Method for detecting antigen content of two-phase oil adjuvant vaccine

Abstract

The invention provides a two-phase oil emulsion vaccine antigen content detection method, which comprises: 1) demulsifying a two-phase oil emulsion vaccine to obtain a demulsification liquid, 2) layering the demulsification liquid to obtain an antigen-containing water phase, and filtering to obtain a water phase filtrate, 3) precipitating the water phase filtrate by using cold acetone to obtain an antigen, redissolving an antigen precipitate, and carrying out freeze drying to obtain the antigen content of the two-phase oil emulsion vaccine. And 4) carrying out total protein concentration detection on the reconstitution fluid by adopting a protein quantitative detection kit. The detection method provided by the invention has relatively low structural requirements on the vaccine antigen protein, does not need a single structure, is also applicable to some multi-conformation proteins, is wide in application, and is stable and accurate.

Description

Technical Field

[0001] The invention relates to a method for detecting the antigen content of a biphasic oil adjuvant vaccine. Background Art

[0002] Biphasic oil adjuvant is one of the most important adjuvants currently used in the field of animal vaccines. The vaccine prepared with this adjuvant has the advantages of lower viscosity than traditional oil-in-water adjuvant, good injectability, strong effect in stimulating immune response, and long duration of immunity.

[0003] With the widespread application of genetically engineered subunit vaccines, quality control of the vaccine is particularly important. In order to effectively control the quality of the vaccine, the antigens in the vaccine need to be re-quantified. At present, there are some detection methods for the quantification of antigens in the adjuvanted vaccine, such as high-performance liquid chromatography analysis after vaccine demulsification, or direct cold acetone precipitation of the vaccine and then semi-quantification by SDS-PAGE, but both methods have certain defects. The method applied in this application is applicable to a wider range of protein types and has relatively lower requirements for the structure of the protein, but the test results are accurate and reliable.

[0004] Three methods have been invented for vaccines prepared with biphasic oil adjuvants: the first is to perform high-performance liquid chromatography analysis after the vaccine is demulsified; the second is to directly perform cold acetone precipitation on the vaccine and then perform semi-quantitative analysis by SDS-PAGE; the third is to perform cold acetone precipitation on the vaccine, then re-dissolve it with PBS, and use a protein quantitative kit to detect the antigen content of the re-dissolved solution. The first method has the advantages of high accuracy and good sensitivity, but this method has very high requirements on the structure of the vaccine antigen protein. The structure of the protein should form a single and specific absorption peak when detected on high-performance liquid chromatography. If the antigen protein structure is diverse and forms multiple absorption peaks, this method cannot be used for accurate quantification, and because the analytical column is relatively expensive, it is not suitable for large-scale animal vaccine testing. The second method has lower requirements on the structure of the antigen protein, but this method is a semi-quantitative method, and the accuracy of the detected vaccine antigen content is low, and it is impossible to accurately control the quality of the vaccine. Although the third method can quantify the vaccine antigen content, this method is relatively rough and the results are less reproducible.

[0005] CN201710054090.6 discloses a method of using cold acetone to precipitate subunit proteins in an oil emulsion vaccine, then using SDS-PAGE electrophoresis, and then using a grayscale scanner to perform grayscale scanning on each band, and combining the grayscale scanning results to calculate the content and purity of the subunit proteins in the vaccine. This method is not very accurate and cannot accurately control the quality of the vaccine.

[0006] CN201710054110.X discloses a method of breaking emulsions with benzyl alcohol, then separating an aqueous phase containing subunit proteins, and then detecting standard proteins and the separated aqueous phase by HPLC to obtain the purity of the subunit proteins in the aqueous phase. This method has high detection accuracy and accuracy, but has very high requirements for the structure of vaccine antigen proteins. The structure of the protein should form a single, specific absorption peak when detected on high-performance liquid chromatography. If the antigen protein structure is diverse and forms multiple absorption peaks, this method cannot be used for accurate quantification, and the analytical column used is relatively expensive. Summary of the invention

[0007] In order to solve the above technical problems of the prior art, the present invention provides a method for detecting the antigen content of a biphasic oil adjuvant vaccine, the detection method comprising the following steps:

[0008] 1) demulsifying a biphasic oil emulsion vaccine to obtain a demulsified liquid, wherein the biphasic oil emulsion vaccine is a subunit protein biphasic oil emulsion vaccine;

[0009] 2) separating the demulsified liquid into layers to obtain a water phase containing subunit proteins, and filtering the demulsified liquid to obtain a water phase filtrate;

[0010] 3) precipitating the aqueous phase filtrate with cold acetone to obtain a subunit protein precipitate, and then redissolving the subunit protein precipitate to obtain a reconstituted solution in which the subunit proteins are uniformly dissolved; and

[0011] 4) Using a protein quantitative detection kit to detect the total protein concentration of the reconstituted solution.

[0012] In a preferred technical solution of the present invention, in step 1), the demulsification is performed by using a demulsifier.

[0013] In a preferred technical solution of the present invention, the demulsifier is one of chloroform, n-hexanol, benzyl alcohol, n-butanol and n-pentanol.

[0014] In a preferred technical solution of the present invention, in step 2), the stratification is performed by centrifugal stratification.

[0015] In a preferred technical solution of the present invention, the centrifugal stratification is to separate the emulsion into two or three phases by centrifuging at 4500 r / min for 10 minutes at 4°C.

[0016] In a preferred technical solution of the present invention, in step 2), filtration is performed using a 0.22 μm filter membrane to obtain an aqueous filtrate.

[0017] In a preferred technical solution of the present invention, in step 3), the cold acetone is acetone pre-cooled at -15°C or below.

[0018] In a preferred technical solution of the present invention, in step 3), the subunit protein precipitate is obtained by centrifugation at a speed of 12000 r / min and a centrifugation time of 20 minutes.

[0019] In a preferred technical solution of the present invention, the subunit protein precipitate can be resuspended with cold acetone for multiple times, and then precipitated and centrifuged to obtain the subunit protein precipitate.

[0020] In a preferred technical solution of the present invention, the re-dissolution is carried out by using SDS or PBS.

[0021] In a preferred technical solution of the present invention, the protein quantitative detection kit is a BCA protein quantitative kit or a Bradford protein quantitative kit.

[0022] Compared with the prior art, the detection method of the present invention has the following beneficial effects: 1) This method has relatively low structural requirements for vaccine antigen proteins, does not require a single structure, and is also applicable to certain multi-conformation proteins. 2) This method first demulsifies the vaccine and then performs cold acetone precipitation, which effectively avoids the interference of oil adjuvants on the efficiency of antigen precipitation and also eliminates the interference of oil adjuvants on subsequent antigen detection. 3) After cold acetone precipitation, this method uses SDS or PBS for re-dissolution, which increases the efficiency of re-dissolution and improves the accuracy and stability of subsequent antigen detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the HPLC result of Swine Erysipelas SpaA protein;

[0024] Figure 2 This is the HPLC result of porcine epidemic diarrhea S protein;

[0025] Figure 3 This is the SDS-PAGE test result of swine erysipelas SpaA protein;

[0026] Figure 4 This is the SDS-PAGE test result of porcine epidemic diarrhea S protein;

[0027] Figure 5 The results of SDS test for swine erysipelas SpaA protein vaccine antigen; M is a marker; N is a negative control; P1 is a positive control for swine erysipelas SPAA protein; 1 / 2 / 3 are three batches of vaccines;

[0028] Figure 6 These are the SDS-PAGE results of porcine epidemic diarrhea S protein vaccine antigen; where M is Marker; N is the negative control; P is the porcine epidemic diarrhea S protein positive control; 1, 2, and 3 are three batches of vaccines. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. 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.

[0030] The strains, plasmids and reagents used in the examples of the present invention are all commercially available products.

[0031] The swine erysipelas SpaA protein and its oil emulsion vaccine involved in the embodiments of the present invention are prepared in an Escherichia coli expression system according to the prior application CN2011910442887.2 or other documents.

[0032] The porcine epidemic diarrhea S protein and its oil emulsion vaccine involved in the embodiments of the present invention are prepared by using a mammalian cell or insect baculovirus expression system according to CN201810310540.8 or other documents previously applied by the inventor.

[0033] Example 1: HPLC structural analysis and SDS-PAGE analysis of two types of proteins

[0034] 1.1 Protein HPLC structure analysis

[0035] Take two proteins, swine erysipelas SpaA protein and porcine epidemic diarrhea S protein, and use PBS as the mobile phase for HPLC analysis at a flow rate of 0.5 ml / min. The HPLC results of swine erysipelas SpaA protein are as follows: Figure 1 , the HPLC results of porcine epidemic diarrhea S protein are as follows Figure 2 The results show that the structure of the swine erysipelas SpaA protein is relatively simple, forming a single, specific absorption peak; while the structure of the porcine epidemic diarrhea S protein is diverse, forming multiple absorption peaks.

[0036] 1.2 Protein SDS-PAGE analysis

[0037] Take 5 μl of the resuspended samples of swine erysipelas SpaA protein and porcine epidemic diarrhea S protein, add 5× loading buffer, and perform SDS-PAGE purity detection. The results of SDS-PAGE detection of swine erysipelas SpaA protein are as follows: Figure 3 , the results of SDS-PAGE test of porcine epidemic diarrhea S protein are as follows Figure 4 The results showed that they could form a single band in SDS-PAGE.

[0038] The above experimental analysis shows that for proteins with diverse structures and multiple absorption peaks, the HPLC method has certain limitations, so the SDS-PAGE analysis is relatively more applicable.

[0039] Example 2: Demulsification and acquisition of antigenic phase

[0040] 2.1 Demulsification of Swine Erysipelas SpaA Protein Oil Emulsion Vaccine and Obtaining the Antigenic Phase

[0041] Take 5ml of swine erysipelas SpaA protein oil emulsion vaccine in a 15ml centrifuge tube, add 0.5ml benzyl alcohol, mix upside down 30 times, mix on a rotating incubator at 4℃ for 10 minutes, and then centrifuge at 4500r / min for 10 minutes at 2-8℃ to break the emulsion. After breaking the emulsion, the vaccine is divided into 3 phases, the lower layer is the water phase, which contains antigen protein. Use a 1ml syringe to carefully insert and absorb the lower water phase, filter it through a 0.2μm filter membrane, and then divide it into 1ml / tube for standby use.

[0042] 2.2 Demulsification of porcine epidemic diarrhea S protein oil emulsion vaccine and acquisition of antigenic phase

[0043] Take 5 ml of porcine epidemic diarrhea S protein oil emulsion vaccine in a 15 ml centrifuge tube, add 0.5 ml of n-pentanol, mix upside down 30 times, mix on a rotating incubator at 4 ° C for 10 minutes, and then centrifuge at 4500r / min for 10 minutes at 2-8 ° C to break the emulsion. After breaking the emulsion, the vaccine is divided into 3 phases, the lower layer is the water phase, which contains antigen protein. Use a 1 ml syringe to carefully insert and absorb the lower water phase, filter through a 0.2 μm filter membrane, and then divide into 1 ml / tube for standby use.

[0044] Example 3: Antigen Phase Cold Acetone Precipitation

[0045] Take 100 μl of the filtrate, add 900 μl of cold acetone (acetone was pre-cooled below -15°C in advance), the final concentration of acetone was 90% (v:v), invert to mix, and place below -15°C for precipitation for 2 hours; after precipitation, centrifuge at 12000 r / min, 2-8°C for 20 minutes, discard the supernatant, and keep the precipitate; add 1.0 ml of cold acetone to the precipitate and resuspend it, centrifuge at 12000 r / min, 2-8°C for 20 minutes, discard the supernatant, and keep the precipitate, and repeat this step twice.

[0046] Example 4: Reconstitution

[0047] After the acetone evaporated and dried, the precipitate was resuspended by adding 100 μl of 5% SDS. The results showed that after resuspending with SDS, the precipitated protein was basically dissolved into a uniform state.

[0048] Example 5: Concentration detection

[0049] The total protein concentration of the resuspended SpaA sample was measured by BCA protein content assay, and the total protein concentration of the resuspended S sample was measured by Bradford protein content assay.

[0050] Example 6: Purity detection

[0051] Take 5 μl of the resuspended sample, add 5× loading buffer, and perform SDS-PAGE purity detection.

[0052] The specific method is as follows:

[0053] 6.1 Preparation of polyacrylamide gel

[0054] 6.1.1 Glass plate selection (Spacer, i.e., selection of gel thickness): plate spacing 1.5 mm, 10-hole gel comb specification, maximum sample loading volume 40 μl.

[0055] 6.1.2 Select 10% gel concentration.

[0056] 6.1.3 Assembly Select appropriate glass plates, wipe the glass plates clean, and assemble the glass plates.

[0057] 6.1.4 Prepare the separation gel. Refer to the formula in Table 1 and select a gel preparation system with an appropriate volume according to the required sample loading amount.

[0058] Table 1 SDS-PAGE separation gel formula

[0059]

[0060] 6.1.5 Quickly shake the separation gel solution prepared in step 1.5.2.1.4, and quickly pour the separation gel along the gap of the glass glue plate (note: do not inject bubbles when pouring the gel). After pouring the separation gel, quickly seal the liquid surface with ultrapure water.

[0061] 6.1.6 After the separation gel solidifies, pour out the upper layer of water and use filter paper to absorb the remaining water.

[0062] 6.1.7 Prepare concentrated gel according to the formula in Table 2. After the concentrated gel is prepared, shake it quickly to mix well, and quickly pour the concentrated gel along one side of the gap between the glass plates to avoid introducing bubbles. Quickly insert the matching tooth comb and let it stand until the concentrated gel is completely solidified.

[0063] Table 2 SDS-PAGE stacking gel (5% Acrylamide) formula

[0064]

[0065] 6.2 Sample treatment Take 20 μl of the sample to be tested and put it into a 1.5 ml EP tube, add 5 μl of reducing 5× loading buffer, boil for 5 to 10 minutes and set aside.

[0066] 6.3 Loading Samples Add the sample to be tested into the loading well.

[0067] 6.4 Electrophoresis: Cover the lid correctly according to the red / black color indicator, run electrophoresis at 80V for 30 minutes, and then increase the voltage to 120V. When the bromophenol blue indicator runs to 5mm below the bottom of the electrophoresis plate, turn off the power and stop electrophoresis.

[0068] 6.5 Staining Carefully peel the gel from the glass plate, add Coomassie Brilliant Blue staining solution, heat in a microwave for 1 minute, and then shake on a shaker for 30 minutes.

[0069] 6.6 Pour the decolorized staining solution into the recovery bottle and add the decolorizing solution. After heating for 1 minute, shake on a shaker to decolorize for 1 hour.

[0070] 6.7 Imaging Use a gel imager to save the data, set the grayscale of the lane of the sample to be tested to 100%, and use the analysis software to obtain the grayscale ratio of the target band in the lane, which is the purity.

[0071] 6.8 Clear the area and the lab bench and put the lab instruments in order.

[0072] Example 7: Vaccine antigen concentration detection

[0073] The antigen concentration in the vaccine is total protein concentration × vaccine antigen purity × 0.46.

[0074] This method was used to detect the antigen content in the biphasic oil emulsion vaccine prepared from the two types of proteins. For detailed results, see Table 3 (antigen content detection results of the biphasic oil emulsion vaccine prepared from swine erysipelas SpaA protein), Table 4 (antigen content detection results of the biphasic oil emulsion vaccine prepared from porcine epidemic diarrhea S protein), Figure 5 (SDS test results of swine erysipelas SpaA protein vaccine antigen, where M is Marker; N is negative control; P1 is swine erysipelas SpaA protein positive control; 1, 2, 3 are three batches of vaccines) and Figure 6 (SDS-PAGE results of porcine epidemic diarrhea S protein vaccine antigen, where M is Marker; N is negative control; P is porcine epidemic diarrhea S protein positive control; 1, 2, and 3 are three batches of vaccines). The results showed that the antigen content in the vaccine detected by this method for swine erysipelas SpaA protein vaccine can reach an average of more than 90%, and the repeatability between different detection times is good and the deviation is small; the antigen content in the porcine epidemic diarrhea S protein vaccine detected by this detection method can reach an average of more than 90%, and the deviation between different detection times is small, which proves that this method is applicable to the antigen content detection method of vaccines made of two types of proteins, and the method has high accuracy and good repeatability.

[0075] Table 3 Antigen content test results of biphasic oil emulsion vaccine prepared from swine erysipelas SpaA protein

[0076]

[0077] Table 4 Antigen content test results of biphasic oil emulsion vaccine prepared from porcine epidemic diarrhea S protein

[0078]

[0079] Comparative Example:

[0080] Take 100 μl of swine erysipelas SpaA protein oil emulsion vaccine and porcine epidemic diarrhea S protein oil emulsion vaccine respectively, add 900 μl of cold acetone (acetone is pre-cooled below -15°C in advance), the final concentration of acetone is 90% (v:v), invert and mix well, and place below -15°C for precipitation for 2 hours; after precipitation, centrifuge at 12000r / min, 2-8°C for 20 minutes, discard the supernatant, and keep the precipitate; add 1.0ml of cold acetone to the precipitate for resuspending, centrifuge at 12000r / min, 2-8°C for 20 minutes, discard the supernatant, and keep the precipitate, and repeat this step 2 times. After the precipitate is evaporated and dried by acetone, add 100 μl of 5% SDS for resuspending. The subunit protein after resuspending is detected with a protein concentration detection kit. Combined with the purity detection result, the obtained vaccine antigen concentration is lower than that of the present invention, and the deviation between different detection times is also larger, as shown in Table 5 and Table 6.

[0081] Table 5 Antigen content test results of biphasic oil emulsion vaccine prepared from swine erysipelas SpaA protein

[0082]

[0083] Table 6 Antigen content test results of biphasic oil emulsion vaccine prepared from porcine epidemic diarrhea S protein

[0084]

[0085]

[0086] In summary, by comparing this detection method with the previous method of directly cold acetone precipitation of oil emulsion, it is proved that this method effectively avoids the interference of oil adjuvant on antigen precipitation efficiency, and also eliminates the interference of oil adjuvant on subsequent antigen detection, making the final test results more accurate and reliable.

[0087] The present invention is illustrated by the above examples, however, it should be understood that the present invention is not limited to the specific embodiments and embodiments described herein. The purpose of including these specific embodiments and embodiments here is to help those skilled in the art to practice the present invention. Any person skilled in the art will readily make further improvements and perfections without departing from the spirit and scope of the present invention, and therefore the present invention is only limited by the content and scope of the claims of the present invention, which is intended to cover all alternatives and equivalents included in the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A method for detecting the antigen content of a two-phase oil emulsion vaccine, characterized in that: The detection method comprises the following steps: 1) demulsifying a biphasic oil emulsion vaccine to obtain a demulsified liquid, wherein the biphasic oil emulsion vaccine is a subunit protein biphasic oil emulsion vaccine; 2) separating the demulsified liquid into layers to obtain a water phase containing subunit proteins, and filtering the demulsified liquid to obtain a water phase filtrate; 3) precipitating the aqueous phase filtrate with cold acetone to obtain a subunit protein precipitate, and then redissolving the subunit protein precipitate to obtain a reconstituted solution in which the subunit protein is uniformly dissolved; as well as 4) Using a protein quantitative detection kit to detect the total protein concentration of the reconstituted solution.

2. The detection method according to claim 1, characterized in that: In step 1), the demulsification is performed by using a demulsifier.

3. The detection method according to claim 2, characterized in that: The demulsifier is one of chloroform, n-hexanol, benzyl alcohol, n-butanol and n-pentanol.

4. The detection method according to claim 1, characterized in that: In step 2), the stratification is performed by centrifugal stratification.

5. The detection method according to claim 4, characterized in that: The centrifugal separation is carried out at 4°C and 4500 r / min for 10 minutes to separate the emulsion into two or three phases.

6. The detection method according to claim 1, characterized in that: In step 2), filtering is performed using a 0.22 μm filter membrane to obtain an aqueous filtrate.

7. The detection method according to claim 1, characterized in that: In step 3), the cold acetone is acetone pre-cooled at -15°C or below.

8. The detection method according to claim 1, characterized in that: In step 3), the subunit protein precipitate is obtained by centrifugation at a speed of 12000 r / min for 20 minutes.

9. The detection method according to claim 8, characterized in that: The subunit protein precipitate can be resuspended with cold acetone for multiple times, and then centrifuged to obtain the subunit protein precipitate.

10. The detection method according to claim 1, characterized in that: The re-dissolution is carried out by SDS re-dissolution or PBS re-dissolution.

11. The detection method according to claim 1, characterized in that: The protein quantitative detection kit is a BCA protein quantitative kit or a Bradford protein quantitative kit.

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