High-purity purification method of porcine rotavirus and application thereof

Abstract

The application discloses a high-purity purification method of a porcine rotavirus and application thereof, and belongs to the technical field of biotechnology. The method comprises the following steps: proliferating the virus in a basic culture medium containing a small peptide and collecting supernatant; after being concentrated through first ultrafiltration, non-denaturing PAGE gel electrophoresis is carried out, and crude pure liquid is obtained by directly sucking residual liquid in a sample hole; then, agarose gel electrophoresis (the sample hole is pre-sealed) is carried out, and fine pure liquid is obtained by sucking residual liquid in the hole; and finally, high-purity virus is obtained by replacing liquid through second ultrafiltration. Through the cooperation of in-hole recovery of double electrophoresis and ultrafiltration, the method effectively removes impure proteins and free nucleic acids, the product is high in purity and complete in structure, and the technical problems of insufficient purity, complicated operation, high cost and easy virus inactivation in the prior art are effectively solved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a high-purity purification method for porcine rotavirus and its application. Background Technology

[0002] Porcine rotavirus (PoRV) belongs to the genus Rotavirus in the family Reoviridae. Its genome consists of 11 segments of double-stranded RNA, encoding 6 structural proteins and 5-6 non-structural proteins. This virus is the core pathogen causing acute intestinal infectious diseases in piglets, primarily affecting piglets under 8 weeks of age, exhibiting a significant winter peak in incidence, while adult pigs are often asymptomatically infected. Clinically, the core manifestation is gastrointestinal dysfunction, with infected pigs exhibiting watery diarrhea, vomiting, and severe dehydration. The mortality rate in newborn piglets is extremely high. Currently, there is no specific treatment for this disease; prevention and control mainly rely on vaccination and symptomatic supportive therapy. Existing vaccine types include trivalent live vaccines and inactivated vaccines. Whether in vaccine development, quality control evaluation, or basic virological research, the preparation of high-purity, highly active virus particles is indispensable; therefore, virus purification is a crucial preliminary step in related research.

[0003] Virus purification refers to the process of isolating target virus particles from complex biological samples while removing as many host cell components, extraneous proteins, nucleic acid fragments, and other impurities as possible, while maintaining virus recovery rate, infectivity, and structural integrity. Currently, commonly used virus purification methods in laboratories and industrial production include differential centrifugation, density gradient centrifugation, ultracentrifugation, polyethylene glycol (PEG) precipitation, chromatography, and filtration. However, these traditional methods generally have many limitations in practical applications: on the one hand, the purity of the purified products is often insufficient, retaining a significant amount of host cell proteins, free nucleic acids, and culture medium components, making it difficult to meet the requirements of high-precision research or high-quality vaccine production; on the other hand, most methods rely on expensive equipment such as ultracentrifuges and chromatography systems, and the operation procedures are complex, require highly skilled technicians, and are time-consuming, resulting in high purification costs and making them unsuitable for general laboratories to carry out efficient purification of small quantities of viruses.

[0004] The limitations of traditional purification methods are more pronounced due to the characteristics of porcine rotavirus. While its non-enveloped, icosahedral structure provides some environmental stability, it is still susceptible to aggregation, damage, or inactivation during purification due to mechanical shearing, osmotic pressure fluctuations, or the effects of chemical reagents. For example, while PEG precipitation is simple to operate, it produces many co-precipitated impurities, making subsequent impurity removal difficult; sucrose or cesium chloride density gradient centrifugation can achieve high purity, but the steps are complex, the centrifugation time is long, and high-concentration solutes may damage virus particles; chromatography is suitable for large-scale production, but the high cost of media and long method development cycle make it unsuitable for the rapid purification of small-scale samples.

[0005] Therefore, there is an urgent need to develop a new method for purifying porcine rotavirus that is simple to operate, requires no special or expensive instruments, is suitable for general laboratory conditions, and can achieve high-purity separation while ensuring the integrity and infectivity of the virus, so as to fill the gap in the field of efficient purification of small amounts of virus in existing technologies. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention provides a high-purity purification method for porcine rotavirus and its application. By adopting the process of "small peptide-assisted culture + double electrophoresis in-well recovery + ultrafiltration medium exchange", a small amount of porcine rotavirus is separated efficiently and with high purity under mild conditions, which effectively solves the technical problems of insufficient purity, complicated operation, high cost and easy inactivation of virus in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for high-purity purification of porcine rotavirus, comprising the following steps: S1. Virus proliferation: Cell culture was carried out by adding a small amino acid peptide solution with a molecular weight of less than 1 kDa to the basic culture medium, inoculating with porcine rotavirus, and collecting the cell culture supernatant after the virus had fully multiplied. Cell debris was removed by low-speed centrifugation, and the cell culture supernatant was collected. S2. First ultrafiltration concentration: The supernatant from step S1 is added to an ultrafiltration tube with a molecular weight cutoff of 10~150K and centrifuged to concentrate the virus solution. S3. Non-denaturing PAGE recovery: The virus concentrate from step S2 is subjected to non-denaturing polyacrylamide gel electrophoresis (PAGE). After electrophoresis, no staining is performed, and the residual liquid in the sample well is directly aspirated to obtain the crude pure virus solution. S4. Nucleic acid electrophoresis recovery: The crude virus solution from step S3 is subjected to agarose gel electrophoresis. The sample wells of the agarose gel are pre-sealed with agarose solution. After electrophoresis, no staining is performed. The residual liquid in the sample wells is directly aspirated to obtain the purified virus solution. S5. Second ultrafiltration solution exchange: Add the purified virus solution from step S4 to an ultrafiltration tube with a molecular weight cutoff of 10~150K for ultrafiltration solution exchange, and collect the retentate, which is high-purity porcine rotavirus.

[0008] Furthermore, in step S1, the basal culture medium is DMEM (Dürbeco modified Eagle medium), and the medium does not contain fetal bovine serum or only contains dialyzed serum; the final concentration of the amino acid peptides in the basal culture medium is 1~10 mg / ml; more preferably 1 mg / ml, 5 mg / ml or 10 mg / ml. Peptides within this concentration range can regulate the culture environment during the viral proliferation phase, reduce non-specific secretion of host proteins, reduce impurity load from the source, and at the same time, do not affect the infectivity and structural integrity of the virus.

[0009] Furthermore, in step S2, the centrifugation concentration conditions are: rotation speed 4000~6000 rpm, time 30~60 min; the concentration factor is 20~50 times. This combination of parameters can efficiently remove small molecule impurities in the culture medium while avoiding mechanical damage to virus particles, providing a suitable sample concentration for subsequent electrophoretic purification.

[0010] Furthermore, in step S3, the gel concentration for the non-denaturing PAGE electrophoresis is 4%–8%, the electrophoresis voltage is 80V, and the electrophoresis time is 90 min. Under these conditions, intact virus particles remain trapped in the sample wells because their particle size is much larger than the gel pore size, while most host proteins can enter the separating gel and be separated, achieving preliminary purification based on particle size. The non-staining operation avoids damage to the virus caused by dye binding or fixatives, ensuring the activity of the recovered virus.

[0011] Furthermore, in step S4, the concentration of the agarose gel is 0.5%~1%, the electrophoresis voltage is 120V, and the electrophoresis time is 50~60min. The sealing operation of the sample loading wells can prevent the sample from leaking out from the bottom of the wells during electrophoresis, ensuring the complete recovery of trace amounts of virus samples; this step uses a nucleic acid electrophoresis system to further separate nucleic acid-protein complexes that are similar in size to virus particles, making up for the insufficient ability of the above-mentioned non-denaturing PAGE to remove nucleic acid impurities.

[0012] Furthermore, in step S5, the concentration of the PBS buffer is 1 mM and the pH is 7.2~7.4; the number of buffer changes is 2~3 times.

[0013] When the final concentration of the small peptide in step S1 is 1 mg / ml or 5 mg / ml, an ultrafiltration tube with a molecular weight cutoff of 30 K can be used for the second ultrafiltration solution change; when the final concentration of the small peptide is 10 mg / ml, an ultrafiltration tube with a molecular weight cutoff of 100 K can be used to maximize the removal of residual small peptides and salt ions while ensuring the virus retention rate.

[0014] The present invention also provides a high-purity porcine rotavirus, which is prepared by the purification method described above.

[0015] Furthermore, the total protein concentration of the high-purity porcine rotavirus is 1-3 mg / ml.

[0016] This invention also provides the application of the above-described porcine rotavirus purification method in the preparation of high-purity porcine rotavirus vaccine antigens, diagnostic reagent standards, or viral seed solutions for cell infection experiments.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) High purity and thorough removal of impurities: This invention uses a dual in-well recovery mechanism of "non-denaturing PAGE + nucleic acid electrophoresis" to remove host protein, free nucleic acid and small molecule impurities step by step based on protein particle size and nucleic acid charge characteristics. The final product is verified by electrophoresis to have no visible impurities, and its purity is significantly better than that of traditional PEG precipitation method and differential centrifugation method.

[0018] (2) Good integrity and activity of the virus: The present invention adopts non-denaturing conditions throughout the process to avoid damaging operations such as staining, organic solvent precipitation, high-density solute and long-term high-speed centrifugation; small peptide-assisted culture improves the quality of the virus from the source, and double electrophoresis well recovery reduces the time the virus is exposed to adverse environments, thus ensuring the structural integrity and infectivity of the virus particles.

[0019] (3) Simple operation, low cost and strong applicability: The present invention only requires a conventional benchtop centrifuge, electrophoresis apparatus and ultrafiltration tube to complete all operations; the process is highly standardized and has low technical requirements for operators, making it particularly suitable for general laboratories to carry out rapid purification of small amounts of virus samples, filling the technical gap in the efficient purification of small amounts of porcine rotavirus.

[0020] (4) Parameter synergy and wide adaptability: The small peptide concentration, electrophoresis conditions and ultrafiltration pore size of this invention form a synergistically optimized parameter system, which can be flexibly adjusted according to different experimental needs, taking into account both the high recovery rate of low background samples and the deep purification of high impurity samples, and has good application scalability. Attached Figure Description

[0021] Figure 1 The images show the non-denaturing PAGE electrophoresis results of Examples 1-3 and the comparative examples of this invention; the left side of the images shows the protein markers, and the samples from Examples 1, 2, 3 and Comparative Example 1 are shown from left to right. Figure 2 The images show the nucleic acid electrophoresis diagrams of Examples 1-3 and the comparative examples of the present invention; the left side of the image shows the DNA Marker, and the samples from Examples 1, 2, 3 and Comparative Example 1 are shown from left to right. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0024] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0025] 1. Confirmation of raw material specifications Cell supernatant: Cell culture supernatant infected with porcine rotavirus (original solution to be purified).

[0026] Additive: A solution of small amino acid peptides with a molecular weight ≤1 kDa.

[0027] Basic culture medium: DMEM.

[0028] Ultrafiltration tubes: 2ml 100K molecular weight cutoff ultrafiltration tube (for the first step of concentration); 2ml 30K / 100K molecular weight cutoff ultrafiltration tube (for the second step of liquid replacement).

[0029] Electrophoresis reagents: non-denaturing PAGE gel preparation reagent, agarose, 1×TAE buffer, 5× non-denaturing loading buffer, nucleic acid loading buffer.

[0030] PBS buffer: 1 mM PBS (pH 7.2~7.4).

[0031] Protein concentration assay kit: BCA method kit.

[0032] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments: Example 1 This example demonstrates purification using a low-concentration small peptide (1 mg / ml) + 4% PAGE + 30K buffer exchange.

[0033] The specific steps are as follows: S1. Virus proliferation: Add a small amino acid peptide solution with a molecular weight ≤1 kDa to DMEM basal medium to a final concentration of 1 mg / ml, inoculate with porcine rotavirus, and collect the cell culture supernatant after the virus has fully multiplied. Centrifuge at low speed to remove cell debris, and take the supernatant as the original solution to be purified.

[0034] S2. First ultrafiltration concentration: Add the supernatant from step S1 to a 2ml ultrafiltration tube with a molecular weight cutoff of 100K, centrifuge at 4000rpm for 60min; discard the filtrate, repeat the process of adding the original solution and centrifuging until the concentration factor reaches 20 times, and obtain the virus concentrate.

[0035] S3. Non-denaturing PAGE recovery: Prepare a 4% non-denaturing PAGE gel, mix the virus concentrate from step S2 with 5× non-denaturing loading buffer, load the sample, and electrophores at a constant voltage of 80V for 90min; after electrophoresis, do not stain, immediately use a sterile pipette tip to carefully insert into the loading well, aspirate the residual liquid in the well, and obtain the crude pure virus solution.

[0036] S4. Nucleic acid electrophoresis recovery: Prepare a 1% agarose gel and seal the sample wells with a small amount of agarose solution. Mix the crude virus solution from step S3 with the nucleic acid loading buffer and spot it into the sealed wells. Electrophoresis is performed at a constant voltage of 120V for 50 minutes. After electrophoresis, do not stain. Immediately aspirate the residual liquid in the sample wells into a new centrifuge tube to obtain the purified virus solution.

[0037] S5. Second ultrafiltration solution change: Add the purified virus solution from step S4 to an ultrafiltration tube with a molecular weight cutoff of 30K, add 1mM PBS to the full mark, centrifuge at 4000rpm for 12min, repeat the solution change 3 times, and collect the retentate, which is the high-purity porcine rotavirus.

[0038] Example 2 This example demonstrates purification using a high-concentration small peptide (10 mg / ml) + 8% PAGE + 100K buffer exchange.

[0039] The specific steps are as follows: S1. Virus proliferation: Add a small amino acid peptide solution with a molecular weight ≤1K Da to DMEM basal medium to a final concentration of 10 mg / ml, inoculate with porcine rotavirus, and collect the cell culture supernatant after the virus has fully multiplied. Centrifuge at low speed to remove cell debris, and take the supernatant as the original solution to be purified.

[0040] S2. First ultrafiltration concentration: Add the supernatant from step S1 to a 2ml ultrafiltration tube with a molecular weight cutoff of 100K, centrifuge at 6000rpm for 30min; discard the filtrate, repeat the process of adding the original solution and centrifuging until the concentration factor reaches 50 times, and obtain the virus concentrate.

[0041] S3. Non-denaturing PAGE recovery: Prepare an 8% non-denaturing PAGE gel. Take the virus concentrate from step S2, mix it with 5× non-denaturing loading buffer, and load the sample. Perform electrophoresis at 80V constant voltage for 90 min. After electrophoresis, do not stain. Immediately insert a sterile pipette tip into the loading well and aspirate the residual liquid in the well to obtain the crude pure virus solution.

[0042] S4. Nucleic acid electrophoresis recovery: Prepare a 0.5% agarose gel and seal the sample wells with a small amount of agarose solution. Mix the crude virus solution from step S3 with the nucleic acid loading buffer and spot it into the sealed wells. Electrophoresis is performed at a constant voltage of 120V for 60 minutes. After electrophoresis, do not stain. Immediately aspirate the residual liquid in the sample wells into a new centrifuge tube to obtain the purified virus solution.

[0043] S5. Second ultrafiltration solution change: Add the purified virus solution from step S4 to an ultrafiltration tube with a molecular weight cutoff of 100K, add 1mM PBS to the full mark, centrifuge at 6000 rpm for 15 min, repeat the solution change 3 times, and collect the retentate, which is the high-purity porcine rotavirus.

[0044] Example 3 This example demonstrates purification of a medium-concentration small peptide (5 mg / ml) using 4% PAGE and 30K buffer exchange.

[0045] The specific steps are as follows: S1. Virus proliferation: Add a small amino acid peptide solution with a molecular weight ≤1K Da to DMEM basal medium to a final concentration of 5 mg / ml, inoculate with porcine rotavirus, and collect the cell culture supernatant after the virus has fully multiplied. Centrifuge at low speed to remove cell debris, and take the supernatant as the original solution to be purified.

[0046] S2. First ultrafiltration concentration: Add the supernatant from step S1 to a 2ml ultrafiltration tube with a molecular weight cutoff of 100K, centrifuge at 5000rpm for 30min; discard the filtrate, repeat the process of adding the original solution and centrifuging until the concentration factor reaches 50 times, and obtain the virus concentrate.

[0047] S3. Non-denaturing PAGE recovery: Prepare a 4% non-denaturing PAGE gel, mix the virus concentrate from step S2 with 5× non-denaturing loading buffer, load the sample, and electrophores at a constant voltage of 80V for 90min; after electrophoresis, do not stain, immediately use a sterile pipette tip to carefully insert into the loading well, aspirate the residual liquid in the well, and obtain the crude pure virus solution.

[0048] S4. Nucleic acid electrophoresis recovery: Prepare a 1% agarose gel and seal the sample wells with a small amount of agarose solution. Mix the crude virus solution from step S3 with the nucleic acid loading buffer and spot it into the sealed wells. Electrophoresis is performed at a constant voltage of 120V for 60 minutes. After electrophoresis, do not stain. Immediately aspirate the residual liquid in the sample wells into a new centrifuge tube to obtain the purified virus solution.

[0049] S5. Second ultrafiltration solution change: Add the purified virus solution from step S4 to an ultrafiltration tube with a molecular weight cutoff of 30K, add 1mM PBS to the full mark, centrifuge at 5000 rpm for 15 min, repeat the solution change 3 times, and collect the retentate, which is the high-purity porcine rotavirus.

[0050] Comparative Example 1 This comparative example uses the traditional PEG precipitation + sucrose density gradient centrifugation method, and the specific steps are as follows: (1) Culture medium preparation and virus proliferation: The same culture medium (DMEM + 10 mg / mL small peptide) as in Example 2 was used, and the same porcine rotavirus was inoculated. The culture was carried out at 37°C and 5% CO2 for 72 h. The supernatant was collected, and the initial culture volume was the same as in Example 2.

[0051] (2) PEG precipitation: Add PEG8000 to the culture supernatant to a final concentration of 8%, and let stand overnight at 4°C to precipitate the virus.

[0052] (3) Centrifugation collection: Centrifuge at 4000 rpm for 30 min, discard the supernatant, and resuspend the precipitate with an appropriate amount of PBS.

[0053] (4) Sucrose density gradient centrifugation: Spread the resuspended liquid on the surface of a continuous sucrose density gradient liquid of 20%~60% and centrifuge at 25000rpm for 2 h.

[0054] (5) Dialysis to remove sugar: Aspirate the visible viral bands, put them into a dialysis bag, and dialyze with 1mM PBS buffer for 24 hours. Change the external dialysis solution every 6 hours during this period to completely remove sucrose and residual small peptides. The internal dialysis solution is then collected as the purified sample.

[0055] Test case The final virus samples obtained in Examples 1-3 and the comparative example (using the traditional PEG precipitation method) were subjected to total protein concentration determination, non-denaturing PAGE electrophoresis analysis, and nucleic acid electrophoresis analysis. The specific operations and results are as follows: (1) Determination of total protein concentration Take 20-50 μL of each sample and perform detection using the BCA protein concentration assay kit. After mixing the sample with the BCA working solution, incubate at 37℃ for 30 min, and measure the absorbance at 562 nm (A562) using a microplate reader. Calculate the total protein concentration by referring to the BSA standard curve. The detection results are shown in Table 1: Table 1 As shown in Table 1, the total protein concentration of the sample was 10.0 mg / mL. Because the comparative example used the same high-concentration small peptide culture medium (10 mg / mL) as Example 2, and the traditional PEG-sucrose gradient method could not effectively remove co-precipitated small peptides and host proteins, the final product's total protein concentration was significantly higher than that of Example 2. Examples 1-3 showed lower total protein concentrations, and almost all of their proteins were target viral structural proteins with extremely low impurity content, indicating that the method of this invention has a significant advantage in removing non-target proteins.

[0056] (2) Non-denaturing PAGE electrophoresis analysis Take appropriate amounts of samples from Examples 1-3 and Comparative Example 1, add non-denaturing loading buffer, mix well, and load onto a 4%-8% non-denaturing PAGE gel. Perform electrophoresis at 80V for 90 minutes. After electrophoresis, perform Coomassie brilliant blue staining (staining for 30 minutes and destaining for 1 hour) and take photos to record the gel images.

[0057] like Figure 1 As shown, the left side is the protein marker, and from left to right are the sample lanes of Example 1, Example 2, Example 3 and Comparative Example 1.

[0058] The results showed that lanes 1-3 of Examples only showed a single main band at the location of the complete porcine rotavirus particle, with no visible contaminating protein bands; in lane 1 of Comparative Example, in addition to the main viral band, multiple contaminating protein bands with widely distributed molecular weights were also visible, indicating that the traditional method failed to effectively remove host cell proteins and culture medium impurities.

[0059] (3) Nucleic acid electrophoresis analysis Take appropriate amounts of samples from Examples 1-3 and Comparative Example 1, add nucleic acid loading buffer, mix well, and load onto a 1% agarose gel (containing EB nucleic acid dye). Electrophoresis is performed at a constant voltage of 120V for 40 minutes. After electrophoresis, observe and photograph the gel under a UV gel imaging system.

[0060] like Figure 2 As shown, the left side is the DNA Marker, and from left to right are the sample lanes of Example 1, Example 2, Example 3 and Comparative Example 1.

[0061] The results showed that no free nucleic acid bands or tailing were observed in lanes 1-3 of Examples, indicating that the virus particles were intact and there was no free RNA / DNA residue. In lane 1 of Comparative Example, obvious nucleic acid tailing and diffuse bands were observed, suggesting that the virus particles were partially damaged during the traditional purification process, resulting in the release of genomic RNA and failure to effectively remove free nucleic acid impurities.

[0062] In summary, the purification method described in this invention, which combines "small peptide-assisted culture + dual electrophoresis well recovery + ultrafiltration medium exchange," has significant advantages over the traditional PEG precipitation method: After dual verification by non-denaturing PAGE and nucleic acid electrophoresis, no contaminating protein bands or free nucleic acid bands were detected in the obtained samples, and the product purity met electrophoretic grade standards. Simultaneously, the nucleic acid electrophoresis showed no tailing or diffusion, confirming that the virus particles remained intact throughout the purification process, maintaining their structural integrity. Through the synergistic effect of size exclusion within the dual electrophoresis wells and differentiated ultrafiltration medium exchange, deep removal of impurities was achieved while ensuring virus recovery rate, truly achieving a purification state of "low total amount, high purity, and high integrity."

[0063] The above provides a detailed description of a high-purity purification method for porcine rotavirus and its applications. Specific examples have been used to illustrate the principles and implementation methods of this invention. The descriptions of these examples are merely illustrative and are intended to aid in understanding the method and its core concepts. It should be noted that those skilled in the art can make various improvements and modifications to this invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims.

Claims

1. A method for high-purity purification of porcine rotavirus, characterized in that, Includes the following steps: S1. Virus proliferation: Cell culture was carried out by adding a small amino acid peptide solution with a molecular weight of less than 1 kDa to the basic culture medium, inoculating with porcine rotavirus, and collecting the cell culture supernatant after the virus had fully multiplied. Cell debris was removed by low-speed centrifugation, and the cell culture supernatant was collected. S2. First ultrafiltration concentration: The supernatant from step S1 is added to an ultrafiltration tube with a molecular weight cutoff of 10~150K and centrifuged to concentrate the virus solution. S3. Non-denaturing PAGE recovery: The virus concentrate from step S2 is subjected to non-denaturing PAGE gel electrophoresis. After electrophoresis, no staining is performed, and the residual liquid in the sample well is directly aspirated to obtain the crude pure virus solution. S4. Nucleic acid electrophoresis recovery: The crude virus solution from step S3 is subjected to agarose gel electrophoresis. The sample wells of the agarose gel are pre-sealed with agarose solution. After electrophoresis, no staining is performed. The residual liquid in the sample wells is directly aspirated to obtain the purified virus solution. S5. Second ultrafiltration solution exchange: Add the purified virus solution from step S4 to an ultrafiltration tube with a molecular weight cutoff of 10~150K for ultrafiltration solution exchange, and collect the retentate, which is high-purity porcine rotavirus.

2. The purification method according to claim 1, characterized in that, In step S1, the basal culture medium is DMEM culture medium, and no fetal bovine serum or only dialyzed serum is added to the culture medium; the final concentration of the amino acid peptide in the basal culture medium is 1~10 mg / ml.

3. The purification method according to claim 2, characterized in that, In step S1, the final concentration of the amino acid peptide is 1 mg / ml, 5 mg / ml, or 10 mg / ml.

4. The purification method according to claim 1, characterized in that, In step S2, the conditions for centrifugal concentration are: rotation speed 4000~6000 rpm, time 30~60 min; the concentration factor is 20~50 times.

5. The purification method according to claim 1, characterized in that, In step S3, the gel concentration for the non-denaturing PAGE electrophoresis is 4%~8%, the electrophoresis voltage is 80V, and the electrophoresis time is 90min.

6. The purification method according to claim 1, characterized in that, In step S4, the concentration of the agarose gel is 0.5%~1%, the electrophoresis voltage is 120V, and the electrophoresis time is 50~60min.

7. The purification method according to claim 1, characterized in that, In step S5, the concentration of the PBS buffer is 1 mM and the pH is 7.2-7.4; the number of buffer changes is 2-3 times.

8. A high-purity porcine rotavirus, characterized in that, It is prepared by the purification method according to any one of claims 1-8.

9. The high-purity porcine rotavirus according to claim 8, characterized in that, The total protein concentration of the high-purity porcine rotavirus was 1-3 mg / ml.

10. The application of the porcine rotavirus purification method according to any one of claims 1-7 in the preparation of high-purity porcine rotavirus vaccine antigens, diagnostic reagent standards, or viral seed solutions for cell infection experiments.

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