Porcine delta coronavirus capable of efficiently reproducing on suspension cells and application of porcine delta coronavirus

Abstract

The invention relates to a porcine delta coronavirus capable of efficiently reproducing on suspension cells and application of the porcine delta coronavirus, and belongs to the technical field of veterinary biology. The porcine delta coronavirus strain is named as a porcine delta coronavirus strain DC19, and the porcine delta coronavirus strain can be stably proliferated on suspension cells to produce virus liquid with high virus content and high immunogenicity. The porcine delta coronavirus can be cultured in a full-suspension manner under the serum-free condition, so that the production scale of virus liquid can be expanded conveniently, and the technical requirements of GMP production can be met; the vaccine prepared from the produced virus liquid has a good immune effect and has a better market prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary biology, and specifically relates to a porcine delta coronavirus that can be efficiently propagated on suspension cells and its application in the production of veterinary vaccines. Background Art

[0002] Porcine delta coronavirus (PDCoV) is a new type of porcine enteric coronavirus that can cause diarrhea and vomiting in suckling piglets aged 5 to 15 days, leading to rapid dehydration and exhaustion, with a morbidity and mortality rate of 50% to 100%. In addition, PDCoV is often co-infected with diarrhea viruses such as porcine epidemic diarrhea virus (PEDV) and porcine transmissible gastroenteritis virus (TGEV), which poses a great challenge to the prevention and treatment of the disease. In recent years, PDCoV has begun to spread widely in various parts of my country, seriously hindering the development of the pig industry. Since there is currently no commercial vaccine or specific drug, strengthening disease prevention is still an important means to control the spread of the disease in my country.

[0003] The suspension culture process has been widely used in the field of biopharmaceuticals and has shown obvious advantages in the field of biopharmaceuticals. Compared with the rotary bottle culture process, the biggest advantages of the suspension culture technology are: first, the number of effective working cells per unit area increases; second, the fully enclosed, pipelined system production process and process automation monitoring and control technology not only reduce the chance of cell contamination, but also reduce the labor intensity while reducing the impact of human operation factors; third, the expansion of the bioreactor volume improves the uniformity of the end product, and combined with the later purification process, the product output and quality are significantly improved; fourth, the labor and workshop, water, electricity, raw materials, energy and other costs used in vaccine production are much lower than the traditional rotary bottle culture process, and the overall cost is greatly reduced.

[0004] The full suspension culture process is a cell culture process that does not rely on serum and carriers. With the increasing degree of intensiveness of the animal husbandry industry and the expansion of breeding scale, vaccines that are both low-cost and high-quality will inevitably conform to the needs of the times. The development of porcine delta coronavirus suitable for cell suspension culture for vaccine production is an inevitable trend in the development of the veterinary biological products industry. Summary of the invention

[0005] In order to solve the problems in the prior art, the patent of the present invention designs a porcine delta coronavirus that can be efficiently propagated on suspension cells. The strain has a high proliferation titer and good immunogenicity, and can be applied to the production process of full suspension culture, thereby reducing production costs, improving the efficiency of downstream purification, rapidly and stably expanding the production scale, and achieving balanced and stable antigen quality.

[0006] The technical solution adopted by the present invention is:

[0007] The present invention provides a porcine delta coronavirus that can be efficiently propagated on suspension cells. The porcine delta coronavirus is the porcine delta coronavirus DC19 strain. The recommended classification name of the porcine delta coronavirus DC19 strain is Porcinedeltacoronavirus. It is deposited in the General Microbiology Center of the China Culture Collection Administration, with the address at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No.46220, and the deposit date is November 15, 2024.

[0008] The method for adapting a porcine delta coronavirus that is efficiently propagated on suspension cells to serum-free suspension ST cell culture as described above is characterized in that the specific steps of the method are:

[0009] (1) Take ST cells grown into a monolayer, discard the culture medium, wash twice with PBS, take porcine delta coronavirus and dilute it with serum-free DMEM culture medium containing 7.5 μg / mL pancreatin, incubate at 37°C for 1 hour, and gently shake the cell bottle once every 20 minutes to mix once; then discard the virus solution, add 5 mL of serum-free DMEM culture medium containing 7.5 μg / mL pancreatin, and culture in a 37°C, 5% CO2 incubator for 3 to 5 days, then freeze and thaw once at -20°C, harvest the virus solution and aliquot for storage; blindly propagate in the same way until cytopathic effects occur, and collect the culture; blindly propagate the virus solution on ST cells for 4 generations, and when the cells develop lesions, propagate the virus to the 8th generation, freeze and thaw once, harvest the virus solution and aliquot for storage;

[0010] (2) ST cells were cultured into a monolayer in a 6-well plate, the nutrient solution was discarded, and the cells were washed twice with Hank's solution. The porcine delta coronavirus suspension obtained in step (1) was diluted with a serum-free maintenance solution, and the diluted virus solution was inoculated into ST cells at an inoculation volume of 1 / 10 of the original nutrient solution. The cells were cultured at 37° C. for 1 hour, the virus solution was aspirated, and 3 ml of a mixture of 2 times the serum-containing nutrient solution and 1.6% agarose was added to each well. The cells were cultured at 37° C. The cell morphology was observed daily until plaques appeared. The positions of the plaques were marked under an inverted microscope. The plaques were aspirated with a sterilized pipette in a biological safety cabinet and dissolved in 800 μl of PBS.

[0011] (3) The virus solution obtained in step (2) was inoculated into ST suspension cells and subcultured for 5 generations. The virus solution was taken and diluted with serum-free DMEM culture medium containing 7.5 μg / mL trypsin, and then inoculated into ST cells that had been washed twice with serum-free DMEM culture medium containing 7.5 μg / mL trypsin, and cultured in a serum-free DMEM culture medium containing 7.5 μg / mL trypsin at 37°C and 5% CO2 in an incubator for 96 hours.

[0012] The porcine delta coronavirus strain efficiently propagated on suspension cells provided by the present invention can be used in the preparation of porcine delta coronavirus vaccines and in the preparation of therapeutic drugs for diseases caused by porcine delta coronavirus infection in piglets.

[0013] The present invention also provides a method for preparing a virus solution of a porcine delta coronavirus that can be efficiently propagated on suspension cells as described above, wherein the preparation method comprises placing ST suspension cells in Aplo ST suspension culture medium, culturing on a shaker in an incubator at 37°C and containing 5% CO2 for 72 hours, and setting the shaker speed to 140 r / min. When the ST suspension cell viability is >95%, the cell growth is stable, and the number of viable cells reaches 6×10 6 Afterwards, the virus inoculation amount MOI was 0.01, and the venom of porcine delta coronavirus DC19 strain adapted to suspension culture was inoculated. After adsorption for 1 hour, Aplo ST medium was added as the maintenance medium; then the cells were placed in a 37°C CO2 incubator with a volume percentage of 5% and a culture speed of 140r / min. Samples were taken for cell counting 72 hours after culture to harvest the virus liquid.

[0014] The method for preparing an inactivated vaccine of the porcine delta coronavirus efficiently propagated on suspension cells provided by the present invention is: adding the virus liquid obtained by the aforementioned virus liquid preparation method to a BEI solution with a final concentration of 0.01 mol / L, inactivating it at 37°C for 24 hours after sufficient mixing, and emulsifying the inactivated virus liquid and adjuvant in a ratio of 1:1 (mass ratio). After complete emulsification, the inactivated vaccine is obtained.

[0015] Furthermore, the immune adjuvant is one of aluminum adjuvant, propolis adjuvant, and oil-water biphasic adjuvant.

[0016] Compared with the prior art, the present invention patent designs a strain of porcine delta coronavirus that can be efficiently propagated on suspension cells and its application in the production of veterinary vaccines: the present invention successfully separates and domesticates a strain of porcine delta coronavirus that is highly adaptable to suspension cells. The porcine delta coronavirus of the present invention does not use a serum-containing culture medium in the production of vaccines in serum-free suspension culture, reduces downstream processing procedures, has a high virus propagation rate, and can greatly improve the yield of biological products. Therefore, the porcine delta coronavirus that is adapted to suspension cell culture and efficiently propagates provided by the present invention can significantly reduce the production cost of the virus solution, while improving the efficiency of downstream purification, and can quickly and stably expand the production scale, the quality is easy to achieve, and it is balanced and stable.

[0017] The results of the immunogenicity study of the PDCoV DC19 strain that is highly adapted to suspension cells of the present invention showed that the neutralizing antibodies induced by the inactivated vaccine of the PDCoV DC19 strain reached 1:608 to 1:1024, indicating that the PDCoVDC19 strain isolated, identified and screened by the present invention has excellent immunogenicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are the cytopathic images of porcine delta coronavirus isolated using ST cells in Example 1; A represents the cell image of the uninfected group, and B represents the cell image of the infected group.

[0019] Figure 2 This is a fluorescence quantitative PCR detection diagram of porcine delta coronavirus in Example 2.

[0020] Figure 3 These are immunofluorescence (IFA) detection images of porcine delta coronavirus in Example 2; A represents the cellular immunofluorescence image of the uninfected group, and B represents the cellular immunofluorescence image of the infected group.

[0021] Figure 4 This is a comparison chart of the virus content determination of different porcine delta coronavirus clones in Example 3.

[0022] Figure 5 This is a graph showing the growth and decline of neutralizing antibodies in immunized pigs in Example 4. DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments. The technical solutions in the embodiments of the present invention are clearly and completely described, and the described embodiments are only embodiments of a part of the present invention, not all of them. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Example 1

[0025] During the isolation and identification of porcine delta coronavirus, the DMEM culture medium and trypsin used were purchased from Gibco.

[0026] Step 1: Collection and processing of diseased materials

[0027] Aseptically collect the small intestinal contents and intestines of piglets that are clinically PDCoV-positive by RT-PCR, crush them, add an equal volume of serum-free DMEM medium, mix well, freeze and thaw repeatedly at -80°C for 3 times, centrifuge at 8000r / min for 10min, take the supernatant into another sterile centrifuge tube, filter and sterilize with a 0.22μm filter membrane to obtain a diseased material suspension, store below -70°C or directly inoculate cells.

[0028] Step 2 Virus isolation and passaging

[0029] Take the ST cells grown into a monolayer, discard the culture medium, wash twice with PBS, take the above-treated diseased sample, dilute and inoculate with serum-free DMEM culture medium containing 7.5μg / mL pancreatin, incubate at 37℃ for 1 hour, and gently shake the cell bottle once every 20 minutes to mix once; then discard the virus solution, add 5mL of serum-free DMEM culture medium containing 7.5μg / mL pancreatin, and culture in a 37℃, 5% CO2 incubator for 3-5 days, then freeze and thaw once at -20℃, harvest the virus solution and store it in aliquots. Blindly propagate in the same way until cell lesions occur. Collect the culture and perform RT-PCR identification. Blindly propagate the virus solution on ST cells for 4 generations, and the cells develop lesions. The virus is propagated to the 8th generation. 48 hours after inoculation, the cells appear rounded, stretched, broken and detached ( Figure 1 B), no cytopathic effect was observed in normal cells ( Figure 1 A). After freezing and thawing once, the virus solution was harvested and stored in aliquots.

[0030] Example 2

[0031] Reagents used in the identification of porcine delta coronavirus: PDCoV monoclonal antibody; fluorescent secondary antibody Goat anti-Mouse IgG (H+L) Superclonal TM Secondary Antibody, Alexa Fluor 488 (Invitrogen TM ), purchased from Thermo Fisher Scientific. qPCR primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. 2× Taq PCR Master Mix was purchased from Takara.

[0032] Step 1 Fluorescence quantitative PCR identification

[0033] The virus that proliferated stably on ST cells was identified by fluorescent quantitative PCR. The reaction system was as follows: 200 μL of the virus solution prepared in Example 1 was taken, RNA was extracted using a DNA / RNA extraction kit, 1 μg of RNA was reverse transcribed into cDNA according to the instructions of the reverse transcription kit, and PCR amplification was performed using PDCoV fluorescent quantitative PCR primers:

[0034] qPDCoV-F(5'-CGACCACATGGCTCCAATTC3')

[0035] qPDCoV-R(5'-CAGCTCTTGCCCATGTAGCTT-3')

[0036] qPDCoV-P(5'-FAM-CACACCAGTCGTTAAGCATGGCAAGC-TAMRA-3')

[0037] The PCR reaction system is a fluorescent PCR system, totaling 25 μl

[0038] 2×TaqPCRMasterMix (enzyme) 12.5μl <![CDATA[ddH2O]]> 7.5μl Upstream primer: qPDCoV-F 1μl Downstream primer: qPDCoV-R 1μl Sample DNA / cDNA 2μl Fluorescent probe: qPDCoV-P 1μl

[0039] Reaction conditions: 95℃ pre-denaturation for 10min; 95℃ denaturation for 15s; 60℃ extension for 60s and collection of fluorescence signals. A total of 40 cycles. After the test, the results were determined based on the collected fluorescence curves and Ct values.

[0040] Result determination: The negative control had no Ct value and no amplification curve, indicating that the sample was negative for PDCoV nucleic acid.

[0041] Positive: Ct value ≤ 30.0, and a typical amplification curve appears, indicating that the sample is positive for PDCoV nucleic acid.

[0042] Suspicious: Samples with Ct values ​​> 30 are recommended to be retested. If there is no Ct value in the retest result, it is negative, otherwise it is positive.

[0043] The results showed that the PDCoV virus infection group had a typical amplification curve and a CT value of less than 30 when the isolated virus was amplified by fluorescent quantitative PCR using PDCoV fluorescent quantitative PCR primers. Figure 2 shown.

[0044] Step 2: Identification by immunofluorescence (IFA)

[0045] The virus that proliferates stably on ST cells is identified by indirect immunofluorescence (IFA) method, and the method is as follows: the porcine delta coronavirus liquid prepared in Example 1 is inoculated into the ST cells in suspension culture, and after culturing for 48 hours, the inoculated sample is washed twice with PBS, and a small amount of supernatant is left to suspend the cells, and the suspended cells are spread in a 96-well cell culture plate, and after drying; 80% acetone is used to fix for 30 minutes at 4°C; PBS is added to the 96-well cell culture plate at 200 μl / well and washed twice; 100 μl / well of PDCoV monoclonal antibody diluted 100 times is added to each well, and incubated in a 37°C incubator for 1 hour; PBS is added to the 96-well cell culture plate at 200 μl / well and washed three times; 100 μl / well of fluorescent secondary antibody diluted 400 times is added to each well, and incubated in a light-proof 37°C incubator for 1 hour; PBS is added to the 96-well cell culture plate at 200 μl / well and washed three times; and observed under a fluorescence microscope. The results showed that ST cells inoculated with porcine delta coronavirus had obvious fluorescence, while cells not inoculated with the virus had no fluorescence, indicating that the isolated virus can react specifically with the monoclonal antibody against PDCoVN protein. Figure 3 shown).

[0046] Example 3

[0047] Reagents used in the screening process of highly adapted porcine delta coronavirus in suspension cells: agarose was purchased from Biowest; DMEM and trypsin were purchased from Gibco.

[0048] Step 1 Screening of highly adapted porcine delta coronaviruses in suspension cells

[0049] ST cells were cultured in a 6-well plate as a monolayer, the nutrient solution was discarded, and the cells were washed twice with Hank's solution. The three strains of porcine delta coronavirus suspensions extracted by the above process were diluted 10 times in a serum-free maintenance solution, and 10 -5 , 10 -6 , 10 -7 , 3 dilutions, 3 wells were inoculated for each dilution, and the inoculation volume of each well was 1 / 10 of the original nutrient solution. Incubate at 37°C for 1 hour to aspirate the virus solution, add 3 ml of a mixture of 2 times serum-containing nutrient solution and 1.6% agarose to each well, and culture at 37°C. Observe the cell morphology every day until plaques appear. Mark the position of the plaques under an inverted microscope, and in a biosafety cabinet, aspirate the plaques with a sterilized pipette and dissolve them in 800 μl of PBS. The purified virus was used for virus expansion. A total of 15 virus clones were obtained.

[0050] Step 2 Determination of the viral content of highly adapted porcine delta coronavirus in suspension cells The clones were inoculated into ST suspension cells and passaged for 5 generations to determine the viral content TCID 50 Determination of TCID 50 The specific operation steps are as follows: dilute the virus solution 10 times in a row in an EP tube or penicillin bottle with serum-free DMEM culture medium containing 7.5 μg / mL trypsin, starting from 10 -1 Up to 10 -9 ; Wash the ST cell monolayer in the 96-well plate twice with serum-free DMEM culture medium containing 7.5 μg / mL trypsin, and take 10 -4 Up to 10 -9 The diluted virus solution was inoculated into the washed ST cell wells, and each dilution was inoculated into a vertical row of 8 wells, with 100 μL in each well; at the same time, 8 wells of normal cell control were set up, and the ST cell monolayer in the cell control wells was also washed twice with serum-free DMEM culture medium containing 7.5 μg / mL trypsin, and then 100 μL of serum-free DMEM culture medium containing 7.5 μg / mL trypsin was added to each well; the 96-well plate was placed in an incubator at 37°C and 5% CO2 for 96 hours, and the cell lesions were observed and recorded every day. TCID was calculated according to the Reed-Muench method 50 The results showed that the virus content of A-5 clone strain was not less than 10 8.5 TCID 50 / ml, and the results are shown in Table 1. The porcine delta coronavirus cell-adapted strain was named porcine delta coronavirus DC19 strain.

[0051] Table 1 Results of determination of porcine delta coronavirus virus content at different generations

[0052]

[0053] Step 3 RT-PCR identification and sequencing of porcine delta coronavirus

[0054] The DC19 strain of porcine delta coronavirus liquid that was stably proliferated on ST suspension was used to extract RNA using a DNA / RNA extraction kit, and 1 μg RNA was reverse transcribed into cDNA according to the instructions of the reverse transcription kit, PCR amplified using PDCoV fluorescent quantitative PCR primers, and sent to BGI for sequencing. The sequencing result is shown in SEQ ID NO.1, and sequence alignment shows that the sequence is the porcine delta coronavirus S gene sequence.

[0055] Step 4 Study on the stability of passage of highly adapted porcine delta coronavirus in suspension cells

[0056] The porcine delta coronavirus DC19 strain that was stably propagated on ST suspension cells and the untamed isolate were subcultured on ST suspension cells for 20 generations, and the virus content was determined for each generation. The results showed that the virus content of the untamed isolate after 20 generations was 10 6.5 ~10 7.5 TCID 50 / ml, the virus content of the domesticated and screened porcine delta coronavirus DC19 strain was no less than 10 8.5 TCID 50 / ml, indicating that the DC19 strain grew stably after domestication and the virus content was significantly higher than that of the undomesticated strain. The results are shown in Table 2.

[0057] Table 2 Results of determination of porcine delta coronavirus virus content at different generations

[0058]

[0059] Example 4

[0060] Reagents used in the preparation of the porcine delta coronavirus inactivated vaccine (DC19 strain):

[0061] 1. Virus: Porcine delta coronavirus inactivated vaccine (DC19 strain).

[0062] 2. Cells: Whole suspension ST cells, provided and stored by Beijing Dingchi Biotechnology Co., Ltd.

[0063] 3. Culture medium: Aplo ST suspension medium and DMEM medium were supplied by Zhejiang Yishengke Biotechnology Co., Ltd.

[0064] 4.Seppic MONTANIDE TM ISA 201VG adjuvant was purchased from Seppic

[0065] Step 1 Preparation of porcine delta coronavirus antigen

[0066] The ST suspension cells were placed in Aplo ST suspension medium and cultured on a shaker in an incubator at 37°C and 5% CO2 for 72 h. The shaker speed was set at 140 r / min. When the ST suspension cell viability was >95%, the cell growth was stable, and the number of viable cells reached 6×10 6 After that, the virus was inoculated with a dose of 0.01 of the inactivated porcine delta coronavirus vaccine (DC19 strain) adapted to suspension culture. After adsorption for 1 hour, Aplo ST medium was added as the maintenance medium; then the cells were placed in a 37°C 5% CO2 incubator and cultured at a speed of 140 r / min. After 72 hours of culture, samples were taken for cell counting, and the antigen solution was harvested and TCID 50 The virus content of the three batches of antigen liquid was 10 8.7 TCID 50 / ml, 10 8.82 TCID 50 / ml, 10 8.81 TCID 50 / ml, which meets the requirements for seedling matching.

[0067] Step 2 Preparation of porcine delta coronavirus inactivated vaccine (DC19 strain)

[0068] Three batches of PDCoV DC19 suspension culture virus liquid were added to BEI solution with a final concentration of 0.01 mol / L, mixed thoroughly and inactivated at 37°C for 24 hours. The inactivated virus liquid was mixed with Montanide ISA 201 (purchased from SEPPIC, France) adjuvant at a ratio of 1:1 (mass ratio) and emulsified. After complete emulsification, it was an inactivated vaccine, which was packaged in 20 mL vaccine bottles and stored at 2-8°C after capping. The appearance, dosage form, stability, viscosity and sterility tests of the prepared vaccine all met the requirements.

[0069] Step 3 Vaccine safety

[0070] Healthy piglets aged 3 to 5 days born by sows with negative PDCoV antigen and antibody were randomly divided into 3 groups, 5 in each group. 2 ml of three batches of PDCoV DC19 inactivated vaccines were inoculated through the neck muscle, and 5 piglets were not inoculated as controls. After vaccination, the clinical manifestations of piglets and changes in the inoculation site were observed continuously for 14 days. The results showed that the mental state, body temperature, appetite, defecation, etc. of piglets were normal after vaccination, and there were no adverse reactions such as swelling and necrosis at the injection site of the vaccine, indicating that the three prepared PDCoV inactivated vaccines are safe for 3-5 day old piglets.

[0071] Step 4 Vaccine immunogenicity

[0072] Ten healthy piglets aged 7 to 10 days with negative PDCoV antigen and antibody were selected and randomly divided into two groups, with 5 pigs in each group. The first group was vaccinated with PDCoV DC19 strain inactivated vaccine by neck intramuscular injection, with a dose of 1 mL / head, and a booster immunization with the same dose 14 days later; the second group was not vaccinated as a blank control. Blood was collected on the 7th, 14th, 28th, 35th, and 42nd day after the first vaccination, and serum was separated to determine PDCoV neutralizing antibodies.

[0073] The results of neutralizing antibody detection are shown in Table 3: PDCoV DC19 inactivated vaccine induced a high level of neutralizing antibodies 14 days after immunization of piglets, and the neutralizing antibody level increased significantly 14 days after booster immunization (28 days after the first immunization), and reached a peak 28 days after booster immunization (42 days after the first immunization), with antibody values ​​of 1:608-1:1024. The above results indicate that PDCoVDC19 has good immunogenicity.

[0074] Table 3 Changes of porcine epidemic diarrhea antibodies in the experimental groups

[0075]

[0076] In summary, the embodiment of the present invention uses porcine delta coronavirus adapted to suspension cell culture as the target cell for the preparation of porcine delta coronavirus inactivated vaccine, and adopts serum-free, fully suspended culture medium to prepare porcine delta coronavirus vaccine, which has the following advantages: 1) low cost: no serum, no carrier, low labor, low purification area; 2) simple operation, high stability; 3) low side effects: low serum, low impurity protein; 4) high antibody titer after vaccine immunization, good immune effect; (5) suitable for large-scale production: automated bioreactor. Using ST suspension cells to culture porcine delta coronavirus in suspension can effectively promote the upgrading of the vaccine industry and has a good application prospect.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to the embodiments, it should be understood by those skilled in the art that any modification or equivalent replacement of the technical solutions of the present invention does not depart from the spirit and scope of the technical solutions of the present invention and should be included in the scope of the claims of the present invention.

Claims

1. A porcine delta coronavirus that can be efficiently propagated in suspension cells, characterized in that: The porcine delta coronavirus is the porcine delta coronavirus DC19 strain, and the recommended classification name of the porcine delta coronavirus DC19 strain is Porcine deltacoronavirus. It is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.46220 and a deposit date of November 15, 2024.

2. The method for adapting a porcine delta coronavirus that can be efficiently propagated on suspension cells to serum-free suspension ST cell culture as claimed in claim 1, characterized in that: The specific steps of the method are: (1) Take ST cells grown into a monolayer, discard the culture medium, wash twice with PBS, take porcine delta coronavirus and dilute it with serum-free DMEM culture medium containing 7.5 μg / mL pancreatin, incubate at 37°C for 1 hour, and gently shake the cell bottle once every 20 minutes to mix once; then discard the virus solution, add 5 mL of serum-free DMEM culture medium containing 7.5 μg / mL pancreatin, and culture in a 37°C, 5% CO2 incubator for 3 to 5 days, then freeze and thaw once at -20°C, harvest the virus solution and aliquot for storage; blindly propagate in the same way until cytopathic effects occur, and collect the culture; blindly propagate the virus solution on ST cells for 4 generations, and when the cells develop lesions, propagate the virus to the 8th generation, freeze and thaw once, harvest the virus solution and aliquot for storage; (2) ST cells were cultured into a monolayer in a 6-well plate, the nutrient solution was discarded, and the cells were washed twice with Hank's solution. The porcine delta coronavirus suspension obtained in step (1) was diluted with a serum-free maintenance solution, and the diluted virus solution was inoculated into ST cells at an inoculation volume of 1 / 10 of the original nutrient solution. The cells were cultured at 37° C. for 1 hour, the virus solution was aspirated, and 3 ml of a mixture of 2 times the serum-containing nutrient solution and 1.6% agarose was added to each well. The cells were cultured at 37° C. The cell morphology was observed daily until plaques appeared. The positions of the plaques were marked under an inverted microscope. The plaques were aspirated with a sterilized pipette in a biological safety cabinet and dissolved in 800 μl of PBS. (3) The virus solution obtained in step (2) was inoculated into ST suspension cells and subcultured for 5 generations. The virus solution was taken and diluted with serum-free DMEM culture medium containing 7.5 μg / mL trypsin, and then inoculated into ST cells that had been washed twice with serum-free DMEM culture medium containing 7.5 μg / mL trypsin, and cultured in a serum-free DMEM culture medium containing 7.5 μg / mL trypsin at 37°C and 5% CO2 in an incubator for 96 hours.

3. Use of a porcine delta coronavirus that can be efficiently propagated on suspension cells as claimed in claim 1 in the preparation of a porcine delta coronavirus vaccine.

4. The method for preparing a virus solution of a porcine delta coronavirus that can be efficiently propagated on suspension cells according to claim 1, characterized in that: The preparation method comprises placing ST suspension cells in Aplo ST suspension culture medium, culturing on a shaker in an incubator at 37°C and containing 5% CO2 for 72 hours, with the shaker speed set at 140 r / min, and culturing until the ST suspension cell viability is greater than 95%, the cell growth is stable, and the number of viable cells reaches 6×10 6 Afterwards, the virus inoculation amount MOI was 0.01, and the venom of porcine delta coronavirus DC19 strain adapted to suspension culture was inoculated. After adsorption for 1 hour, Aplo ST medium was added as the maintenance medium; then the cells were placed in a 37°C CO2 incubator with a volume percentage of 5% and a culture speed of 140r / min. After 72 hours of culture, samples were taken for cell counting and the virus liquid was harvested.

5. The method for preparing an inactivated vaccine of a porcine delta coronavirus efficiently propagated on suspension cells as claimed in claim 1, characterized in that: The method comprises: adding the virus liquid obtained by the preparation method described in claim 4 to a BEI solution with a final concentration of 0.01 mol / L, inactivating the solution at 37° C. for 24 hours after being fully mixed, mixing the inactivated virus liquid with an immune adjuvant in a mass ratio of 1:1, and emulsifying the mixture. After the emulsification is complete, the inactivated vaccine is obtained.

6. The method for preparing an inactivated vaccine of a porcine delta coronavirus efficiently propagated on suspension cells according to claim 5, characterized in that: The immune adjuvant is one of aluminum adjuvant, propolis adjuvant and oil-water biphasic adjuvant.

7. Use of a porcine delta coronavirus that can be efficiently propagated on suspension cells as claimed in claim 1 in the preparation of a therapeutic drug for diseases caused by porcine delta coronavirus infection in piglets.

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