Detection method of African swine fever two-component oil emulsion vaccine

Abstract

The invention provides a detection method of an African swine fever two-component oil emulsion vaccine, which comprises the following steps: demulsifying the African swine fever two-component oil emulsion vaccine by using a demulsifier to obtain a demulsified solution; taking a water phase at the lower layer of the demulsified liquid after demulsification; enabling the water phase to pass through an affinity chromatography filler 1 so as to collect an antigen 1 antigen liquid, and enabling the antigen 1 to be African swine fever outer envelope CD2V protein; the water phase passing through the affinity chromatography filler 1 continuously passes through the affinity chromatography filler 2 to collect antigen 2 antigen liquid, and the antigen 2 is African swine fever outer capsid p72 protein; carrying out concentration detection on the antigen 1 by using a Bradford method; and detecting the concentration of the antigen 2 by using a BCA method. According to the detection method, n-amyl alcohol is adopted for demulsification, the antigen is recycled through affinity chromatography, repeatability is good, and accuracy is high; and a certain amount of protein loss can be generated after the vaccine is demulsified.

Description

Technical Field

[0001] The present invention belongs to the field of veterinary biological products, and in particular, relates to a method for detecting an African swine fever two-component oil emulsion vaccine. Background Art

[0002] African swine fever (ASF) is a highly contagious hemorrhagic infectious disease caused by African swine fever virus (ASFV). It can infect domestic pigs and wild boars of all breeds and ages at the same time, causing high morbidity and mortality in domestic pigs and wild boars. Pigs infected with African swine fever virus show symptoms such as high fever, depression, anorexia, cyanosis, congestion, and decrease in peripheral platelets and lymphocytes. The clinical manifestations of African swine fever range widely, including acute, subacute, and chronic. The acute course is short, with a mortality rate of 100%. The World Organization for Animal Health classifies it as a Class A epidemic disease, and my country also classifies it as a Class I animal infectious disease.

[0004] The development of African swine fever vaccine began in the 1960s. Due to the complexity of the biology of African swine fever virus, there is no safe and effective commercial vaccine to date. Natural attenuated strains and recombinant attenuated live vaccines with knocked-out virulence genes of African swine fever virus have good protection, but there are significant biosafety issues and persistent infection. Traditional inactivated vaccines have been proven to be ineffective. In addition, attenuated ASFV live vaccines lack stable cell lines for production because ASFV can only replicate in primary monocytes / macrophages. For these reasons, ASFV subunit vaccines have been explored as a viable alternative. In particular, subunit vaccines are highly safe and therefore have greater potential and advantages in the development of African swine fever vaccines.

[0005] ASFV is the only member of the genus Asfivirus in the family Asfarviridae and is the only known arbovirus with a DNA genome. The viral genome is a linear, covalently enclosed double-stranded DNA (dsDNA) molecule. The genome of ASFV isolates from different regions has different specificities and lengths, with the length of different isolates ranging from approximately 170 to 190 kb. ASFV contains 151 to 167 open reading frames (ORFs), encoding 150-200 proteins, and mature virus particles contain more than 50 structural proteins. Among them, the proteins encoded by the antigen 1 and antigen 2 genes are the most important structural proteins of ASFV, which can induce the body to produce an immune response and are also important target proteins for the study of genetically engineered vaccines for African swine fever.

[0006] The mammalian cell expression system has accurate post-transcriptional modification functions, and the expressed proteins are closest to natural protein molecules in terms of molecular structure, physicochemical properties and biological functions; it has the ability to efficiently amplify and express recombinant genes, and stably integrate exogenous genes; it rarely secretes endogenous proteins, which is conducive to the purification of exogenous proteins. Our team successfully constructed and screened the CHO-10C3 cell line that efficiently and stably expresses the African swine fever CD2V protein and the CHO-77E7 cell line that expresses the p72 protein. The vaccine prepared by the combination of the expressed CD2V protein and the p72 protein can induce a good immune response in the immunized pigs, and can well resist the attack of the African swine fever virus, providing a new and efficient preventive preparation for African swine fever, which will play an important role in the prevention and control of African swine fever in my country and even around the world.

[0007] In the development of recombinant protein vaccines, the stability of antigen protein after adsorption with adjuvant is one of the necessary testing indicators, which requires effective separation of antigen protein and adjuvant, and accurate determination of antigen content in finished vaccine. The finished vaccine is mostly a colloidal complex after the adsorption of antigen protein and adjuvant. How to use appropriate methods to completely dissociate protein antigen and adjuvant without destroying the properties of antigen is a difficult problem that needs to be solved. Summary of the invention

[0008] In order to solve the above-mentioned problems, the present invention provides a detection method for a two-component oil emulsion vaccine for African swine fever. Antigen identification testing and content determination are carried out in the inspection of the finished product of the genetically engineered subunit vaccine for African swine fever, and the antigen and adjuvant of the vaccine need to be separated. Specifically, the detection method includes the following steps: 1) Demulsifying the two-component oil emulsion vaccine for African swine fever with a demulsifier to obtain a demulsified liquid; 2) Taking the aqueous phase at the bottom of the demulsified liquid after demulsification; 3) Passing the aqueous phase through affinity chromatography filler 1 to collect antigen liquid 1, wherein the antigen 1 is the African swine fever outer envelope CD2V protein; 4) The aqueous phase that has passed through affinity chromatography filler 1 continues to pass through affinity chromatography filler 2 to collect antigen liquid 2, wherein the antigen 2 is the African swine fever outer capsid p72 protein; 5) Using the Bradford method to detect the concentration of the antigen 1 in the antigen liquid 1; and 6) Using the BCA method to detect the concentration of the antigen 2 in the antigen liquid 2. Please refer to Figure 1 Flow chart of the detection method of the present invention is shown.

[0009] In the preferred technical scheme of the detection method of the present invention, preferably, the African swine fever two-component oil emulsion vaccine comprises an aqueous phase composed of the antigen liquid 1 and the antigen liquid 2, and an oil adjuvant; the oil adjuvant is ISA201VG.

[0010] In the preferred technical scheme of the detection method of the present invention, preferably, in the African swine fever two-component oil emulsion vaccine, the volume ratio of the oil adjuvant to the aqueous phase composed of the antigen liquid 1 and the antigen liquid 2 is 54:46.

[0011] In the preferred technical scheme of the detection method of the present invention, preferably, in step 1), the volume ratio of the demulsifier to the African swine fever two-component oil emulsion vaccine is 1:5-12, preferably 1:10.

[0012] In the preferred technical solution of the detection method of the present invention, preferably, in step 1), the demulsifier is benzyl alcohol, chloroform, or n-pentanol.

[0013] In the preferred technical solution of the detection method of the present invention, preferably, in step 3), the affinity chromatography filler 1 is A; In step 4), the affinity chromatography filler 2 is Anti-DYKDDDDK G1 Affinity Resin.

[0014] In the preferred technical solution of the detection method of the present invention, preferably, the detection method also includes WB verification of the antigen.

[0015] In the preferred technical scheme of the detection method of the present invention, preferably, the antigen WB verification includes: adding the collected antigen liquid 1 and antigen liquid 2 to the loading buffer 5×SDS-PAGE Loading Buffer, treating in boiling water for 10 minutes, electrophoresing in SDS-PAGE, wet transfer, blocking, incubating with African swine fever positive serum and sheep anti-pig enzyme-labeled secondary antibody labeled with horseradish peroxidase, developing by ECL method, and finally scanning and identifying with a gel imaging system.

[0016] The present invention uses n-pentanol demulsification and combines it with affinity chromatography to recover different antigens, and the recovered antigens are subjected to WB specific identification and content determination. The results of the present invention show that: 1) the use of n-pentanol demulsification and affinity chromatography to recover antigens has good repeatability and high accuracy; 2) a certain amount of protein loss will occur after the vaccine is demulsified. In the test of the present invention, the content loss of antigen 1 after demulsification is 7 μg / head portion, and the content loss of antigen 2 is 9 μg / head portion. Therefore, the protein content determination standard of the African swine fever genetic engineering subunit vaccine after demulsification is set as antigen 1 ≥ 43 μg / head portion; antigen 2 ≥ 41 μg / head portion. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a flow chart of the detection method of the present invention;

[0018] Figure 2A and Figure 2B The SDS-PAGE and WB results of antigen solution 1 are as follows: Figure 2A The results of SDS-PAGE detection are shown in Figure 2. Figure 2B is the WB test result; M is Marker; N is negative control; P is positive control; 1 / 2 / 3 is antigen 1 in three batches of vaccines;

[0019] Figure 3A and Figure 3B The SDS-PAGE and WB results of antigen solution 2 were recovered, among which, Figure 3A The results of SDS-PAGE detection are shown in Figure 2. Figure 3B is the WB test result; M is Marker; N is the negative control; P is the positive control; 1 / 2 / 3 are antigen 2 in three batches of vaccines. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with embodiments and drawings. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention, but are not intended to limit the present invention.

[0021] Example 1: Determination of antigen detection method

[0022] Methods: It is known that the concentration of antigen 1 is 1500μg / ml and the concentration of antigen 2 is 800μg / ml. In order to accurately quantify the antigen 1 and antigen 2 recovered from the finished vaccine demulsification, it is necessary to explore the quantitative method of antigen 1 and antigen 2. The BCA method, Bradford method, Lowry method and spectrophotometry were used to detect the content of antigen 1 and antigen 2, and the best detection method was determined by analyzing the accuracy and standard deviation.

[0023] Results: The concentration of antigen 1 was detected by BCA method, Bradford method and Lowry method, and each method was repeated 20 times. The accuracy and standard deviation were calculated, as shown in Table 1.

[0024] Table 1. Results of different methods for detecting antigen 1 (1500 μg / ml)

[0025]

[0026] The concentration of antigen 2 was detected by BCA method, Bradford method and Lowry method respectively. Each method was repeated 20 times, and the average concentration and standard deviation were calculated, as shown in Table 2.

[0027] Table 2. Results of different methods for detecting antigen 2 (800 μg / ml)

[0028]

[0029] Analysis and conclusion: Analysis of the detection results of the three protein concentration detection methods for the concentrations of antigen 1 and antigen 2 showed that when antigen 1 was detected by BCA method and Lowry method, the average concentrations were 3104.75μg / ml and 1807.01μg / ml, respectively, and the standard deviations were 8.75 and 13.33. The concentrations detected by these two methods were both high. The average concentration detected by Bradford method was 1503.65μg / ml, and the standard deviation was 5.11. The detection results of this method were closest to the true concentration, and the detection results were stable with small deviations. Therefore, the concentration detection method for antigen 1 was determined to be Bradford method. When antigen 2 was detected by Bradford method and Lowry method, the average concentrations were 706.70μg / ml and 905.53μg / ml, respectively, with standard deviations of 6.37 and 7.87. The concentrations detected by these two methods deviated greatly from the true concentrations. The average concentration detected by BCA method was 810.02μg / ml, with a standard deviation of 4.29. The detection results of this method were closest to the true concentration, and the detection results were stable with small deviations. Therefore, the concentration detection method for antigen 2 was determined to be the BCA method.

[0030] Example 2: Dilution and detection of antigen concentration before vaccination

[0031] According to the adjuvant manufacturer's recommendation, the oil adjuvant: aqueous phase (v:v) = 54:46 ratio, the preparation of antigen 1 and antigen 2 protein concentrations of 25μg / ml (2ml / head) vaccine requires the dilution concentration of antigen liquid 1 and antigen liquid 2 to be 54.35μg / ml. Since the African swine fever two-component oil emulsion vaccine is a two-component vaccine mixed with two antigens, the concentrations of antigen liquid 1 and antigen liquid 2 should be doubled, each of which is 108.70μg / ml. Take antigen liquid 1 and antigen liquid 2, dilute them with PBS to the above concentrations, and name them C1, D1; C2, D2; C3, D3, C1, C2, C3 are tested for concentration by Bradford method, and D1, D2, D3 are tested for concentration by BCA method.

[0032] Example 3: Vaccine preparation

[0033] Using ISA 201VG oil adjuvant, C1 and D1 are defined as the 202002a batch of African swine fever two-component oil emulsion vaccine antigen phase; C2 and D2 are defined as the 202002b batch of African swine fever two-component oil emulsion vaccine antigen phase; C3 and D3 are defined as the 202002c batch of African swine fever two-component oil emulsion vaccine antigen phase, respectively, according to the ratio of oil adjuvant: aqueous phase (v:v) = 54:46, that is, in the African swine fever two-component oil emulsion vaccine, the volume ratio of the oil adjuvant to the aqueous phase composed of the antigen liquid 1 and the antigen liquid 2 is 54:46. Here, the "African swine fever two-component oil emulsion vaccine" is prepared according to Chinese Patent Application No. 202110752873.8 (Publication No. CN113940992A). Adjust the rotation speed to 250-400 r / min, stir at 33±1°C for 30 minutes, and emulsify into a two-phase oil emulsion vaccine.

[0034] Example 4: Vaccine sample testing

[0035] 4.1 Demulsifier screening and optimization

[0036] Select demulsifiers: benzyl alcohol, chloroform, n-pentanol and other demulsifiers. The volume ratio of demulsifier to African swine fever two-component oil emulsion vaccine is 1:5-12, preferably 1:10. The ratio of African swine fever two-component oil emulsion vaccine to demulsifier is screened as: 5:1, 9:1, 10:1, 11:1, 12:1. Demulsification time: 5 minutes, 10 minutes, 15 minutes, 20 minutes. Centrifugation time: 10 minutes, 20 minutes, 30 minutes. Screen out the best demulsifier and the best demulsification method.

[0037] 4.2 Antigen recovery and concentration determination

[0038] The vaccine to be tested was demulsified with n-pentanol (the ratio of n-pentanol to vaccine was 1:10 (v:v)) and centrifuged at 4500r / min for 15 minutes to accelerate the demulsification. The lower aqueous phase was taken and antigen liquid 1 was first collected through affinity chromatography filler 1 and then antigen liquid 2 was collected through affinity chromatography filler 2. The concentration of antigen liquid 1 was detected by Bradford method and the concentration of antigen liquid 2 was detected by BCA method. The corresponding sample concentration was calculated as follows: antigen concentration in vaccine = antigen liquid concentration × 0.46 × 2.

[0039] Example 5: Antigen WB test

[0040] 5.1 Add the collected antigen liquid (antigen liquid 1 and antigen liquid 2) into 5×SDS-PAGE Loading Buffer, treat in boiling water for 10 minutes, electrophoresed in SDS-PAGE, wet transfer, block, incubate with African swine fever positive serum and goat anti-swine enzyme-labeled secondary antibody labeled with horseradish peroxidase, develop with ECL method, and finally scan and identify with gel imaging system.

[0041] 5.2 Test results

[0042] Please refer to Table 3 for the concentration results of the detected antigen phases C1, D1; C2, D2; C3, D3.

[0043] Table 3

[0044]

[0045] Example 6: Vaccine antigen WB test

[0046] The recovered antigens were subjected to SDS-PAGE (under reducing conditions) and WB detection (under reducing conditions). The test results showed that the affinity chromatography filler The size of the antigen collected by A is about 95 kDa, and the WB test is positive, so the collected antigen is 1. The size of the antigen collected by affinity chromatography medium Anti-DYKDDDDK G1 Affinity Resin is about 80 kDa, and the WB test is positive, so the collected antigen is antigen 2. Figure 2A , Figure 2B , Figure 3A , Figure 3B .

[0047] Example 7: Results of antigen recovery concentration test after vaccine demulsification

[0048] The antigen 1 and antigen 2 antigens in the three groups of finished vaccines (202002a, 202002b, 202002c) were recovered by affinity chromatography. The average concentrations of the recovered antigen 1 detected by Bradford method were 43.84μg / head portion, 43.59μg / head portion, and 43.00μg / head portion, respectively; the SD values ​​were 1.35, 0.50, and 0.50, respectively; the average concentrations of the antigen 2 antigen detected by BCA method were 41.28μg / head portion, 41.82μg / head portion, and 41.85μg / head portion, respectively; the SD values ​​were 1.40, 1.16, and 1.51, respectively. Detailed results are shown in Tables 4, 5, and 6. A total of 30 repeated experiments were statistically analyzed, and the results are shown in Table 7.

[0049] Table 4 Test results of 202002a batch vaccine

[0050]

[0051] Table 5 Testing results of 202002b batch of vaccines

[0052]

[0053] Table 6 Test results of 202002c batch vaccine

[0054]

[0055] Table 7 Statistical analysis results of vaccine antigen content detection methods

[0056]

[0057] According to the results of 30 repeated tests of three batches of vaccines, 202002a, 202002b, and 202002c, the statistical analysis results in Table 7 were obtained. It can be seen that within the 99% confidence interval, the upper limit of the loss of antigen 1 (CD2V antigen) during the demulsification process is 7, the standard deviation is 0.92, and the T value is 2.76. It can be concluded that the lower limit of the content of antigen 1 (CD2V antigen) after the vaccine demulsification is 43μg / head portion; the upper limit of the loss of antigen 2 (p72 antigen) is 9, the standard deviation is 1.35, and the T value is 2.76. It can be concluded that the lower limit of the content of antigen 2 (p72 antigen) after the vaccine demulsification is 41μg / head portion. It can better reflect the actual content of antigens in vaccines and play a good guiding role in the inspection of vaccine antigen content.

[0058] It should be noted that the above invention content and specific implementation methods are intended to demonstrate the practical application of the technical solution provided by the present invention and should not be interpreted as limiting the scope of protection of the present invention. Those skilled in the art can make various modifications, equivalent substitutions, or improvements within the spirit and principles of the present invention.

Claims

1. A method for detecting a two-component oil emulsion vaccine for African swine fever, characterized in that: The detection method comprises the following steps: 1) Demulsifying the African swine fever two-component oil emulsion vaccine with a demulsifier to obtain a demulsified liquid; 2) taking the aqueous phase at the bottom of the demulsified liquid after demulsification; 3) passing the aqueous phase through affinity chromatography filler 1 to collect antigen solution 1, wherein the antigen 1 is African swine fever outer envelope CD2V protein; 4) the aqueous phase that has passed through affinity chromatography filler 1 continues to pass through affinity chromatography filler 2 to collect antigen liquid 2, wherein the antigen 2 is the outer capsid p72 protein of African swine fever; 5) detecting the concentration of the antigen 1 in the antigen solution 1 by Bradford method; and 6) The concentration of the antigen 2 in the antigen solution 2 is detected by the BCA method.

2. The detection method according to claim 1, characterized in that: The African swine fever two-component oil emulsion vaccine comprises an aqueous phase consisting of the antigen liquid 1 and the antigen liquid 2, and an oil adjuvant; the oil adjuvant is ISA201VG.

3. The detection method according to claim 2, characterized in that: In the African swine fever two-component oil emulsion vaccine, the volume ratio of the oil adjuvant to the aqueous phase composed of the antigen liquid 1 and the antigen liquid 2 is 54:

46.

4. The detection method according to claim 1, characterized in that: In step 1), the volume ratio of the demulsifier to the African swine fever two-component oil emulsion vaccine is 1:5-12, preferably 1:

10.

5. The detection method according to claim 4, characterized in that: In step 1), the demulsifier is benzyl alcohol, chloroform, or n-pentanol.

6. The detection method according to claim 1, characterized in that: In step 3), the affinity chromatography filler 1 is A; In step 4), the affinity chromatography filler 2 is Anti-DYKDDDDK G1 Affinity Resin.

7. The detection method according to any one of claims 1 to 6, characterized in that: The detection method also includes WB verification of the antigen.

8. The detection method according to claim 7, characterized in that: The antigen WB verification includes: adding the collected antigen liquid 1 and antigen 2 to the loading buffer 5×SDS-PAGE Loading Buffer, treating in boiling water for 10 minutes, electrophoresing in SDS-PAGE, wet transfer, blocking, incubating with African swine fever positive serum and goat anti-swine enzyme-labeled secondary antibody labeled with horseradish peroxidase, developing with the ECL method, and finally scanning and identifying with a gel imaging system.

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