gel filtration column chromatography purification technology can obtain relatively pure virus antigen, impurities brought by the virus antigen in the culture process directly influence the yield of the virus antigen, and meanwhile, the molecular weight of partial nucleic acid generated by the breakage, lesion, necrosis or apoptosis of host cells is very similar to that of virus particles, and the purity of the virus antigen is directly influenced.
Therefore, the development of a virus purification method with simple purification technical route, high separation efficiency, good separation selectivity and small damage to virus structures is of great significance.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a virus purification method, which can be used for large-scale virus purification, obtaining the virus with high purity and high titer, and reducing the cost investment.
The invention provides a purification method of a virus antigen, wherein the purification method comprises the following steps: removing macromolecular impurities in the proliferated virus liquid by medium-speed centrifugation; treating the virus liquid obtained in the step (1) after removing the macromolecular impurities with nuclease to degrade the nucleic acid of the host cell; step (3) adding EDTA (ethylene diamine tetraacetic acid) into the virus liquid treated by the nuclease in the step (2) to inhibit the nuclease; and (4) purifying and separating the virus liquid of the inhibited nuclease in the step (3) by using gel chromatography to obtain a purified virus antigen.
The purification method of the invention can purify the virus antigen and retain the immunogenicity of the antigen to a greater extent, and the prepared inactivated vaccine has the immunogenicity equivalent to the virus antigen which is not purified.
The treatment process for removing macromolecular impurities by the virus purification method adopts a medium-speed centrifugation mode, has low requirement and mild treatment condition, cannot cause secondary damage to virus particles, and has the characteristics of large treatment capacity and low treatment cost; according to the virus purification method, nuclease is adopted to treat virus liquid, so that the purity of the virus is improved, and the yield of later-stage purified virus is obviously increased; according to the virus purification method, EDTA is adopted to neutralize nuclease, so that continuous damage to a target virus structure is avoided, and the immunogenicity of a virus antigen is ensured to the greatest extent; the virus purification method can treat various virus antigens and has wide applicability.
In one embodiment of the present invention, in the method for purifying a virus antigen of the present invention, the virus solution proliferated in the step (1) is subjected to a concentration pretreatment.
In one embodiment of the present invention, in the method for purifying a viral antigen of the present invention, the centrifugation rate of the medium-speed centrifugation in the step (1) is 7000rpm to 9000rpm, and the centrifugation time is 10min to 20 min.
In a preferred embodiment of the present invention, in the method for purifying a viral antigen of the present invention, the centrifugation rate of the medium-speed centrifugation is 8000rpm, and the centrifugation time is 12 min.
In one embodiment of the present invention, in the method for purifying a virus antigen of the present invention, the nuclease in the step (2) is Benzonase nuclease, the nuclease addition amount is 1.5 to 2U/mL, and the nuclease treatment time is 30 min.
In a preferred embodiment of the present invention, the nuclease is added in an amount of 1.8U/mL in the method for purifying a viral antigen of the present invention.
In one embodiment of the present invention, in the method for purifying a viral antigen of the present invention, the concentration of EDTA in the step (3) is 2 mmol/L.
The invention also provides a virus antigen obtained by the purification method.
The virus antigen of the invention has good immunogenicity, is equivalent to the virus antigen which is not purified, and the prepared vaccine can be completely protected and has small side reaction. The vaccine prepared by purifying the virus by the virus purification method of the invention has the advantages that the virus antigen keeps the original immunogenicity to the maximum extent, the titer of the generated antibody is higher than that of the vaccine prepared by untreated antigen with the same content, the good immune effect is ensured, and the method is suitable for preparing the vaccine in large scale in industry.
As an embodiment of the present invention, the purified virus antigen of the present invention is porcine reproductive and respiratory syndrome virus, porcine circovirus, porcine pseudorabies virus, porcine parvovirus, foot and mouth disease virus, or porcine epidemic diarrhea virus.
The invention also provides a vaccine composition, wherein the vaccine composition comprises an immunizing amount of inactivated viral antigen and a pharmaceutically acceptable carrier.
The vaccine composition has good immunogenicity, can be completely protected, and has small side effect.
As an embodiment of the present invention, in the vaccine of the present invention, the pharmaceutically acceptable carrier is an adjuvant, and the adjuvant includes: (1) white oil, alumina gel adjuvant, saponin, alfudine, DDA; (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a copolymer of a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative; and one or more of RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, Montanide ISA 206 and Gel adjuvant; preferably, the saponin is Quil A, QS-21, GPI-0100; the adjuvant content is 5% -70% V/V.
The adjuvant may be present in an amount selected from the group consisting of 5% V/V, 6% V/V, 7% V/V, 8% V/V, 9% V/V, 10% V/V, 15% V/V, 20% V/V, 25% V/V, 30% V/V, 35% V/V, 40% V/V, 45% V/V, 50% V/V, 55% V/V, 60% V/V, 65% V/V, 66% V/V, 67% V/V, and 70% V/V.
The vaccine compositions of the present invention may be formulated using available techniques, preferably together with pharmaceutically acceptable carriers. For example, the oil may help stabilize the formulation and additionally act as a vaccine adjuvant. The oil adjuvant can be natural source or obtained by artificial synthesis. The term "adjuvant" refers to a substance added to the composition of the present invention to increase the immunogenicity of the composition. Known adjuvants include, but are not limited to: (1) aluminium hydroxide, saponin (saponin) (e.g. QuilA), alfuzidine, DDA, (2) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivatives, (3) vaccines can be made in the form of oil-in-water, water-in-oil or water-in-oil-in-water emulsions, or (4) Montanide TM Gel。
In particular, the emulsion may be based on light liquid paraffin oil, isoprenoid oil, such as squalane or squalene; oils resulting from the oligomerization of olefins, in particular isobutene or decene, esters of acids or alcohols with linear alkyl groups, more in particular vegetable oils, ethyl oleate, propylene glycol di (caprylate/caprate), glycerol tri (caprylate/caprate), propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used with an emulsifier to form an emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of polyoxyethylated fatty acids (e.g.oleic acid), sorbitan, mannitol (e.g.anhydromannitol oleate), glycerol, polyglycerol, propylene glycol and optionally ethoxylated oleic acid, isostearic acid, ricinoleic acid, hydroxystearic acid, ethers of fatty alcohols and polyols (e.g.oleyl alcohol), polyoxypropylene-polyoxyethylene block copolymers, in particular Pluronic R, especially L121 (see Hunter et al, 1995, "The Theory and Practical applications of Advances" (Steward-Tull, D.E.S. eds.) John Wiley and sons, NY, 51-94; Todd et al, Vaccine, 1997, 15, 564 + 570).
In particular, the acrylic or methacrylic acid polymers are crosslinked by polyalkenyl ethers of sugars or polyols. These compounds are known as carbomers.
The amount of adjuvant suitable for use in the compositions of the present invention is preferably an effective amount. By "effective amount" is meant the amount of adjuvant necessary or sufficient to exert their immunological effect in a host when administered in combination with the antigen of the invention without causing undue side effects. The precise amount of adjuvant to be administered will vary depending on factors such as the ingredients used and the type of disease being treated, the type and age of the animal being treated, the mode of administration, and the other ingredients in the composition.
The vaccine composition of the present invention may further comprise other agents added to the composition of the present invention. For example, the compositions of the present invention may also comprise agents such as: drugs, immunostimulants (e.g., alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and interleukin 2(IL2)), antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives. To prepare such compositions, methods well known in the art may be used.
The vaccine composition according to the present invention may be prepared in oral or non-oral dosage forms.
Preferred are non-oral dosage forms that can be administered by intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, or epidural routes.
In one embodiment of the present invention, in the vaccine composition of the present invention, the viral antigen is pre-inactivation 10 6.0 ～10 8.0 TCID 50 The inactivated antigen of the porcine reproductive and respiratory syndrome virus NVDC-JXA1 strain is/ml, and the pharmaceutically acceptable carrier is an oil-in-water emulsion adjuvant.
In one embodiment of the present invention, in the vaccine composition of the present invention, the viral antigen is pre-inactivation 10 7.0 ～10 8.0 TCID 50 The inactivated antigen of the porcine epidemic diarrhea virus HN1303 strain is/ml, and the pharmaceutically acceptable carrier is an alumina gel adjuvant. In one embodiment of the present invention, in the vaccine composition of the present invention, the viral antigen is pre-inactivation 10 7.0 TCID 50 Inactivation of porcine reproductive and respiratory syndrome virus NVDC-JXA1 strain/mlAn antigen, said pharmaceutically acceptable carrier being an oil-in-water emulsion adjuvant.
In one embodiment of the present invention, in the vaccine composition of the present invention, the viral antigen is pre-inactivation 10 8.0 TCID 50 The inactivated antigen of the porcine epidemic diarrhea virus HN1303 strain is/ml, and the pharmaceutically acceptable carrier is an alumina gel adjuvant.
The vaccine composition removes the influence of macromolecular protein and other impurities on immune response in the antigen components, so that the generated antibody titer is higher than that of a vaccine prepared from unpurified antigen with the same content, and the vaccine composition shows good immunogenicity.
Detailed Description
The present invention will be further described with reference to specific embodiments, and advantages and features of the present invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The chemical reagents used in the examples of the invention are analytically pure and purchased from the national pharmaceutical group.
The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.
Example 1 purification of porcine reproductive and respiratory syndrome Virus
1. Removal of macromolecular impurities
Dividing NVDC-JXA1 strain (disclosed in Chinese patent application 200710086549.7) cultured by Marc-145 cells into three parts by concentrated virus liquid, adding the first part into a centrifuge tube, putting the centrifuge tube into a medium-speed centrifuge, centrifuging for 12min at 8000rpm, removing cell debris and partial impurities, and obtaining primary purified virus liquid which is named as A1; adding the second part into a centrifuge tube, placing into a high-speed centrifuge, centrifuging at 12000rpm for 5min, removing cell debris and partial impurities to obtain primary purified virus liquid, which is named as A2; the third portion was untreated and was designated A3.
2. Nuclease treatment
The virus solutions A1, A2 and A3 obtained in the above steps are respectively divided into one part, the Benzonase nuclease is added for treating the virus for 30min, the adding amount is 1.8U/mL, and the treated virus solutions are respectively named as B1, B2 and B3.
EDTA inhibition of nucleases
Splitting virus solutions B1, B2 and B3 obtained in the above steps into parts, adding EDTA to a final concentration of 2mmol/L, inhibiting nuclease, and respectively naming the inhibited virus solutions as C1, C2 and C3.
4. Purifying and separating by gel chromatography
Respectively carrying out fine purification and separation on the virus solutions A1, A2 and A3 obtained in the steps by adopting a gel chromatography, wherein the yield of the virus solution A1 purified by the gel chromatography is 40%, and the virus solution purified by the gel chromatography is named as A11; the yield of the A2 purified by gel chromatography is 41%, and the virus solution purified by gel chromatography is named A21; the yield of A3 after purification by gel chromatography was 38%, and the virus solution after purification by gel chromatography was named A31.
Respectively carrying out fine purification and separation on the virus solutions B1, B2 and B3 obtained in the steps by adopting a gel chromatography, wherein the yield of the purified virus solution B1 is 90% after the purification by the gel chromatography, and the virus solution purified by the gel chromatography is named as B11; the yield of B2 after purification by gel chromatography is 91%, and the virus solution after purification by gel chromatography is named as B21; the yield of B3 after purification by gel chromatography was 40% (the virus solution after purification by gel chromatography was named B31.
Respectively carrying out fine purification and separation on virus solutions C1, C2 and C3 obtained in the steps by adopting a gel chromatography, wherein the yield of the C1 purified by the gel chromatography is 90%, and the virus solution purified by the gel chromatography is named as C11; the yield of the C2 purified by gel chromatography is 91%, and the virus solution purified by gel chromatography is named as C21; the yield of C3 after purification by gel chromatography was 40%, and the virus solution after purification by gel chromatography was named C31.
The results show that the yield is higher after the purification by gel chromatography after the centrifugation and nuclease treatment or the centrifugation, nuclease and EDTA treatment.
Adding the purified virus into 10% (V/V) formaldehyde solution to make the final concentration of formaldehyde be 0.2% (V/V), inactivating at 37 deg.C for 18 hr, stirring 1 time every 4 hr, stirring for 10min each time, and inactivating completely for use.
Example 2 preparation of porcine reproductive and respiratory syndrome Virus vaccine composition
The porcine reproductive and respiratory syndrome virus antigens A3, B11, B21, C11 and C21 prepared in example 1 are inactivated and then respectively mixed with an oil-in-water emulsion adjuvant according to the volume ratio of 50:50, and stirred for 15 minutes at the temperature of 30 ℃ at 120 rpm. The specific ratio is shown in table 1.
TABLE 1 porcine reproductive and respiratory syndrome Virus vaccine composition ratio
Figure BDA0002015669900000071
EXAMPLE 3 porcine reproductive and respiratory syndrome Virus vaccine compositions immunogenicity testing
30 piglets, which were 43 days old and were negative for PRRSV antigen antibody, were randomly divided into 6 groups, 5 groups, and each group, and the vaccines were injected according to table 2, and the control group was inoculated with 2 ml/head of PBS.
TABLE 2 porcine reproductive and respiratory syndrome Virus vaccine composition immunogenicity testing cohort
Group of Injectable vaccines Immunization dose
1 Vaccine 1 2 ml/head
2 Vaccine 2 2 ml/head
3 Vaccine 3 2 ml/head
4 Vaccine 4 2 ml/head
5 Vaccine 5 2 ml/head
6 PBS 2 ml/head
The virus is attacked 28 days after immunization, the porcine reproductive and respiratory syndrome virus NVDC-JXA1 strain is used for attacking the virus, and the dose of the attacking virus is 10 5.5 TCID 50 First, clinical symptoms are observed as shown in table 3.
TABLE 3 porcine reproductive and respiratory syndrome Virus vaccine compositions immunogenicity test results
Figure BDA0002015669900000081
The result shows that the porcine reproductive and respiratory syndrome virus vaccine composition (vaccine 4) prepared by the method disclosed by the invention is used for immunizing piglets, and is attacked by NVDC-JXA1 strain, so that the virus infection (clinical symptoms) can be blocked, and 100% (5/5) protection is provided for the piglets; the piglet is immunized by the porcine reproductive and respiratory syndrome virus vaccine composition (vaccine 1) prepared by a conventional cell culture method, and the NVDC-JXA1 strain is used for counteracting virus, so that the virus infection (clinical symptoms) can be blocked, and 100% (5/5) of limited protection can be provided; the pig breeding and respiratory syndrome virus vaccine composition (vaccine 2) prepared by directly adopting gel chromatography for fine purification and separation or the pig breeding and respiratory syndrome virus vaccine composition (vaccine 3) prepared by directly adopting gel chromatography for immunization of piglets is subjected to virus attack by using NVDC-JXA1 strain, so that virus infection (clinical symptoms) cannot be completely blocked, and 80% (4/5) limited protection can be provided; after high-speed centrifugation, nuclease treatment and EDTA neutralization, the pig breeding and respiratory syndrome virus vaccine composition (vaccine 5) prepared by fine purification and separation by adopting a gel chromatography is used for immunizing piglets, NVDC-JXA1 strain is used for counteracting the virus, the virus infection (clinical symptoms) can not be completely blocked, and 80% (4/5) of limited protection can be provided; after the challenge of the challenge control piglet, all the piglets show clinical symptoms, and part of the piglets die.
The method and the antigen provided conventionally show that the prepared porcine reproductive and respiratory syndrome virus vaccine composition has good immune effect and can provide 100% protection; other methods, such as high-speed centrifugation, nuclease addition, EDTA addition or combination thereof, and fine purification and separation by gel chromatography cannot achieve the effects of the method of the present invention.
Example 4 porcine reproductive and respiratory syndrome Virus vaccine composition safety test
30 litters of PRRSV antigen-antibody negative piglets of 3-4 weeks old are randomly divided into 5 groups and 6 litters/group, and the vaccines are injected according to the table 4.
TABLE 4 porcine reproductive and respiratory syndrome Virus vaccine composition safety test group
Group of Injectable vaccines Immunization dose
7 Vaccine 1 4 ml/head
8 Vaccine 2 4 ml/head
9 Vaccine 3 4 ml/head
10 Vaccine 4 4 ml/head
11 Vaccine 5 4 ml/head
And after immunization, continuously feeding and observing for 21 days, observing the reaction of the immunized animals, dissecting partial piglets, and observing the absorption condition of the vaccine at the injection part. The results are shown in Table 5.
TABLE 5 porcine reproductive and respiratory syndrome Virus vaccine composition safety test results
Figure BDA0002015669900000091
The results show that the porcine reproductive and respiratory syndrome virus vaccine composition (vaccine 4) prepared by the method and the porcine reproductive and respiratory syndrome virus vaccine composition (vaccine 5) prepared by performing fine purification and separation by a gel chromatography method after high-speed centrifugation, nuclease treatment and EDTA neutralization have no adverse reaction and good safety; the porcine reproductive and respiratory syndrome virus vaccine composition (vaccine 1) prepared by a conventional cell culture method is used for immunizing piglets in an overdose manner, and the adverse reaction of about 5 percent occurs; the swine breeding and respiratory syndrome virus vaccine composition (vaccine 2) prepared by directly adopting a gel chromatography for fine purification and separation is treated by adding nuclease after medium-speed centrifugation or the swine breeding and respiratory syndrome virus vaccine composition (vaccine 3) prepared by directly adopting a gel chromatography for fine purification and separation is subjected to super-dose immunization on piglets, and about 31% of adverse reactions occur.
The antigen provided by the fine purification and separation by adopting the gel chromatography after the medium-speed centrifugation or the high-speed centrifugation and the nuclease treatment and the EDTA neutralization, the prepared porcine reproductive and respiratory syndrome virus vaccine composition has better safety, but the immunogenicity of the method is better; vaccines prepared by other purification methods cannot achieve the effects of the methods of the invention in both immunogenicity and safety.
Example 5 purification of porcine epidemic diarrhea Virus
With reference to the method of example 1, concentrated virus solution of porcine epidemic diarrhea virus HN1303 strain (disclosed in Chinese patent application CN106148287A) cultured by Vero cells is put into a medium-speed centrifuge and centrifuged at 8000rpm for 12min to remove cell debris and partial impurities, thus obtaining primary purified virus solution; adding Benzonase nuclease into the virus liquid obtained in the step to treat the virus for 30min, wherein the addition amount is 1.8U/mL; adding EDTA with the final concentration of 2mmol/L to neutralize nuclease; after neutralization, gel chromatography is adopted for fine purification and separation, and the yield after purification is 90%.
And respectively adding the purified virus and the virus which is not purified into beta-propiolactone for inactivation treatment, and emulsifying and culturing the inactivated virus and the alumina gel adjuvant according to the volume ratio of 9: 1. The concrete proportions are shown in Table 6.
TABLE 6 porcine epidemic diarrhea virus vaccine composition ratio
Composition (I) Vaccine 6 (purified) Vaccine 7 (unpurified)
Antigen (TCID) 50 /ml) 10 8.0 10 8.0
Adjuvant content (V/V%) 10 10
Example 6 immunogenicity testing of porcine epidemic diarrhea Virus vaccine compositions
13 heads of PEDV antigen antibody negative piglets aged for 8-10 days are randomly divided into 3 groups, the vaccines are injected according to the table 7, the two times of immunization are carried out at an interval of 2 weeks, and 2ml of PBS is inoculated to a control group.
TABLE 7 immunogenicity test groupings of porcine epidemic diarrhea virus vaccine compositions
Group of Quantity (head) Injectable vaccines Immunization dose
12 5 Vaccine 6 2 ml/head
13 5 Vaccine 7 2 ml/head
14 3 PBS 2 ml/head
Blood was collected weekly beginning 2 weeks after the hyperimmunization and the serum was assayed for PEDV neutralizing antibody titers. The results are shown in Table 8.
TABLE 8 immunogenicity of porcine epidemic diarrhea Virus vaccine compositions test antibody profiles
Figure BDA0002015669900000111
The results show that after the piglets are immunized by the two porcine epidemic diarrhea virus inactivated vaccines, high neutralizing antibodies can be generated, the neutralizing antibodies of all groups of piglets in 3 weeks after immunization are more than 1:32, and the immunity can be maintained for a long time; the vaccine composition prepared from the purified virus antigen has faster generation of neutralizing antibody titer and higher titer.
11 replacement gilts negative for the PEDV antigen antibody were used in this experiment and randomly divided into 3 groups, and the vaccines were injected according to table 9, first-immunised at 7 weeks prenatal, second-immunised at 4 weeks prenatal, and the control group was vaccinated with PBS 2 ml/head.
TABLE 9 porcine epidemic diarrhea virus vaccine composition replacement gilt immunization cohort
Group of Quantity (head) Injectable vaccines Immunization dose
15 4 Vaccine 6 2 ml/head
16 4 Vaccine 7 2 ml/head
17 3 PBS 2 ml/head
Blood was collected weekly beginning 2 weeks after the hyperimmunization and the serum was assayed for PEDV neutralizing antibody titers. The results are shown in Table 10.
TABLE 10 porcine epidemic diarrhea virus vaccine composition replacement gilt immune antibody status
Figure BDA0002015669900000121
The results show that after the replacement gilts are immunized by the two porcine epidemic diarrhea virus inactivated vaccines, high neutralizing antibodies can be generated, the neutralizing antibodies of all groups of piglets in 3 weeks after immunization are larger than 1:32, and the immunity can be maintained for a long time; the vaccine composition prepared from the purified virus antigen has faster generation of neutralizing antibody titer and higher titer.
In summary, the purified antigen of the invention is in the same TCID 50 The content of the produced neutralizing antibody titer is not higher than that of the purified inactivated antigen, because the immune response is influenced by the large molecular impurities and other impurities which are not removed, and the antigen of the invention produces better immune effect by removing the large molecular impurities and other impurities.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (5)

1. A method of purifying a viral antigen, wherein the method of purifying comprises the steps of:
concentrating and pre-treating the virus liquid proliferated in the step (1), and centrifuging at a medium speed to remove macromolecular impurities in the virus liquid proliferated, wherein the centrifugation speed of the medium speed centrifugation is 8000rpm, and the centrifugation time is 12 min;
treating the virus liquid obtained in the step (1) after removing macromolecular impurities with Benzonase nuclease to degrade nucleic acid of host cells; the adding amount of the nuclease is 1.5-2U/mL, and the treatment time of the nuclease is 30 min;
step (3) adding EDTA (ethylene diamine tetraacetic acid) with the concentration of 2mmol/L into the virus liquid treated by the nuclease in the step (2) to inhibit nuclease; and
step (4) purifying and separating the virus liquid of the inhibited nuclease in the step (3) by using gel chromatography to obtain a purified virus antigen;
the virus antigen is porcine reproductive and respiratory syndrome virus or porcine epidemic diarrhea virus.
2. The method for purifying a viral antigen according to claim 1, wherein the nuclease is added in an amount of 1.8U/mL in the step (2).
3. Use of a purification process according to claim 1 or 2 for the preparation of a vaccine composition.
4. Use according to claim 3, wherein the vaccine composition comprises pre-inactivation 10 6.0 ～10 8.0 TCID 50 The inactivated antigen of the porcine reproductive and respiratory syndrome virus NVDC-JXA1 strain and an oil-in-water emulsion adjuvant are/ml; or
The vaccine composition comprises pre-inactivation 10 7.0 ～10 8.0 TCID 50 The inactivated antigen of the porcine epidemic diarrhea virus strain HN1303 and an alumina gel adjuvant are contained in the solution per ml.
5. Use according to claim 3, wherein the vaccine composition comprises pre-inactivation 10 7.0 TCID 50 The inactivated antigen of the porcine reproductive and respiratory syndrome virus NVDC-JXA1 strain and an oil-in-water emulsion adjuvant are/ml; or
The vaccine composition comprises pre-inactivation 10 8.0 TCID 50 The inactivated antigen of the porcine epidemic diarrhea virus strain HN1303 and an alumina gel adjuvant are contained in the solution per ml.
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