Pseudorabies virus inactivated antigen standard substance, preparation method and application thereof, and high performance liquid chromatography detection method for antigen in pseudorabies virus inactivated vaccine

Abstract

The invention provides a pseudorabies virus inactivated antigen standard substance, a preparation method and application thereof, and a high performance liquid chromatography detection method of an antigen in a pseudorabies virus inactivated vaccine, and relates to the technical field of analysis and detection. The invention provides a preparation method of a pseudorabies virus inactivated antigen standard substance, which comprises the following steps: reproducing pseudorabies virus to obtain reproduced virus liquid; sequentially carrying out inactivation, tangential flow purification and concentration and 4FF chromatographic column purification on the breeding virus liquid to obtain a pseudorabies virus inactivated antigen standard substance; an inactivation reagent used for inactivation is diethyleneimine. The prepared pseudorabies virus inactivated antigen standard substance is positive through WB identification, high in purity and good in stability, and accurate quantitative detection of protein concentration in a PRV vaccine semi-finished product or a PRV vaccine can be achieved by taking the pseudorabies virus inactivated antigen standard substance as a standard substance and adopting a high performance liquid chromatography (HPLC) method.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical detection, and particularly relates to a standard product of inactivated pseudorabies virus antigen, a preparation method and application thereof, and a high performance liquid chromatography detection method for antigens in inactivated pseudorabies virus vaccines. Background Art

[0002] Currently, the technologies widely used for virus content detection include real-time fluorescence quantitative PCR method (Real-time fluorescence quantitative polymerase chain reaction, RT-qPCR) and TCID 50 method (Tissue culture infective dose). However, RT-qPCR is a quantification of nucleic acids, and in the production of biological products, it is impossible to determine the content of effective antigen components (proteins). TCID 50 method is used to quantify the number of infectious virus particles, that is, after culturing on a suitable culture system for a certain period of time, the median infective dose of the virus is detected. This method is time-consuming and cannot achieve the determination of the median infective dose for antigens that need to be inactivated in the production of biological products.

[0003] Currently, the semi-finished product antigen of inactivated pseudorabies virus vaccine is measured by TCID 50 method before inactivation, and the effective antigen amount after inactivation cannot be determined. Chinese Patent CN110274982A discloses a quantitative detection method for inactivated rabies virus antigen. First, SDS-PAGE method and TEM method are used for qualitative identification of the target peak; then a protein quantification kit is used to determine the concentration of the standard product; this standard product is serially diluted, and the absorption peak of the standard product at 280 nm is detected by a gel chromatography column on a chromatograph to establish a linear regression equation between the concentration and the peak area; the concentration of inactivated rabies virus protein in the sample to be measured is calculated according to the linear regression equation and the peak area of the sample to be measured. The rabies virus detected by this method is a rhabdovirus with a diameter of about 45 - 100 nm and has 5 main proteins - nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (L). The pseudorabies virus belongs to the herpesvirus, is a large virus with an envelope, has a diameter of 150 - 180 nm, and the mature virus has about 50 proteins. The above method cannot accurately detect the antigen content after inactivation of the pseudorabies virus. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a standard product of inactivated pseudorabies virus antigen, a preparation method and application thereof, and a high performance liquid chromatography detection method for antigens in inactivated pseudorabies virus vaccines. The standard product of inactivated pseudorabies virus antigen prepared by the present invention can accurately detect the antigens in inactivated pseudorabies virus vaccines.

[0005] To achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0006] The present invention provides a preparation method of a pseudorabies virus inactivated antigen standard product, comprising the following steps:

[0007] Propagate the pseudorabies virus to obtain a propagated virus solution;

[0008] Subject the propagated virus solution to inactivation, tangential flow purification and concentration, and 4FF column chromatography purification in sequence to obtain a pseudorabies virus inactivated antigen standard product; the inactivation reagent used for inactivation is divinylimine.

[0009] Preferably, the virus content of the propagated virus solution ≥ 10 6.50 TCID 50 .

[0010] Preferably, the working concentration of divinylimine in the inactivation process is 0.004 mol / L;

[0011] The temperature of the inactivation is 30 °C, and the time is 28 h. Stir once every 1 h during the inactivation process.

[0012] Preferably, after inactivation, inactivation blocking is also included using an aqueous sodium thiosulfate solution.

[0013] Preferably, the tangential flow purification and concentration is hollow fiber ultrafiltration purification and concentration. The conditions for the hollow fiber ultrafiltration purification and concentration include: the molecular weight cut-off is 300 - 500 kD, the membrane area is 790 - 1600 cm 2 , the concentration multiple is 10 - 20 times; the flow rate of the inactivated virus solution is 80 - 120 mL / min; the concentrated solution is washed, filtered and concentrated with a PBS solution;

[0014] The eluent for the 4FF column chromatography purification is a PBS solution.

[0015] The present invention also provides a pseudorabies virus inactivated antigen standard product prepared by the preparation method described in the above technical solution. The protein concentration of the pseudorabies virus inactivated antigen standard product is 5 - 200 μg / mL, and the purity ≥ 90%.

[0016] The present invention also provides the application of the pseudorabies virus inactivated antigen standard product described in the above technical solution in the detection of pseudorabies virus inactivated vaccines.

[0017] The present invention also provides a high performance liquid chromatography detection method for the antigen in a pseudorabies virus inactivated vaccine, comprising the following steps:

[0018] Perform high performance liquid chromatography detection on the to-be-detected pseudorabies virus inactivated vaccine to obtain the chromatographic peak information of the to-be-detected sample;

[0019] Obtain the purity detection result of the antigen in the inactivated pseudorabies virus vaccine according to the chromatographic peak information of the sample to be tested;

[0020] Obtain the detection result of the protein concentration in the inactivated pseudorabies virus vaccine according to the chromatographic peak information of the sample to be tested and the standard curve; the standard curve uses the concentration of the inactivated pseudorabies virus antigen standard product described in the above technical solution as the independent variable and the ultraviolet absorption peak area as the dependent variable.

[0021] Preferably, the conditions for the high performance liquid chromatography detection include: the chromatographic column is a gel filtration column; the column temperature is 25 °C; the globulin separation range is 2×10 4 ~7×10 6 D; the mobile phase is a phosphate buffer solution, the sample temperature is 4 °C; the injection volume is 100 μL; the detection wavelength is 280 nm; the running time is 35 min.

[0022] Preferably, the solutes in the mobile phase include disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium sulfate; the concentration of disodium hydrogen phosphate in the phosphate buffer solution is 42 mmol / L, the concentration of sodium dihydrogen phosphate is 8 mmol / L, and the concentration of sodium sulfate is 100 mmol / L.

[0023] The present invention provides a preparation method of an inactivated pseudorabies virus antigen standard product, which includes the following steps: propagating pseudorabies virus to obtain a propagated virus solution; sequentially inactivating, tangential flow purifying and concentrating, and purifying with a 4FF chromatography column the propagated virus solution to obtain an inactivated pseudorabies virus antigen standard product; the inactivating reagent used for inactivation is diethyl imine. The inactivated pseudorabies virus antigen standard product prepared by the present invention is identified as positive by WB, has high purity and good stability. Taking it as a standard product, the accurate quantitative detection of the protein concentration in the semi-finished product of the PRV vaccine or the PRV vaccine can be realized by using the high performance liquid chromatography (HPLC) method.

[0024] As shown by the test results of the examples, the inactivated pseudorabies virus antigen standard product prepared by the present invention (denoted as PRV standard product) is identified as positive by WB, the protein concentration is 160 μg / mL, the purity is 92.44%, and after being stored at 2-8 °C for 3 months, the protein concentration of the standard product is 81% of the initial concentration of storage, and the purity remains basically unchanged. Therefore, it can be used as a standard product for testing the protein concentration of the PRV semi-finished product.

[0025] Using the inactivated pseudorabies virus antigen standard product prepared by the present invention, the accurate detection of the antigen in the inactivated acid pseudorabies virus vaccine can be realized by using the high performance liquid chromatography (HPLC) detection method, and the HPLC detection method provided by the present invention has high accuracy, good stability and strong operability. Description of the Drawings

[0026] Figure 1 It is the identification result diagram of the PRV standard product by the WB method. Among them, M: Marker (10 - 180 kDa), 1 - 3: PRV standard product;

[0027] Figure 2 It is the HPLC purity detection result diagram of the PRV standard product;

[0028] Figure 3 It is the HPLC detection result diagram of the PRV standard product stored for 3 months;

[0029] Figure 4 It is the HPLC chromatogram corresponding to different sample loading volumes of the PRV standard product;

[0030] Figure 5 It is the standard curve diagram of the PRV standard product;

[0031] Figure 6 It is the HPLC chromatogram for the repeatability verification of the PRV standard product. Detailed implementation mode

[0032] The present invention provides a preparation method of a pseudorabies virus inactivated antigen standard product, including the following steps:

[0033] Propagate the pseudorabies virus to obtain a propagated virus solution;

[0034] Inactivate, tangential flow purify and concentrate, and purify through a 4FF chromatography column the propagated virus solution to obtain a pseudorabies virus inactivated antigen standard product; the inactivating reagent used for inactivation is divinylimine.

[0035] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0036] The present invention propagates the pseudorabies virus to obtain a propagated virus solution.

[0037] In the present invention, the pseudorabies virus preferably includes the rPRV - 3 strain.

[0038] In the present invention, the propagation preferably includes: inoculating the working seed of the pseudorabies virus into sensitive cells containing a maintenance solution for propagation. In the present invention, the sensitive cells preferably include one or more of baby hamster kidney cells (BHK - 21 cells), porcine kidney cells (PK15 cells), and porcine testicular cells (ST cells); the animal cells are preferably monolayer animal cells. In the present invention, the inoculation amount of the pseudorabies virus is preferably 1 v / v% of the maintenance solution. In the present invention, the maintenance solution is preferably 2% fetal bovine serum BMEM cell nutrient solution. The present invention preferably harvests the propagated virus solution when the animal cell lesions reach more than 75%.

[0039] In the present invention, the virus content of the propagated virus solution is preferably ≥ 10 6.50 TCID 50 . In the present invention, the test method for the virus content preferably includes the Reed-Muench method.

[0040] After obtaining the propagated virus solution, the present invention sequentially inactivates, tangentially flow purifies and concentrates, and purifies with a 4FF chromatography column the propagated virus solution to obtain a standard inactivated antigen of pseudorabies virus; the inactivating reagent used for inactivation is beta-propiolactone.

[0041] In the present invention, the inactivation preferably includes: mixing the propagated virus solution with an aqueous sodium hydroxide solution of beta-propiolactone (BEI), and inactivating the obtained mixture.

[0042] In the present invention, the working concentration of beta-propiolactone during the inactivation process (i.e., the concentration of BEI in the mixture) is preferably 0.004 mol / L.

[0043] In the present invention, the temperature for inactivation is preferably 30 °C, the time is preferably 28 h, and it is preferably stirred once every 1 h during the inactivation process.

[0044] In the present invention, the preparation method of the aqueous sodium hydroxide solution of beta-propiolactone preferably includes the following steps: mixing 2-bromoethylamine hydrobromide (BEA) with an aqueous sodium hydroxide solution, and performing cyclization to obtain the aqueous sodium hydroxide solution of beta-propiolactone. In the present invention, the concentration of 2-bromoethylamine hydrobromide in the aqueous sodium hydroxide solution is preferably 4 g / mL. In the present invention, the concentration of the aqueous sodium hydroxide solution is preferably 0.2 mol / L. In the present invention, the temperature for cyclization is preferably 37 °C, the time is preferably 1 h; it is preferably shaken once every 10 - 15 min during the cyclization process; after cyclization, it preferably further includes subjecting the obtained cyclized system to sterile filtration to obtain the aqueous sodium hydroxide solution of beta-propiolactone.

[0045] After the inactivation, the present invention preferably further includes inactivating and blocking with an aqueous sodium thiosulfate solution. Specifically, mixing the inactivated system with an aqueous sodium thiosulfate solution, and inactivating and blocking the obtained mixture to obtain an inactivated virus solution. In the present invention, the concentration of the aqueous sodium thiosulfate solution is preferably 50 g / mL; the concentration of the aqueous sodium thiosulfate solution in the mixture is preferably 2 v / v%. In the present invention, it is preferably sampled for inactivation inspection during the inactivation and blocking process, that is, the sensitive cells inoculated with the inactivated solution are blindly passaged 3 times, and no specific lesions caused by pseudorabies virus should appear.

[0046] It should be noted that there is an error in the original text. The inactivating reagent mentioned in item should be "beta-propiolactone" instead of "diethylenimine". The above translation has been corrected accordingly.In the present invention, the tangential flow purification and concentration is hollow fiber ultrafiltration purification and concentration. The conditions for the hollow fiber ultrafiltration purification and concentration preferably include: the cut-off molecular weight is 300 - 500 kD, which can be 300 - 500 kD in specific embodiments, and can be 300 kD, 350 kD, 400 kD, 450 kD or 500 kD in specific embodiments; the membrane area is 790 - 1600 cm 2 , which can be 790 cm 2 , 800 cm 2 , 900 cm 2 , 1000 cm 2 , 1100 cm 2 , 1200 cm 2 , 1300 cm 2 , 1400 cm 2 , 1500 cm 2 or 1600 cm 2 ; the concentration multiple is 10 - 20 times, which can be 10 times, 12 times, 14 times, 15 times, 16 times, 18 times or 20 times in specific embodiments; the flow rate of the inactivated virus solution is 80 - 120 mL / min, which can be 80 mL / min, 90 mL / min, 100 mL / min, 110 mL / min or 120 mL / min in specific embodiments; the concentrated solution is washed, filtered and concentrated with PBS solution, and the number of times of the PBS solution washing, filtering and concentrating is 3 - 5 times, which can be 3 times, 4 times or 5 times in specific embodiments; the pH value of the PBS solution is preferably 7.2 - 7.4, which can be 7.2, 7.3 or 7.4 in specific embodiments; the PBS solution preferably includes sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and water; the sodium dihydrogen phosphate preferably includes disodium hydrogen phosphate dodecahydrate; the concentration of sodium dihydrogen phosphate in the PBS solution is preferably 34 mM (mmol / L), the concentration of potassium dihydrogen phosphate is preferably 6 mM, and the concentration of sodium chloride is preferably 145 mM.

[0047] In the present invention, the PBS solution washing, filtering and concentrating preferably includes: adding PBS solution to the concentrated solution and shaking well, then continuing the concentration to obtain the PBS washed, filtered and concentrated solution, and repeating the above steps 2 - 4 times to obtain the tangential flow purified and concentrated solution. In the present invention, the volume ratio of the concentrated solution to the PBS solution used for single PBS solution washing, filtering and concentrating is preferably 1:1.

[0048] In the present invention, the tangential flow purification and concentration is preferably carried out in a hollow fiber ultrafiltration concentration system. In the present invention, before using the hollow fiber ultrafiltration concentration system, it is preferably subjected to airtightness detection and disinfection treatment. The specific operations preferably include: zeroing the system pump pressure, installing the hollow fiber column, cleaning the instrument pipeline and the fiber column with ultrapure water for 10 - 30 min (in specific embodiments, it can be 10 min, 15 min, 20 min, 25 min or 30 min), exposing the inlet end to the air, sucking air into the liquid inlet pipe to drain the water in the pipeline and the hollow fiber column, clamping the return end pipeline with a hemostat. When the inlet pressure is 0.05 - 0.1 MPa (in specific embodiments, it can be 0.05 MPa, 0.06 MPa, 0.07 MPa, 0.08 MPa, 0.09 MPa or 0.1 MPa), stop the pump. Observe that there are no bubbles in the permeate end pipeline and the inlet end pressure is stable without dropping, indicating that the airtightness of the whole system is good and virus concentration can be carried out; place the inlet end, return end and permeate end pipelines in a blue mouth bottle containing 1 - 3 L (in specific embodiments, it can be 1 L, 1.5 L, 2 L, 2.5 L or 3 L) of 0.5 M NaOH aqueous solution, and circulate the whole system for 30 - 60 min (in specific embodiments, it can be 30 min, 40 min, 50 min or 60 min) for pre-concentration cleaning and disinfection treatment.

[0049] In the present invention, the tangential flow purification and concentration carried out in the hollow fiber ultrafiltration concentration system preferably includes the following steps: put the inlet end into the PBS solution, and at the same time, put the return end and the permeate end into the waste liquid tank. Rinse the system with the PBS solution until the pH of the permeate end is 7.2 - 7.4. Then place the inlet end and the return end in the inactivated virus liquid to be concentrated, set the flow rate to 80 - 120 mL / min, and concentrate the sample 10 - 20 times to obtain the concentrated liquid. Add an equal volume of PBS solution to the concentrated liquid and shake well, and continue to concentrate until the volume is the same as that of the concentrated liquid to obtain the PBS washing and filtering concentrated liquid. Repeat the operation of adding an equal volume of PBS solution and shaking well, and continue to concentrate until the volume is the same as that of the concentrated liquid 2 - 4 times, and collect the obtained tangential flow purified concentrated liquid.

[0050] After the tangential flow purification and concentration are completed, the present invention preferably further includes the following operations on the used hollow fiber ultrafiltration and concentration system: washing the hollow fiber column and the system with water for 10 - 30 min (which can be 10 min, 15 min, 20 min, 25 min or 30 min in specific embodiments), then placing the inlet end, reflux end, and permeate end pipelines in a blue mouth bottle containing 1 - 3 L (which can be 1 L, 1.5 L, 2 L, 2.5 L or 3 L) of 0.5 M NaOH aqueous solution, circulating the entire system for 10 - 30 min (which can be 10 min, 15 min, 20 min, 25 min or 30 min in specific embodiments) for post - concentration disinfection treatment, and finally storing the hollow fiber column and the system with 0.1 mol / L NaOH aqueous solution.

[0051] In the present invention, the eluent for the 4FF chromatography column purification is preferably PBS solution, and the PBS solution preferably includes sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and water; the sodium dihydrogen phosphate preferably includes sodium dihydrogen phosphate dodecahydrate; the concentration of sodium dihydrogen phosphate in the PBS solution is preferably 34 mM (mmol / L), the concentration of potassium dihydrogen phosphate is preferably 6 mM, and the concentration of sodium chloride is preferably 145 mM; the pH value of the PBS solution is preferably 7.2 - 7.4, which can be 7.2, 7.3 or 7.4 in specific embodiments; the dosage of the PBS solution is preferably 1 column volume; during the 4FF chromatography column purification process, when the UV signal of the first peak ≥ 20 mAU, start collecting the elution sample to obtain the inactivated pseudorabies virus antigen standard product (containing PBS solution).

[0052] In the present invention, before using the 4FF chromatography column, it is preferably rinsed with PBS solution until the pH = 7.2 - 7.4 and the PBS solution rinsing is stopped when the UV baseline is flat; the flow rate of the PBS solution is preferably 2 mL / min, and the dosage of the PBS solution is preferably 2 - 3 column volumes; the PBS solution preferably includes sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and water; the sodium dihydrogen phosphate preferably includes sodium dihydrogen phosphate dodecahydrate; the concentration of sodium dihydrogen phosphate in the PBS solution is preferably 34 mM (mmol / L), the concentration of potassium dihydrogen phosphate is preferably 6 mM, and the concentration of sodium chloride is preferably 145 mM; the pH value of the PBS solution is preferably 7.2 - 7.4, which can be 7.2, 7.3 or 7.4 in specific embodiments.

[0053] After the 4FF chromatography column is completed, the present invention preferably further includes: rinsing with purified water for 1.5 column volumes, rinsing with 20 v / v% ethanol aqueous solution for 2 column volumes, and storing the 4FF chromatography column in 20 v / v% ethanol aqueous solution.

[0054] The present invention also provides a standard product of inactivated pseudorabies virus antigen prepared by the preparation method described in the above technical solution. The protein concentration of the standard product of inactivated pseudorabies virus antigen is 5-200 μg / mL, and in specific embodiments, it can be 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, 150 μg / mL, 180 μg / mL or 200 μg / mL; the purity of the standard product of inactivated pseudorabies virus antigen is preferably ≥90%.

[0055] The present invention also provides the application of the standard product of inactivated pseudorabies virus antigen described in the above technical solution in the detection of inactivated pseudorabies virus vaccine.

[0056] The present invention also provides a high performance liquid chromatography detection method for antigens in an inactivated pseudorabies virus vaccine, comprising the following steps:

[0057] Performing high performance liquid chromatography detection on the to-be-detected inactivated pseudorabies virus vaccine to obtain chromatographic peak information of the to-be-detected sample;

[0058] Obtaining a purity detection result of the antigen in the inactivated pseudorabies virus vaccine according to the chromatographic peak information of the to-be-detected sample;

[0059] Obtaining a detection result of the protein concentration in the inactivated pseudorabies virus vaccine according to the chromatographic peak information of the to-be-detected sample and a standard curve; the standard curve uses the concentration of the standard product of inactivated pseudorabies virus antigen described in the above technical solution as the independent variable and the ultraviolet absorption peak area as the dependent variable.

[0060] The present invention performs high performance liquid chromatography detection on the to-be-detected inactivated pseudorabies virus vaccine to obtain chromatographic peak information of the to-be-detected sample.

[0061] In the present invention, the inactivated pseudorabies virus vaccine preferably includes a semi-finished product of inactivated pseudorabies virus vaccine or a finished product of inactivated pseudorabies virus vaccine. In the present invention, the inactivated pseudorabies virus vaccine preferably includes an inactivated vaccine with pseudorabies gE gene deletion, and the pseudorabies virus preferably includes rPRV-3 strain.

[0062] In the present invention, the conditions for the high performance liquid chromatography detection preferably include: the chromatographic column is a gel filtration column, and the gel filtration column is preferably a TOSOH TSKgel4000 SWXL high performance liquid chromatography analysis column; the column temperature is 25°C; the globulin separation range is 2×10 4 ~7×10 6D; The mobile phase is phosphate buffer solution, the sample temperature is 4 °C; the injection volume is 100 μL; the detection wavelength is 280 nm; the running time is 35 min. In the present invention, the solutes in the mobile phase preferably include disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium sulfate; the sodium dihydrogen phosphate preferably includes sodium dihydrogen phosphate dihydrate; the concentration of disodium hydrogen phosphate in the phosphate buffer solution is 42 mM, the concentration of sodium dihydrogen phosphate is 8 mM, and the concentration of sodium sulfate is 100 mM.

[0063] After obtaining the chromatographic peak information of the sample to be tested, the present invention obtains the purity detection result of the antigen in the inactivated pseudorabies virus vaccine according to the chromatographic peak information of the sample to be tested. Specifically, the ratio of the peak area of the target substance (antigen) to the total chromatographic peak area of the sample to be tested is the purity detection result of the antigen in the inactivated pseudorabies virus vaccine.

[0064] After obtaining the chromatographic peak information of the sample to be tested, the present invention obtains the detection result of the protein concentration in the inactivated pseudorabies virus vaccine according to the chromatographic peak information of the sample to be tested and the standard curve; the standard curve uses the concentration of the inactivated pseudorabies virus antigen standard product described in the above technical solution as the independent variable and the ultraviolet absorption peak area as the dependent variable.

[0065] In the present invention, the method for obtaining the standard curve preferably includes the following steps: performing high performance liquid chromatography detection on a series of concentrations of inactivated pseudorabies virus antigen standard products under the conditions of the above high performance liquid chromatography detection, using the concentration of the inactivated pseudorabies virus antigen standard product solution as the independent variable and the ultraviolet absorption peak area as the dependent variable, and drawing a standard curve. In a specific embodiment of the present invention, the injection volumes of the series of concentrations of inactivated pseudorabies virus antigen standard products are preferably 5 μL, 10 μL, 20 μL, 40 μL, 60 μL, 80 μL or 100 μL of inactivated pseudorabies virus antigen standard product with a concentration of 160 μg / mL in sequence.

[0066] In order to further illustrate the present invention, the following examples will be used to describe in detail the inactivated pseudorabies virus antigen standard product provided by the present invention and its preparation method and application, and the high performance liquid chromatography detection method for the antigen in the inactivated pseudorabies virus vaccine, but they cannot be understood as limiting the protection scope of the present invention.

[0067] Example 1

[0068] Preparation and Identification of Inactivated Pseudorabies Virus Antigen Standard Product (PRV Standard Product)

[0069] 1 Materials

[0070] The BCA protein concentration detection kit was purchased from Thermp Scientific, with the product number 23227. The reagent Na2HPO4·2H2O, product number S886003-100g; anhydrous NaH2PO4, product number S817785-250g; KH2PO4, product number P816465-250g; all were purchased from MACKLIN. Na2SO4, product number S112276-500g, was purchased from aladdin. The PRV monoclonal antibody was purchased from Shandong Hongdu Biotechnology Co., Ltd., with the product number HD125210220. The rabbit anti-mouse secondary antibody was purchased from Sigma Company of the United States, with the product number A9044. Maintenance solution: 2% fetal bovine serum BMEM cell nutrient solution. PBS solution: The concentration of NaH2PO4·12H2O was 34 mM, the concentration of KH2PO4 was 6 mM, the concentration of NaCl was 145 mM, and pH = 7.2 - 7.4.

[0071] 1.3 Instruments

[0072] 1.3.1 High performance liquid chromatography: Purchased from waters company, the UV detector model is 2489, and the separation component model is 2695.

[0073] 1.3.2 Gel filtration chromatography column: The model is TSKgel G4000SWXL, and the column specification is 7.8×300 mm, purchased from Tosoh (Shanghai) Biotechnology Co., Ltd.

[0074] 2 Preparation of PRV standard

[0075] 2.1 Virus propagation

[0076] Take the rPRV-3 strain working seed, inoculate the well-grown monolayer BHK-21 cells with the maintenance solution (2% fetal bovine serum BMEM cell nutrient solution) at 1 v / v%, incubate at 36.5 ± 0.5 °C for 24 - 48 h, observe the cytopathic effect. When the cytopathic effect reaches more than 75%, harvest the culture to obtain the propagated virus solution.

[0077] 2.2 Virus content (TCID 50 determination)

[0078] Dilute the above propagated virus solution serially 10-fold with the maintenance solution, take 10 -5 、10 -6 、10 -7 、10 -84 dilutions were set, and each dilution was inoculated into a 96-well cell culture plate, 100 μL per well, and 4 wells were inoculated for each dilution, and then 100 μL of cell solution was added to each well. At the same time, 4 wells with normal cells were set as controls, and 100 μL of cell solution was added to each well, and then 100 μL of maintenance solution was added. The cells were cultured in an incubator at 36.5±0.5℃ and 5% CO2 for 72-120 hours, and the cell lesions were observed under a microscope. The number of cell lesions at each dilution was recorded, and the TCID was calculated according to the Reed-Muench method. 50 The virus content in each 0.1mL of virus propagation liquid should not be less than 10 6.50 TCID 50 .

[0079] 2.3 Inactivation

[0080] Cyclization of 2-bromoethylamine hydrobromide (BEA): Add 4 g / mL of BEA to 0.2 mol / L sodium hydroxide solution, place in a 37°C water bath, shake once every 10-15 minutes, and act for 60 minutes to obtain a cyclized solution of diethylenimine (BEI). Sterile filter and set aside.

[0081] Inactivation: Stir the BEI solution and the propagation virus solution thoroughly to obtain a mixed solution (the final concentration of BEI is 0.004 mol / L), invert the can, and start timing when the temperature rises to 30°C. Inactivate for 28 hours, stirring once every 60 minutes.

[0082] Inactivation blocking: After the timing is over, add the filtered and sterilized 50g / mL sodium thiosulfate aqueous solution to the inactivated virus solution to make the final concentration of the sodium thiosulfate aqueous solution 2v / v%. After sufficient mixing, take samples for inactivation test at the same time to obtain the inactivated virus solution.

[0083] 2.4 Purification and concentration: After the inactivated virus solution is purified and concentrated by tangential flow and purified by 4FF chromatography column, the flow-through sample is collected to obtain the PRV standard product. The operation is as follows:

[0084] Tangential flow purification and concentration: The inactivated blocked virus solution was purified and concentrated using a 500 kD hollow fiber ultrafiltration system (membrane area of ​​790 cm 2)Perform 10-fold concentration. The specific operations are as follows: First, zero the pressure of the system pump. Then, install the hollow fiber column according to the pipeline sequence, and wash the instrument pipeline and the fiber column with ultrapure water for 10 min. Next, expose the inlet end to the air, and suck air into the liquid inlet pipe to drain the water in the pipeline and the hollow fiber column. Clamp the return pipeline with a hemostat. When the inlet pressure is 0.05 - 0.1 MPa, stop the pump. Observe that there are no bubbles in the permeate pipeline and the inlet pressure is stable without dropping, indicating that the airtightness of the entire system is good and virus concentration can be carried out. Then, place the inlet pipeline, the return pipeline, and the permeate pipeline into a blue-mouth bottle containing 1 L of 0.5 M NaOH aqueous solution, and circulate the entire system for 30 - 60 min for pre-concentration cleaning and disinfection treatment. Then, put the inlet end into the PBS solution, and at the same time, put the return end and the permeate end into the waste liquid tank, and rinse the system with PBS until the pH of the permeate end is 7.2 - 7.4. Then, place the inlet end and the return end into 1 L of inactivated blocking virus solution to be concentrated, set the flow rate to 80 mL / min. When the sample is concentrated to 100 mL, add an equal volume of PBS solution, shake well. When it is concentrated to 100 mL again, the operations of adding an equal volume of PBS solution, shaking well, and concentrating to 100 mL are carried out 3 times in total, and collect the obtained tangential flow purified and concentrated virus solution. Then, wash the hollow fiber column and the system with water for 10 min. Next, place the inlet pipeline, the return pipeline, and the permeate pipeline into a blue-mouth bottle containing 1 L of 0.5 M NaOH aqueous solution, and circulate the entire system for 30 min for post-concentration disinfection treatment. Finally, store the hollow fiber column and the system with 0.1 mol / L NaOH aqueous solution.

[0085] Purification by 4FF chromatography column: Wash the 4FF chromatography column (Bogolong, 16×85 cm, column volume 170 mL) with PBS solution at a flow rate of 2 mL / min for 2 - 3 column volumes until the pH = 7.2 - 7.4, and at the same time, require the UV baseline to be flat. Add 8 mL of tangential flow purified and concentrated virus solution, and perform elution with PBS solution at a flow rate of 2 mL / min. Observe the UV signal. When the UV signal of the first peak ≥ 20 mAU, start to collect the elution sample solution, and this sample solution is the PRV standard product. After PBS elutes for 1 column volume, then wash with purified water for 1.5 column volumes, and finally wash with 20 v / v% ethanol aqueous solution for 2 column volumes, and store the system in 20 v / v% ethanol aqueous solution.

[0086] Detection of 3 PRV standard products

[0087] 3.1 Protein identification: Weigh 10 g of sucrose, 5 g of sodium dodecyl sulfate (SDS), 0.05 g of bromophenol blue, 2.5 mL of 1 M Tris-HCl solution (pH = 8.0), and 2.5 mL of mercaptoethanol into a 50 mL centrifuge tube. Add purified water to 50 mL, mix well to dissolve, and obtain a 5× reducing loading buffer. Mix 30 μL of the PRV standard with 8 μL of the 5× reducing loading buffer evenly, place it in a 100 °C metal bath for 10 min for WB detection. The primary antibody is a PRV monoclonal antibody, and the secondary antibody is a rabbit anti-mouse secondary antibody.

[0088] Figure 1 The identification result of the PRV standard (WB method), where M: Marker (10 - 180 kDa), 1 - 3: PRV standard. From Figure 1 it can be seen that the test result is positive.

[0089] 3.2 Concentration detection: Use a BCA protein concentration detection kit (operate according to the kit instructions). Two laboratory testers repeat the detection 3 times at different times, and all the detection results are averaged as the fixed concentration. The results are shown in Table 1, and the concentration of the PRV standard is finally determined to be 160 μg / mL.

[0090] Table 1 Results of the PRV standard concentration detection (BCA method)

[0091]

[0092] 3.3 Purity detection: The antigen purity of the PRV standard is determined by HPLC method.

[0093] 3.3.1 Preparation of the mobile phase (42 mM Na2HPO4·2H2O, 8 mM NaH2PO4, 100 mM Na2SO4): Weigh 7.48 g of Na2HPO4·2H2O, 0.96 g of NaH2PO4, and 14.2 g of Na2SO4 accurately with a ten-thousandth balance into a sample bottle, add an appropriate amount of ultrapure water to dissolve, adjust the pH to 7.2 with KH2PO4 and make up the volume to 1 L.

[0094] Filter through a 0.45 μm filter and sonicate for 10 min.

[0095] 3.3.2 Sample preparation: Take 200 μL of the PRV standard and place it in a chromatographic vial containing an inner insert tube.

[0096] 3.3.3 Instrument operation

[0097] 3.3.3.1 Turn on the switches of the computer, high-performance liquid chromatograph, ultraviolet detector, column oven, etc. in sequence, wait for the instrument to warm up and the self-check to end.

[0098] 3.3.3.2 Clean the system

[0099] 3.3.3.2.1 Place Pump A in the freshly prepared mobile phase and Pump B in fresh ultrapure water. Click Menu on the operation panel, (set the degasser to on and the Col Htr to off), set Pump A to 100%, select Direct function, wetprime, and displace the old mobile phase in Pump A; set Pump B to 100%, select Direct function, wetprime, and displace the ultrapure water in Pump B.

[0100] 3.3.3.2.2 Set Pump B to 100% and the flow rate to 1 mL / min. Flush the entire system with ultrapure water for 15 min, then change to Pump A at 100% and flush the system with the mobile phase for 10 min.

[0101] 3.3.3.3 Column Connection and System Equilibration

[0102] Connect the chromatographic column: Change the flow rate to 0.5 mL / min. Connect the TSK SWxl guard column and the TSK G4000 SWxl analytical column in sequence from front to back according to the direction on the analytical column, and equilibrate for 30 - 50 min until the baseline is stable. The baseline stability can be checked by clicking "Monitor" after opening the software.

[0103] 3.3.3.4 Injection and Data Acquisition Method Editing

[0104] Select the newly created instrument method, select System HPLC 2489 - 2695, and set:

[0105] General - Needle depth: 2 mm;

[0106] Flow - Upper limit: 400 psi, pump mode: isocratic, total flow 0.5, 100% A;

[0107] Temperature - Column temperature 25 °C, sample temperature: 4 °C;

[0108] Mobile phase flow rate - 0.5 mL / min;

[0109] W2498 - Detection wavelength: 280 nm.

[0110] 3.3.3.5 Save the Newly Created Instrument Method

[0111] 3.3.3.6 Sample Group Method Editing

[0112] Create a new sample group and set respectively: Injection volume: 100 μL; Injection times: 1 time; Input of sample name, function setting, method group: PRV, running time: 35 min.

[0113] 3.3.3.7 Save the newly created sample group and start running the samples. When the sample group needs to be changed, right-click and select "Modify Current Sample Group Information". After the modification is completed, click "Save", and then click "Run".

[0114] 3.3.3.8 Sample analysis

[0115] After the determination is completed, open the file results at 280 nm, and select the peak area for integration near the tangents on both sides of the absorption peak.

[0116] 3.3.3.9 Chromatographic column preservation and system post-treatment

[0117] After the detection is completed, remove the chromatographic column and the guard column from back to front in sequence, plug them with stoppers, and store them at room temperature. Replace the mobile phase with ultrapure water, rinse the entire system at 0.5 mL / min for 30 min, and then rinse with 10% methanol for 30 min, and then turn off the instrument and the computer.

[0118] Calculate the purity based on the proportion of the target protein peak area to all peak areas. The results are shown in Table 2 and Figure 2 , from Table 2 and Figure 2 it can be seen that the purity of the PRV standard is 92.44%.

[0119] Table 2 HPLC detection results of PRV standard

[0120] Peak Retention Time / min Peak Area Area / % Peak Height 1 9.682 401 0.06 17 2 10.825 621337 92.44 15598 3 24.792 50448 7.51 494

[0121] 3.4 Stability detection: Store the PRV standard at 2 - 8 °C, and perform protein concentration and antigen purity detection on the standards stored for 0 months and 3 months respectively. The results are shown in Table 3 and Figure 3 , Figure 3 the peak information of

[0122] Table 3 Stability detection results of PRV standard

[0123] Storage Time (months) Concentration (μg / mL) Purity (%) 0 160 mL 92.44 3 129 mL 86.33

[0124] Table 4 Comprehensive HPLC component table

[0125]

[0126]

[0127] 4 Conclusion

[0128] The purified and concentrated PRV standard product was identified as positive by WB, with a protein concentration of 160 μg / mL and a purity of 92.44%. The results showed that after being stored at 2-8 °C for 3 months, the protein concentration of the standard product was 81% of the initial concentration, and the purity remained basically unchanged. Therefore, it can be used as a standard product for testing the protein concentration of the PRV semi-finished product.

[0129] Example 2

[0130] Establishment of a method for testing the semi-finished product of pseudorabies virus (HPLC)

[0131] 1 Materials

[0132] 1.1 The standard product for testing is the PRV concentrated and purified antigen, the PRV standard product prepared in Example 1.

[0133] 1.2 Instruments

[0134] 1.2.1 High performance liquid chromatography instrument, purchased from Waters.

[0135] 1.2.2 The chromatographic column model is TSKgel G4000SWXL, the column specification is 7.8×300 mm, and the separation range is globulin 2×10 4 ~7×10 6 D, purchased from Tosoh (Shanghai) Biotech Co., Ltd.

[0136] 1.3 Reagents Na2HPO4·2H2O, product number S886003-100g; anhydrous NaH2PO4, product number S817785-250g; KH2PO4, product number P816465-250g; all purchased from MACKLIN. Na2SO4, product number S112276-500g, purchased from aladdin.

[0137] 2 Methods and test results

[0138] 2.2 Establishment of the PRV standard curve

[0139] The injection volumes were 5 μL, 10 μL, 20 μL, 40 μL, 60 μL, 80 μL, and 100 μL in sequence. Other operations were the same as those in 3.3 Purity detection in Example 1.

[0140] The PRV standard product with a BCA quantitative detection of 160 μg / mL was analyzed by loading samples with different volumes. The loading volumes were 5 μL, 10 μL, 20 μL, 40 μL, 60 μL, 80 μL, and 100 μL in sequence. Taking the loading volume as the abscissa and the peak area as the ordinate, a standard curve was drawn. The results of the standard curves drawn in different loading volume ranges are shown in Table 5, Figure 4 and Figure 5 , Figure 4The heights of the middle peaks correspond to injection volumes of 5 μL, 10 μL, 20 μL, 40 μL, 60 μL, 80 μL, and 100 μL in ascending order from low to high. It can be seen that the R 2 value of the standard curve is 0.999, and R 2 > 0.99, indicating good linearity and good repeatability, which shows that the standard curve is successfully established.

[0141] Table 5 Areas corresponding to PRV chromatographic peaks

[0142] Sample Loading Volume / μL 5 10 20 40 60 80 100 Peak Area 38512 84286 183344 391186 621337 865015 1058885

[0143] 2.3 Repeatability verification of the HPLC detection method

[0144] The PRV standard product with a protein concentration of 160 μg / mL was repeatedly injected 3 times with an injection volume of 80 μL, and repeatability verification was carried out according to the method in 2.1, and the relative standard deviation (RSD) was calculated. The results are shown in Table 6 and Figure 6 , and it can be seen that the peak emergence time and peak area are basically consistent. The RSD of the peak area is 1.1%, and the RSD of the retention time is 0.2%, indicating that the detection method provided by the present invention has good repeatability.

[0145] Table 6 Chromatographic peak areas corresponding to different injection times in repeatability verification

[0146] Retention Time / min Peak Area Peak Height Peak 1 10.876 814093 19398 Peak 1 10.834 831607 19016 Peak 1 10.831 824684 18824 Average 823461.169 19079.073 Standard Deviation 8820.575 292.263 RSD / % 1.1 1.5

[0147] 3 Conclusion

[0148] 3.1 Identification of standard product purity: The purity of the previously prepared PRV standard product with a protein concentration of 160 μg / mL was determined by HPLC to reach 92.44%, which can be used for the establishment of the standard curve.

[0149] 3.2 Establishment of the standard curve: The results show that the R 2 value of the standard curve is 0.999, and R 2 > 0.99, indicating good linearity and good repeatability, which shows that the present invention successfully established the PRV standard curve.

[0150] 3.3 Establishment of the HPLC method: The repeatability test confirmed that the HPLC detection method has high accuracy, good stability and strong operability, and can be used as a detection method for the antigen content and protein concentration of the semi-finished or finished product of the inactivated pseudorabies virus vaccine.

[0151] Example 3

[0152] Research report on the formulation of the quality control standards (protein concentration and purity) for the semi-finished product of the inactivated pseudorabies gE gene-deleted vaccine (rPRV-3 strain, suspension culture)

[0153] 1 Materials

[0154] 1.1 Main equipment: FerMac 320 cell reactor (10L), purchased from Electrolab Company in the UK; purification and concentration system: VERSAflux benchtop membrane filtration system, 500KD hollow fiber column, both purchased from GE Company in the US; high-speed refrigerated centrifuge, purchased from Thermo Fisher Scientific Co., Ltd.

[0155] 1.2 Cells: BHK21 suspension cells, prepared, identified and preserved by Jinyu Baoling Pharmaceutical Co., Ltd.

[0156] 1.3 Seed virus: Pseudorabies rPRV-3 strain, F18 generation, virus content 10 7.5 TCID 50 / 0.1mL.

[0157] 1.4 Culture medium: Cell nutrient solution B21-JY, purchased from Invitrogen (Shanghai) Trading Co., Ltd.; virus maintenance medium B21-JY, purchased from Invitrogen (Shanghai) Trading Co., Ltd.

[0158] 1.5 Newborn bovine serum: Purchased from Inner Mongolia Jinyuankang Bioengineering Co., Ltd. After inspection, the PRV serum neutralizing antibody is not higher than 1:4, the gB antibody ELASA test is negative, and the PRV antigen test is negative.

[0159] 1.6 Other chemical reagents: Inactivator 2-bromoethylamine hydrobromide (BEA) purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., PEG6000 purchased from BBI Life Sciences. PBS solution: The concentration of NaH2PO4·12H2O is 34 mM, the concentration of KH2PO4 is 6 mM, the concentration of NaCl is 145 mM, pH = 7.2 - 7.4.

[0160] 2 Methods

[0161] 2.1 Manufacture of five batches of semi-finished products prepared by laboratory processes

[0162] 2.1.1 Preparation of virus solution in 10L reactor For each batch, 1 - 2 vials of suspended BHK-21 seed cells are resuscitated. After centrifugation, the cells are added to the cell culture medium and transferred to a shake flask. Suspension culture is carried out in a 5% CO2 incubator at a temperature of 36.5 ± 0.5 °C. When the cell concentration reaches more than 2.5×10 6 cells / mL, stepwise expansion culture of suspended cells is carried out, and the cell state is observed regularly. When the number of suspended cells reaches 2.0×10 6When the concentration is 0 cells / mL, inoculate 1 v / v% of the production seed virus maintenance solution into the suspended BHK-21 cells, set the culture parameters (pH = 7.0, DO value = 40%, stirring speed is 50 r / min), and perform suspension culture at a temperature of 36.5 ± 0.5 °C. Observe under the microscope that the cell viability is less than 50% or harvest the propagated virus solution after culturing for 48 - 60 h.

[0163] 2.1.2 Inactivation: The operation is the same as "2.3 Inactivation" in Example 1 to obtain the inactivated virus solution.

[0164] 2.1.3 Concentration and purification: According to the tangential flow purification and concentration operation in 2.4 of Example 1, purify and concentrate the above inactivated virus solution 8 - 12 times with a 500 KD hollow fiber ultrafiltration concentration system, then purify it with 11 v / v% PEG6000, and resuspend it with a sterile PBS solution (0.04 M, pH value 7.2 - 7.4) to the same volume as the inactivated virus solution to obtain five batches of rPRV-3 strain antigen solution (semi-finished product of pseudorabies gE gene-deleted inactivated vaccine).

[0165] 2.2 Determination of protein concentration and purity of five batches of semi-finished products

[0166] Determine the protein concentration and antigen purity of the five batches of rPRV-3 strain antigen solution prepared according to the method of Example 1.

[0167] 3 Results

[0168] 3.1 Manufacture and inspection of five batches of rPRV-3 strain antigen solution prepared by the laboratory process

[0169] 3.1.1 The antigen production situation is shown in Table 7. A total of five batches of rPRV-3 strain antigen solution were produced, and the harvest time was 48 - 54 h, and the cell viability was lower than 50% for all.

[0170] Table 7 Summary of the preparation of five batches of rPRV-3 strain antigen solution

[0171]

[0172] 3.1.2 The results of the determination of the virus content in the five batches of rPRV-3 strain antigen solution before inactivation are shown in Table 8. It can be seen that the virus solution content in the five batches of rPRV-3 strain antigen solution before inactivation all meets the requirements of the semi-finished product.

[0173] Table 8 Results of the determination of the virus content in the five batches of rPRV-3 strain antigen solution before inactivation

[0174]

[0175] 3.1.3 The inactivation inspection results of the five batches of rPRV-3 strain antigen solution are shown in Table 9. It can be seen that the five batches of inactivated rPRV-3 strain antigen solution are all completely inactivated.

[0176] Table 9 Inactivation test results of five batches of rPRV-3 strain antigen solution

[0177]

[0178] 3.1.4 The protein concentration measurement results of five batches of rPRV-3 strain antigen solution are shown in Table 10. The test results show that the protein concentration of the rPRV-3 strain antigen solution is 103-146 μg / mL.

[0179] Table 10 Protein concentration measurement results of five batches of rPRV-3 strain antigen solution

[0180]

[0181]

[0182] 3.1.6 The antigen purity measurement results of five batches of rPRV-3 strain antigen solution are shown in Table 11. The test results show that the antigen purity of the semi-finished product after purification and concentration of the five batches of rPRV-3 strain is 61-67%, and the antigen purity of the five batches of rPRV-3 strain antigen solution is not less than 60%.

[0183] Table 11 Antigen purity measurement results of five batches of rPRV-3 strain antigen solution

[0184]

[0185] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a pseudorabies virus inactivated antigen standard, comprising the following steps: Propagating the pseudorabies virus to obtain a propagation virus fluid; The propagated virus liquid is sequentially inactivated, purified and concentrated by tangential flow and purified by 4FF chromatography column to obtain a pseudorabies virus inactivated antigen standard; the inactivation reagent used in the inactivation is diethyleneimine.

2. The preparation method according to claim 1, characterized in that: The virus content of the virus breeding liquid is ≥10 6.50 TCID 50 .

3. The preparation method according to claim 1, characterized in that: The concentration of diethyleneimine in the inactivation process is 0.004 mol / L; The inactivation temperature is 30° C. and the time is 28 h. The inactivation process is stirred once every 1 h.

4. The preparation method according to claim 1 or 3, characterized in that: After the inactivation, the method further comprises using a sodium thiosulfate aqueous solution for inactivation blocking.

5. The preparation method according to claim 1, characterized in that: The tangential flow purification and concentration is hollow fiber ultrafiltration purification and concentration, and the conditions of the hollow fiber ultrafiltration purification and concentration include: a molecular weight cutoff of 300 to 500 kD, a membrane area of ​​790 to 1600 cm 2 , the concentration multiple is 10 to 20 times; the flow rate of the inactivated virus solution is 80 to 120 mL / min; the concentrate is concentrated by filtration with PBS solution; The eluent for the 4FF chromatography column purification is a PBS solution.

6. The inactivated pseudorabies virus antigen standard product prepared by the preparation method according to any one of claims 1 to 5, wherein the protein concentration of the inactivated pseudorabies virus antigen standard product is 5 to 200 μg / mL and the purity is ≥90%.

7. Use of the inactivated pseudorabies virus antigen standard product according to claim 6 in the detection of inactivated pseudorabies virus vaccine.

8. A high performance liquid chromatography method for detecting antigens in an inactivated pseudorabies virus vaccine, comprising the following steps: The inactivated pseudorabies virus vaccine to be tested is subjected to high performance liquid chromatography to obtain chromatographic peak information of the sample to be tested; Obtaining a purity test result of the antigen in the inactivated pseudorabies virus vaccine according to the chromatographic peak information of the sample to be tested; The detection result of the protein concentration in the pseudorabies virus inactivated vaccine is obtained according to the chromatographic peak information of the sample to be tested and the standard curve; the standard curve takes the concentration of the pseudorabies virus inactivated antigen standard according to claim 6 as the independent variable and the ultraviolet absorption peak area as the dependent variable.

9. The detection method according to claim 8, characterized in that: The conditions of the high performance liquid chromatography detection include: the chromatographic column is a gel filtration column; the column temperature is 25°C; the globulin separation range is 2×10 4 ~7×10 6 D; the mobile phase was phosphate buffer, the sample temperature was 4°C; the injection volume was 100 μL; the detection wavelength was 280 nm; and the running time was 35 min.

10. The detection method according to claim 9, characterized in that: The solutes in the mobile phase include disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium sulfate; the concentration of disodium hydrogen phosphate in the phosphate buffer is 42 mmol / L, the concentration of sodium dihydrogen phosphate is 8 mmol / L, and the concentration of sodium sulfate is 100 mmol / L.

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