Method for detecting content of virus-like particles of porcine circovirus type II Cap protein

Abstract

The invention provides a method for detecting the content of porcine circovirus type II Cap protein virus-like particles, and relates to the technical field of biology, the method comprises the following steps: providing a pretreatment sample, and carrying out sucrose density gradient ultracentrifugation on the sample to be detected to obtain a centrifugal sample; carrying out polyacrylamide gel electrophoresis, membrane transfer, sealing, antibody incubation and color development on the centrifugal sample to obtain a WB picture; and calculating to obtain the virus-like particle content of the porcine circovirus type II Cap protein according to the particle stripe gray value and the total gray value of the porcine circovirus type II Cap protein on the WB picture. According to the technical scheme, different self-assembled polymers of the Cap protein are separated from a to-be-detected product through sucrose density gradient ultracentrifugation, distribution of the different self-assembled polymers of the Cap protein in each sucrose density gradient is detected through a WB method, gray analysis is conducted on the different self-assembled polymers, and detection of the ratio of the baculovirus expressed porcine circovirus type II Cap protein VLP particles is successfully achieved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for detecting the content of porcine circovirus type II Cap protein virus-like particles. Background Art

[0002] Porcine circovirus type II (PCVII) belongs to the genus Porcine Circovirus. Its virions are 14-17 nm in diameter, exhibit icosahedral symmetry, lack an envelope, and consist of a covalently closed, single-stranded, circular, negative-sense DNA. The genome is approximately 1.7 kb in size. Four reported genotypes of PCVII have been reported. PCVII can co-infect pigs with a variety of viruses and bacteria, exacerbating clinical manifestations. PCVII has been confirmed to be one of the primary pathogens of porcine circovirus-associated disease (PCVAD), a disease prevalent worldwide and causing significant economic losses to the swine industry. Vaccination is the primary means of preventing the disease, and genetically engineered subunit vaccines are highly favored due to their excellent safety profile. The icosahedral capsid protein (Cap protein), encoded by the PCVII ORF2 gene, is the primary antigen of the virus. Subunit vaccines prepared by recombinantly expressing the Cap protein have gained market acceptance.

[0003] Virus-like particles (VLPs) are morphologically identical or similar to actual virus particles, have a good safety profile, and can strongly stimulate the immune system, producing a protective immune response, attracting widespread attention. The porcine circovirus type 2 Cap protein (PCVII-Cap) monomers can self-assemble into icosahedral VLPs, and subunit vaccines prepared from them have been widely used due to their excellent safety and protective efficacy.

[0004] In the prior art, antigen production for PCVII-Cap subunit vaccines can be achieved using a variety of expression systems. Semi-finished antigens for vaccine formulation are often quantified using various methods such as Elisa and BCA. These detection methods all assume that recombinantly expressed PCVII-Cap monomers are uniform, effective, and can completely self-assemble into VLPs. However, the spatial structure of PCVII-Cap monomers expressed in different expression systems cannot be guaranteed to be completely consistent with the theoretical design, nor can it be guaranteed that they can all self-assemble into VLP particles. Furthermore, differences in the spatial structure of antigens cultured from different batches within the same expression system can lead to differences in self-assembly efficiency, further affecting differences between vaccine batches and resulting in inaccurate quantitative detection results for VLP particles. Summary of the Invention

[0005] The main purpose of the present invention is to propose a method for detecting the content of porcine circovirus type II Cap protein virus-like particles, aiming to solve the problem of inaccurate quantitative detection results of VLP particles in the existing technology.

[0006] To achieve the above object, the present invention provides a method for detecting the content of porcine circovirus type II Cap protein virus-like particles, comprising the following steps:

[0007] Providing a pretreated sample, performing sucrose density gradient ultracentrifugation on the sample to be tested, and obtaining a centrifuged sample;

[0008] The centrifuged samples were subjected to polyacrylamide gel electrophoresis, transferred to a membrane, blocked, incubated with antibodies, developed, and obtained Western blotting images;

[0009] The content of virus-like particles of porcine circovirus type II Cap protein was obtained based on the gray value of the particle band and the total gray value of the porcine circovirus type II Cap protein on the WB image.

[0010] In one embodiment, the step of providing a pre-treated sample comprises:

[0011] The sample to be tested is taken, filtered, and the concentration is determined, and mixed with a diluent to 1-10 μg / mL to obtain a pretreated sample.

[0012] In one embodiment, the diluent includes a PBS solution, and the concentration of the PBS solution is 0.1-0.01 mol / L and the pH is 7.2-7.4.

[0013] In one embodiment, the sample to be tested is subjected to sucrose density gradient centrifugation to obtain the centrifuged sample, comprising:

[0014] Adding sucrose of different concentrations into an ultracentrifuge tube in order from high concentration to low concentration, and adding the pretreated sample to the top layer of the ultracentrifuge tube;

[0015] The centrifuged sample was obtained by centrifugation at 20,000 to 30,000 r / min and 4 to 8°C for 4 to 6 hours.

[0016] In one embodiment, the steps of performing polyacrylamide gel electrophoresis on the centrifuged sample, transferring to a membrane, blocking, incubating with antibodies, and developing to obtain WB bands include:

[0017] Take samples from the centrifuged sample from top to bottom, mix with electrophoresis buffer, and place in a boiling water bath for 8 to 12 minutes to obtain the electrophoresis pretreatment sample;

[0018] Perform polyacrylamide gel electrophoresis on the electrophoresis pretreated samples to obtain electrophoresis bands;

[0019] Transfer the Cap protein on the electrophoresis band to a PVDF membrane;

[0020] Block, react with monoclonal antibody (primary antibody) and HRP-labeled secondary antibody respectively;

[0021] Commercially available ultrasensitive colorimetric solution was prepared and developed using a developer to obtain WB images.

[0022] In one embodiment, when sampling the centrifuged sample from top to bottom, a 1 mL pipette is used to sample from top to bottom.

[0023] In one embodiment, in the step of obtaining the content of porcine circovirus type II Cap protein virus-like particles according to the grayscale value and total grayscale value of the particle band of porcine circovirus type II Cap protein on the WB strip:

[0024] Content of porcine circovirus type II Cap protein virus-like particles = (gray value of porcine circovirus type II Cap protein particle band / total gray value) × 100%.

[0025] In one embodiment, when sampling the centrifuged sample from top to bottom, a total of 12 tubes are sampled, each tube having 1 mL;

[0026] Porcine circovirus type II Cap protein virus-like particle content = (sum of gray values of tubes 6 to 12 / sum of gray values of tubes 1 to 12) × 100%.

[0027] In the technical solution of the present invention, by using sucrose density gradient ultracentrifugation to separate different self-assembled aggregates of Cap protein from the sample to be tested, the distribution of different self-assembled aggregates of Cap protein in different sucrose density gradients is detected by WB method, and grayscale analysis is performed, the proportion of VLP particles of porcine circovirus type II Cap protein expressed by baculovirus is successfully detected; by adopting the mature WB technology commonly used in molecular biology, using highly specific monoclonal antibodies as the reactants for detection, and cooperating with the amplification effect of commercial HRP-labeled secondary antibodies, the particle proportion of Cap protein can be more accurately reflected. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 The graph shows the concentration and grayscale value of the PCVII-Cap protein in the present invention;

[0030] Figure 2 is a linear fitting graph of the concentration and gray value of the PCVII-Cap protein in the present invention;

[0031] Figure 3 This is an IFA identification diagram of the Cap protein expressed by baculovirus in the present invention;

[0032] Figure 4 This is a diagram for determining the optimal sucrose gradient concentration during ultracentrifugation of the PCVII-Cap protein in the present invention;

[0033] Figure 5 The WB band diagrams of the PCVII-Cap protein in the present invention are shown in the supernatant tubes 1 to 12 during sucrose density gradient centrifugation;

[0034] Figure 6 For the present invention Figure 5 Electron micrographs corresponding to super separation samples 1 to 12;

[0035] Figure 7 This is an analysis chart of the antigen content of particles in Example 1 of the present invention.

[0036] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0037] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0038] It should be noted that, in the embodiments, those without specifying specific conditions, are carried out according to conventional conditions or the conditions recommended by the manufacturer. Those for reagents or instruments used that do not specify the manufacturer are conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes. Taking "A and / or B" as an example, it includes schemes A, B, or A and B that meet the same time. In addition, the technical solutions between the various embodiments can be combined with each other, but must be based on the ability of those of ordinary skill in the art to achieve. When the combination of the technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work belong to the scope of protection of the present invention.

[0039] In the prior art, antigen production for PCVII-Cap subunit vaccines can be achieved using a variety of expression systems. Semi-finished antigens used in vaccine formulation are often quantified using various methods such as Elisa and BCA. These detection methods all assume that recombinantly expressed PCVII-Cap monomers are uniform, effective, and can completely self-assemble into VLPs. However, the spatial structure of PCVII-Cap monomers expressed in different expression systems cannot be guaranteed to be completely consistent with the theoretical design, nor can it be guaranteed that they can all self-assemble into VLP particles. Furthermore, differences in the spatial structure of antigens cultured from different batches within the same expression system can lead to differences in self-assembly efficiency, further affecting differences between vaccine batches and resulting in inaccurate quantitative detection results for VLP particles.

[0040] In view of this, the present invention proposes a method for detecting the content of porcine circovirus type II Cap protein virus-like particles, comprising the following steps:

[0041] Providing a pretreated sample, performing sucrose density gradient ultracentrifugation on the sample to be tested, and obtaining a centrifuged sample;

[0042] The centrifuged samples were subjected to polyacrylamide gel electrophoresis, transferred to the membrane, blocked, incubated with antibodies, and developed to obtain WB bands;

[0043] The content of virus-like particles of porcine circovirus type II Cap protein was calculated based on the gray value of the particle band and the total gray value of the porcine circovirus type II Cap protein on the WB image.

[0044] In the technical solution of the present invention, by using sucrose density gradient ultracentrifugation to separate different self-assembled aggregates of Cap protein from the sample to be tested, the distribution of different self-assembled aggregates of Cap protein in each sucrose density gradient is detected by WB method, and grayscale analysis is performed, the proportion of VLP particles of porcine circovirus type II Cap protein expressed by baculovirus is successfully detected; by adopting the mature WB technology commonly used in molecular biology, using highly specific monoclonal antibodies as the reactants for detection, and cooperating with the amplification effect of commercial HRP-labeled secondary antibodies, the particle proportion of Cap protein can be more accurately reflected.

[0045] It should be noted that the WB image is a Western Blot image (protein blot), and the analysis method of the grayscale value on the WB image is not limited. Specifically, in some embodiments of the present invention, ImageJ is used for analysis. By adopting the same grayscale acquisition method, the method error that may be caused when the software calculates the grayscale data is avoided, the consistency of the analysis data is ensured, and the accuracy of the detection is thereby improved.

[0046] In one embodiment, the step of providing a pre-treated sample includes: taking a sample to be tested, filtering the sample to be tested, measuring the concentration of the sample, and mixing the sample with a diluent to a concentration of 1 to 10 μg / mL to obtain a pre-treated sample.

[0047] In the technical solution of the present invention, the concentration of the pretreated sample was adjusted to 1-10 μg / mL so that WB quantification could form a good linear range. The experimental results were verified as follows: Figure 1 and Figure 2 WB detection uses specific monoclonal antibodies verified by immunofluorescence assay (IFA) to ensure the objectivity and accuracy of the test results. The verification results are shown in Figure 3 PCVII-Cap protein samples were ultracentrifuged using different sucrose density gradients to detect the distribution of PCVII-Cap protein virus-like particles in 30% to 60% sucrose solutions. The test results are shown in Figure 4 PCVII-Cap protein samples were ultracentrifuged using 15% and 60% sucrose concentrations, and each sample tube was simultaneously observed by Western blotting and electron microscopy. It was found that the PCVII-Cap protein virus-like particles were consistent with the results shown by Western blotting bands. The results are as follows: Figure 5 and Figure 6 .

[0048] It should be noted that the specific method used in the filtration step is not limited and can be filtration at normal pressure or at reduced pressure. The specifications of the filter paper or filter membrane are also not limited. Specifically, in some embodiments of the present invention, a 0.45 μm filter membrane is used for filtration; the concentration determination refers to using the purification elution buffer as a blank and using Nanodrop one to determine the protein concentration of the sample to be tested. In this setting, the Cap protein concentration is determined using Nanodrop one, diluted to 3 mg / ml, and then appropriately diluted (20-fold to 3000-fold) before WB detection. After confirming the protein concentration and linear range of the Cap protein detected by WB, the Nanodrop one device can be used to perform protein determination of the sample to be tested, which is fast, simple, and efficient.

[0049] In one embodiment, the diluent comprises a PBS solution having a concentration of 0.1 to 0.01 mol / L and a pH of 7.2 to 7.4. Specifically, in some embodiments of the present invention, the PBS solution has a concentration of 0.01 mol / L and a pH of 7.2 to 7.4.

[0050] In one embodiment, the sample to be tested is subjected to sucrose density gradient centrifugation to obtain the centrifuged sample, comprising:

[0051] Adding sucrose of different concentrations into an ultracentrifuge tube in order from high concentration to low concentration, and adding the pretreated sample to the top layer of the ultracentrifuge tube;

[0052] The centrifuged sample was obtained by centrifugation at 20,000 to 30,000 r / min and 4 to 8°C for 4 to 6 hours.

[0053] In one embodiment, the steps of performing polyacrylamide gel electrophoresis on the centrifuged sample, transferring to a membrane, blocking, incubating with antibodies, and developing to obtain WB bands include:

[0054] Take samples from the centrifuged sample from top to bottom, mix with electrophoresis buffer, and place in a boiling water bath for 8 to 12 minutes to obtain the electrophoresis pretreatment sample;

[0055] Perform polyacrylamide gel electrophoresis on the electrophoresis pretreated samples to obtain electrophoresis bands;

[0056] Transfer the Cap protein on the electrophoresis band to a PVDF membrane;

[0057] Block, react with monoclonal antibody (primary antibody) and HRP-labeled secondary antibody respectively;

[0058] Commercially available ultrasensitive colorimetric solution was prepared and developed using a developer to obtain WB images.

[0059] In one embodiment, when centrifuged sample is sampled from top to bottom, 1mL pipette is adopted to sample from top to bottom. Specifically, in some embodiments of the present invention, when 1mL pipette is adopted to sample from top to bottom, the supporting gun tip tip bevel of 1mL pipette can be cut off about 3mm with the horizontal at about 30 °, when drawing last 1mL, if there is precipitation at the bottom of the ultracentrifuge tube, the bottom sediment can be resuspended and then sucked out together. So set, on the one hand, the observation when bevel is conducive to sampling, and the height of the arm raised when reducing sampling is achieved, which achieves the purpose of saving effort, on the other hand, the sharp angle is cut off, and the contact area between the pipette suction surface and the ultra-desorption surface is increased when sampling, and the suction force when reducing imbibition is on the influence of the lower floor's surface to be sucked out, which reduces detection error; when drawing last 1ml, the bottom sediment can be resuspended and then sucked out together, which avoids sample loss, to reduce detection error.

[0060] In one embodiment, in the step of calculating the content of porcine circovirus type II Cap protein virus-like particles according to the grayscale value and total grayscale value of the particle band of porcine circovirus type II Cap protein on the WB image:

[0061] Content of porcine circovirus type II Cap protein virus-like particles = (gray value of porcine circovirus type II Cap protein particle band / total gray value) × 100%.

[0062] In one embodiment, when sampling the centrifuged sample from top to bottom, a total of 12 tubes are sampled, each tube having 1 mL;

[0063] Porcine circovirus type II Cap protein virus-like particle content = (sum of gray values of tubes 6 to 12 / sum of gray values of tubes 1 to 12) × 100%.

[0064] In the technical solution of the present invention, when selecting Cap protein VLP particle tubes, based on the simultaneous verification of multiple rounds of electron microscopy and WB that Cap protein VLP particle tubes exist in 5 to 12 tubes, in order to more objectively evaluate the particle proportion of Cap protein, tubes 6 to 12 are used as baculovirus-expressed porcine circovirus type II Cap protein VLP particles, avoiding the unclear interface between VLP particles and non-particles caused by differences in operation and other factors, thereby ensuring the objectivity and accuracy of Cap protein VLP particles.

[0065] The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0066] Experimental methods

[0067] The SDS-PAGE (polyacrylamide gel electrophoresis) operation method was carried out according to the standard operating procedures of "Molecular Biology Experimental Techniques, 3rd Edition". This detection method used a 15-well gel with a thickness of 1.0 mm and a polyacrylamide content of 12% of the separating gel.

[0068] Furthermore, the pretreatment process of the electrophoresis sample mainly includes: adding 5× electrophoresis loading buffer to the electrophoresis sample, centrifuging for about 5 seconds, mixing with a vibrator for about 5 seconds, boiling in a water bath for 10 minutes, centrifuging for about 5 seconds, and mixing with a vibrator for about 5 seconds.

[0069] Furthermore, the precise loading of electrophoresis samples mainly includes: loading samples using a micro-sample needle, loading a volume of 5 μL / well, and controlling the sample volume in each well. The sample needle must be rinsed before loading the sample, and after loading each sample, it must be washed three times before loading the next sample. After all samples are loaded, the sample needle must be rinsed with clean water and stored.

[0070] Furthermore, electrophoresis is performed as soon as possible after the sample is added, and the voltage is controlled within 100 V in the first half hour, and then gradually adjusted to about 120 V. The electrophoresis is terminated according to the position of bromophenol blue.

[0071] Furthermore, the transfer process includes:

[0072] Label the PVDF membrane; pretreat the PVDF; assemble the transfer sandwich, remove all air bubbles between the PVDF membrane and the gel, and transfer the membrane under refrigeration.

[0073] Furthermore, the blocking solution is TBS containing 5% skimmed milk powder.

[0074] Furthermore, the primary antibody is a verified porcine circovirus type II Cap protein monoclonal antibody (such as Figure 3 shown).

[0075] Furthermore, the secondary antibody used is a commercial goat anti-mouse secondary antibody labeled with HRP.

[0076] Furthermore, the membrane was washed three times with TBST, each time for 10 min; and washed once more with TBS before color development, for 10 min.

[0077] Example 1

[0078] The embodiment of the present invention provides a method for effectively detecting the proportion of VLP particles expressing porcine circovirus type II Cap protein by baculovirus, comprising the following steps:

[0079] Step S1: Pretreatment of samples to be tested:

[0080] The sample to be tested was filtered with a 0.45 μm filter membrane; the purified eluate was used as a blank, and the protein concentration of the sample to be tested was determined to be 3.42 by Nanodrop one. It was then diluted to 10 μg / mL with 0.01 mol / L PBS using a gradient dilution method.

[0081] Step S2, sucrose density gradient ultracentrifugation:

[0082] Weigh 6 g of sucrose solid pellets, dissolve them in 8 mL of 0.01 mol / L PBS, and dilute to 10 mL to prepare a 60% sucrose solution. Prepare 15 mL of a 15% sucrose solution in the same manner. Add 5 mL of the 60% sucrose solution to a 13 mL ultracentrifuge tube, then add 6 mL of the 15% sucrose solution on top of the 60% sucrose solution. Add 1 mL of the sample diluted to 10 μg / mL to the top layer, ensuring a clear interface between the layers. Add sucrose to another ultracentrifuge tube in the same manner. Balance both tubes to ±0.0005 using an electronic scale. Pre-chill a Beckman ultracentrifuge with an SW41 rotor and perform ultracentrifugation according to standard ultracentrifugation procedures, selecting "SLOW" for both speeds, maintaining a temperature of 4°C, and centrifuging at 25,000 rpm for 5 hours at 4°C.

[0083] Step S3, sample separation after ultracentrifugation and SDS-PAGE electrophoresis sample preparation:

[0084] After ultracentrifugation, carefully remove the centrifuge tube from the sleeve, cut off about 3mm of the tip of the 1mL pipette at an angle of about 30° to the horizontal, and sample from the ultracentrifuge tube from top to bottom, 1mL per tube. When aspirating the last 1mL, pipette several times to the bottom of the ultracentrifuge tube and aspirate all the samples together. A total of 12 tubes are labeled 1 to 12 respectively; mix the aspirated samples from each tube, sample 200μL from each tube, add 50μL of 5× Loading buffer, and label them 1 to 12 respectively. Centrifuge for about 5 seconds, shake and mix on a vibrator for about 5 seconds, boil in a water bath for 10 minutes, centrifuge the samples for about 5 seconds, and shake and mix on a vibrator for about 5 seconds.

[0085] According to the SDS-PAGE operation method of "Molecular Biology Experimental Techniques, 3rd Edition", a gel with a thickness of 1.0 mm and a polyacrylamide content of 12% was prepared, and a 15-hole comb was used to prepare the stacking gel.

[0086] Step S4: Separate the Cap proteins in each ultracentrifugation sample by SDS-PAGE in Western blotting:

[0087] The sample was loaded using a loading needle at a volume of 5 μL / well, and 1× Loading buffer was loaded into the blank well.

[0088] After loading the sample, voltage-limited electrophoresis was started. The voltage was 100 V for the first half hour and then adjusted to 120 V. The electrophoresis was terminated when the bromophenol blue was about 5 mm from the bottom.

[0089] Step S5: Transfer the Cap protein from the electrophoresis gel to the PVDF membrane by Western blotting:

[0090] Cut the PVDF membrane into a size slightly larger than the electrophoresis gel and mark the WB sample information on the membrane;

[0091] After electrophoresis is completed, cut off the electrophoresis, take out the electrophoresis gel, pry open the glass plate, remove the accumulated gel, and completely remove the separation gel;

[0092] Remove the PVDF outer packaging paper and immerse the membrane completely in methanol;

[0093] Soak the transfer fixture, filter paper, and sponge in pre-chilled transfer buffer. Assemble the transfer sandwich in the order of cathode carbon plate + sponge + filter paper + electrophoresis gel + membrane + filter paper + sponge + anode carbon plate, removing all air from between the PVDF membrane and gel. Place the assembled transfer sandwich in the transfer tank and add transfer buffer.

[0094] Set the electrophoresis conditions and transfer the membrane at a current limit of 200 mA / cell for 2 hours. Transfer the membrane under refrigeration conditions.

[0095] Step S6, Western Blot blocking and incubation:

[0096] Weigh 5 g of skim milk powder and dissolve it in 100 mL of TBS;

[0097] After transfer, the membrane was placed in TBS containing 5% skim milk powder and blocked at room temperature for 1 h;

[0098] Primary antibody preparation: weigh 0.25 g skim milk powder, dissolve in 10 mL TBST, add 10 μL of the corresponding monoclonal antibody, mix well and set aside;

[0099] Primary antibody incubation: The blocked PVDF membrane was completely immersed in the primary antibody and incubated at room temperature for 2 h on a shaker at 60 rpm.

[0100] Membrane washing: After incubation, the PVDF membrane was completely immersed in TBST and shaken at 60 rpm for 10 min. Then, the membrane was washed with fresh PBST for a total of 3 times in the same manner.

[0101] Preparation of secondary antibody: Weigh 0.25 g skim milk powder, dissolve in 10 mL TBST, add 10 μL HPR-labeled secondary antibody, mix well and set aside;

[0102] Secondary antibody incubation: After washing, the PVDF membrane was completely immersed in the secondary antibody and incubated at room temperature for 1 h on a shaker at 60 rpm.

[0103] Membrane washing: After incubation, completely immerse the PVDF membrane in TBST and shake on a shaker at 60 rpm for 10 min. Wash the membrane three times in the same manner using fresh PBST. Wash the membrane once with TBS in the same manner for 10 min.

[0104] Step S7, WB color development:

[0105] Turn on the developer power supply about 5 minutes in advance and start pre-cooling the device (relevant operations are performed according to the device manual);

[0106] Start the computer, open the color development software, and connect the device to the software;

[0107] Prepare the chemiluminescent colorimetric solution by following the instructions for use of the ECL ultrasensitive colorimetric solution and mix the two components of the colorimetric solution in equal volumes.

[0108] Immerse the PVDF membrane completely in the color developing solution for 1 min;

[0109] Take out the PVDF membrane in the center of the color development plate and place it in the color development device;

[0110] Click the software to display the device color and take a photo for grayscale analysis. The WB banding diagram is as follows: Figure 5 As shown;

[0111] Step S8, calculating the proportion of porcine circovirus type II Cap protein VLP particles to be tested; specifically,

[0112] Import the WB photos into ImageJ and perform grayscale analysis according to Appendix 1. The analysis results are as follows: Figure 7 As shown, it is calculated according to the following formula:

[0113] Cap protein VLP particle ratio = (sum of gray values of tubes 6 to 12 / sum of gray values of tubes 1 to 12) × 100%.

[0114] The calculation results are shown in Table 1.

[0115] Table 1 VLP particle content detection results in Example 1

[0116] VLP particle gray value Gray value of non-VLP particles Total grayscale value VLP particle ratio 44950.789 2881.836 47832.625 93.98％

[0117] Example 2

[0118] According to Example 1, two people simultaneously used the invented method three times to test the proportion of three batches of baculovirus-expressed porcine circovirus type II Cap protein VLP particles. The test results are shown in Table 2.

[0119] Table 2 Virus content detection results in Example 2

[0120]

[0121]

[0122] As shown in the table, in Example 2, two operators simultaneously and three times used the inventive method to test the percentage of three batches of Cap protein VLP particles. The results showed that the coefficient of variation (CV%) between the results of single-person repeated testing and multi-person simultaneous testing was within 10%, indicating that the specificity, sensitivity, and repeatability of the method can meet the requirements of the test. This shows that the inventive method can effectively solve the technical problem of being unable to detect the percentage of circovirus type II Cap protein VLP particles expressed by a baculovirus expression system constructed through genetic engineering means.

[0123] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. All equivalent structural transformations made by using the contents of the present invention specification under the inventive concept of the present invention, or direct / indirect application in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A method for detecting the content of porcine circovirus type II Cap protein virus-like particles, characterized in that: The following steps are involved: Providing a pretreated sample, performing sucrose density gradient ultracentrifugation on the sample to be tested, and obtaining a centrifuged sample; The centrifuged samples were subjected to polyacrylamide gel electrophoresis, transferred to a membrane, blocked, incubated with antibodies, developed, and obtained Western blotting images; The content of virus-like particles of porcine circovirus type II Cap protein was calculated based on the gray value of the particle band and the total gray value of the porcine circovirus type II Cap protein on the WB image.

2. The method for detecting the content of porcine circovirus type II Cap protein virus-like particles according to claim 1, wherein The steps for providing a pre-treated sample include: The sample to be tested is taken, filtered, and the concentration is determined, and mixed with a diluent to 1-10 μg / mL to obtain a pretreated sample.

3. The method for detecting the content of porcine circovirus type II Cap protein virus-like particles according to claim 2, wherein: The diluent includes a PBS solution, wherein the concentration of the PBS solution is 0.1 to 0.01 mol / L and the pH value is 7.2 to 7.

4.

4. The method for detecting the content of porcine circovirus type II Cap protein virus-like particles according to claim 1, wherein The sample to be tested is subjected to sucrose density gradient centrifugation, and the steps of obtaining the centrifuged sample include: Adding different concentrations of sucrose into an ultracentrifuge tube in order from high concentration to low concentration, and adding the pretreated sample to the top layer of the ultracentrifuge tube; The centrifuged sample was obtained by centrifugation at 20,000 to 30,000 r / min and 4 to 8°C for 4 to 6 hours.

5. The method for detecting the content of porcine circovirus type II Cap protein virus-like particles according to claim 1, wherein The steps of performing polyacrylamide gel electrophoresis on the centrifuged sample, transferring to a membrane, blocking, incubating with antibodies, and developing to obtain WB bands include: Take samples from the centrifuged sample from top to bottom, mix with electrophoresis buffer, and place in a boiling water bath for 8 to 12 minutes to obtain the electrophoresis pretreatment sample; Perform polyacrylamide gel electrophoresis on the electrophoresis pretreated samples to obtain electrophoresis bands; Transfer the Cap protein on the electrophoresis band to a PVDF membrane; Block, react with monoclonal antibody (primary antibody) and HRP-labeled secondary antibody respectively; Commercially available ultrasensitive colorimetric solution was prepared and developed using a developer to obtain WB images.

6. The method for detecting the content of porcine circovirus type II Cap protein virus-like particles according to claim 5, wherein: When sampling centrifuged samples from top to bottom, use a 1 mL pipette to sample from top to bottom.

7. The method for detecting the content of porcine circovirus type II Cap protein virus-like particles according to claim 1, wherein In the step of obtaining the content of porcine circovirus type II Cap protein virus-like particles according to the gray value and total gray value of the particle band of porcine circovirus type II Cap protein on the WB image: Content of porcine circovirus type II Cap protein virus-like particles = (gray value of porcine circovirus type II Cap protein particle band / total gray value) × 100%.

8. The method for detecting the content of porcine circovirus type II Cap protein virus-like particles according to claim 7, wherein: When sampling centrifuged samples from top to bottom, a total of 12 tubes were sampled, with 1 mL per tube; Porcine circovirus type II Cap protein virus-like particle content = (sum of gray values of tubes 6 to 12 / sum of gray values of tubes 1 to 12) × 100%.

Images