CN111073863B

CN111073863B - Porcine epidemic diarrhea and porcine delta coronavirus bivalent attenuated vaccine and preparation method thereof

Info

Publication number: CN111073863B
Application number: CN201911360567.9A
Authority: CN
Inventors: 徐高原; 周明光; 张华伟; 陈波; 郝根喜; 金建云
Original assignee: Wuhan Keqian Biological Co ltd
Current assignee: Wuhan Keqian Biological Co ltd
Priority date: 2019-12-25
Filing date: 2019-12-25
Publication date: 2022-03-01
Anticipated expiration: 2039-12-25
Also published as: CN111073863A

Abstract

The invention provides a porcine epidemic diarrhea and porcine delta coronavirus bivalent attenuated vaccine and a preparation method thereof. The invention provides a safe and reliable porcine epidemic diarrhea and porcine delta coronavirus bigeminal attenuated vaccine for emergency vaccination, which has high immune protection rate, good stability and high yield by adopting a cell continuous passage method and a cell suspension culture technology, and provides an effective means for the prevention and control of PEDV and PDCoV.

Description

Porcine epidemic diarrhea and porcine delta coronavirus bivalent attenuated vaccine and preparation method thereof

Technical Field

The invention relates to the field of biotechnology and preventive veterinarian, in particular to a porcine epidemic diarrhea and porcine delta coronavirus bigeminal attenuated vaccine and a preparation method thereof.

Background

Porcine Epidemic Diarrheal Virus (PEDV) belongs to the order nidongdales, genus coronavirus, and viral nucleic acid is infectious, linear single-stranded positive-strand non-segmented RNA. Porcine Epidemic Diarrhea Virus (PEDV) is one of the major pathogens of porcine viral diarrhea outbreaks.

Porcine Deltacoronavirus (PDCoV) belongs to the genus delta coronavirus of the subfamily coronaviridae, the family nidonodae, and the viral nucleic acid is a single-stranded positive-stranded non-segmented RNA. PDCoV is a novel enterocoronavirus infecting pigs newly discovered in 2012, can infect pigs of different ages of days and show clinical symptoms of diarrhea, vomiting, dehydration and the like, and the death rate of sick piglets is 30-40%. The disease outbreaks in the U.S. pig farm in 2014, in China inland in the same year, and spreads worldwide in the next few years, which brings huge economic loss to the pig industry. At present, a series of detection methods are established aiming at PDCoV, and the detection result shows that PDCoV has single infection and mixed infection with other porcine enteroviruses, however, the research aiming at the pathogenic mechanism of PDCoV is less, the understanding of the disease is not comprehensive, the commercial vaccine is lacked, and the effective vaccine is lacked for preventing and controlling the disease.

PEDV and PDCoV belong to coronavirus families, have similar antigen forms, and diseases caused by the two viruses are extremely similar in epidemiology and pathological anatomy, but have no common antigenicity and no cross reaction in immunology and serology. Therefore, it is very important to prevent and control two diseases simultaneously. At present, diseases caused by mixed infection of PEDV, PDCoV and the like are a big problem in the global pig industry, and related research on PEDV and PDCoV combined attenuated vaccine is not available in China. The attenuated vaccine has the advantages of multiple immunization ways, small immunization dose, long immunization duration, no need of adjuvant, no need of immune cells after immunizing animals, capability of enabling immune cells to quickly reach respiratory tract mucous membranes and digestive tract mucous membranes to take corresponding action with pathogens, immunoprotection, rejection of continuous infection and invasion of the pathogens, capability of killing the pathogens by immunoregulation, and the like. Therefore, the development of the safe and reliable porcine epidemic diarrhea and porcine delta coronavirus bigeminal cell attenuated vaccine with good immune effect has important significance for purification of positive pig farms and prevention and control of domestic PEDV and PDCoV.

Disclosure of Invention

The invention aims to provide a safe and reliable porcine epidemic diarrhea and porcine delta coronavirus bigeminal attenuated vaccine with good immune effect and a preparation method thereof.

In order to achieve the object of the present invention, in a first aspect, the present invention provides a porcine epidemic diarrhea virus KQ 2018F 120 strain, which is attenuated by the cell passage of the porcine epidemic diarrhea virus, and which is now deposited in the chinese collection of type cultures, address: wuhan, Wuhan university, post code 430072, preservation number CCTCC NO: V202007, preservation date 2019, 12 months and 19 days.

In a second aspect, the present invention provides a porcine delta coronavirus KQ 2018F 120 strain, which is attenuated by passage of porcine delta coronavirus cells, and is deposited in the chinese type culture collection at the address: wuhan, Wuhan university, post code 430072, preservation number of V202008, preservation date of 2019, 12 months and 19 days.

Wherein, the porcine epidemic diarrhea virus cell passage induced attenuated virus seed virus is separated in a certain pig farm in Hubei, the pathologic map and the PCR identification map are shown in figure 1, the PCR primer aims at the PEDV S protein gene, the target fragment is 651bp, the upstream primer is PEDV-F: 5'-TTCTGAGTCACGAACAGCCA-3', and the sequence of the downstream primer is PEDV-R: 5'-CATATGCAGCCTGCTCTGAA-3' are provided. After separating to obtain the porcine epidemic diarrhea virus, carrying out passage 120 generations by the adherent vero cells to obtain the attenuated virus caused by passage of the porcine epidemic diarrhea virus cells.

The porcine delta coronavirus cell passage attenuated virus seed virus is separated from a certain pig farm in Henan, a lesion map and a PCR identification map of the porcine delta coronavirus cell passage attenuated virus seed virus are shown in figure 2, a PCR primer aims at a PDCoV N protein gene, a target fragment is 329bp, an upstream primer is PDCoV-F: 5'-CCAAACGCAACCCCAACAATCC-3', the sequence of the downstream primer is PDCoV-R: 5'-CTTCTCAGTGTCTGCAGAGCCG-3' are provided. After separating to obtain the porcine epidemic diarrhea virus, carrying out passage 120 generations by adherent ST cells to obtain the porcine delta coronavirus cell passage attenuated virus.

In a third aspect, the invention provides an application of the porcine epidemic diarrhea virus KQ 2018F 120 strain and/or the porcine delta coronavirus KQ 2018F 120 strain in vaccine preparation.

In a fourth aspect, the present invention provides a composition comprising the porcine epidemic diarrhea virus and/or the porcine delta coronavirus KQ 2018F 120 strain and a pharmaceutically acceptable carrier.

In a fifth aspect, the invention provides a porcine epidemic diarrhea and porcine delta coronavirus bigeminal attenuated vaccine, which is obtained by respectively inoculating the porcine epidemic diarrhea virus KQ 2018F 120 strain and the porcine delta coronavirus KQ 2018F 120 strain to susceptible cells (such as ST cells) for culture, mixing the obtained virus solutions in proportion, adding a freeze-drying protective agent, uniformly mixing, and then carrying out freeze-drying.

The virus titer of the virus liquid of the harvested porcine epidemic diarrhea virus KQ 2018F 120 strain and the virus liquid of the porcine Deltacoronavirus KQ 2018F 120 strain are not lower than 10⁶TCID₅₀/0.1mL。

Preferably, the two virus fluids are mixed in equal volumes.

Preferably, the lyoprotectant is gelatin and sucrose.

More preferably, the lyoprotectant is prepared by adding water into gelatin and sucrose, wherein the gelatin is 8g/L, and the sucrose is 48 g/L.

And (3) mixing the freeze-drying protective agent in a volume ratio of 1: 7 to the virus mixture of PEDV and PDCoV.

In a sixth aspect, the present invention provides a method for preparing the bivalent attenuated vaccine, comprising the following steps:

1) suspending and culturing ST cells in a bioreactor, respectively inoculating the cultured ST cells with a porcine epidemic diarrhea virus KQ 2018F 120 strain and a porcine delta coronavirus KQ 2018F 120 strain, continuously culturing, harvesting the viruses when the cytopathic effect reaches more than 80%, taking the supernatant of the naturally precipitated viruses, filtering and concentrating to obtain virus liquid for preparing the vaccine;

2) and mixing the obtained virus liquid in proportion, adding a freeze-drying protective agent, uniformly mixing, and freeze-drying.

In the above method, the cell culture conditions are: 37 ℃, pH7.2, DO (dissolved oxygen) value of 50%, rotation speed of 75 rpm.

In the present invention, the medium used for suspension culture of ST cells is MEM medium containing 100IU/mL penicillin and 100IU/mL streptomycin.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the invention selects the attenuated virus caused by cell passage of the epidemic strain to prepare the vaccine, thereby ensuring the high immune protection of the vaccine.

The low virulent strain prepared by the invention has good stability, the PEDV and PDCoV low virulent strains keep good stability after cloning and purifying, small plaques within 1mm are selected by cloning and purifying, and the diameter and morphological characteristics of the plaques are detected for 3 times when the bivalent low virulent vaccine is prepared, so that the characteristics of the small plaques are kept.

And thirdly, the low virulent strain provided by the invention has high safety, and the prepared porcine epidemic diarrhea and porcine delta coronavirus bigeminal cell low virulent vaccine has an avirulence reversion phenomenon.

And (IV) the invention selects the suspension ST cells to prepare the virus liquid for vaccine preparation, thereby providing guarantee for high-efficiency vaccine production.

The invention provides material basis and technical support for clinically preventing and controlling the porcine epidemic diarrhea and the porcine delta coronavirus and clearing the porcine epidemic diarrhea virus and the porcine delta coronavirus in a swinery, and has wide application prospect.

Drawings

FIG. 1 shows the pathological change picture of the porcine epidemic diarrhea virus inoculated on Vero cells and the PCR detection result of the virus isolate. In the figure, a is an ungerminated blank cell control; b is the cytopathy after inoculation; c is the PCR detection result of the porcine epidemic diarrhea virus isolate, and in the figure, M: DNA Marker DL 2000; 1: a sample; 2: and (5) negative control.

FIG. 2 shows the pathological changes of the porcine delta coronavirus inoculated onto ST cells and the PCR detection results of the virus isolate. In the figure, a is an ungerminated blank cell control; b is the cytopathy after inoculation; c is the PCR detection result of the porcine delta coronavirus isolate, and in the figure, M: DNA Marker DL 2000; 1: a sample; 2: and (5) negative control.

Detailed Description

The porcine epidemic diarrhea and porcine delta coronavirus bivalent attenuated vaccine provided by the invention can be prepared by adopting the following method:

propagation of virus I

Discarding original culture solution, inoculating 1% of the original culture solution to the adherent ST cells with good monolayer growth, respectively inoculating the porcine epidemic diarrhea virus cells with attenuated virus due to passage or porcine delta coronavirus cells with attenuated virus due to passage, adsorbing for 2h, replacing with DMEM maintenance solution containing penicillin and streptomycin with 100IU/mL respectively, adding pancreatin with final concentration of 10ug/mL at 37 deg.C and 5% CO₂Concentration ofCulturing in an incubator for 24-72 h, harvesting virus when cytopathic effect reaches more than 80%, repeatedly freezing and thawing twice in a refrigerator at-80 ℃, and performing sterile inspection, mycoplasma inspection and exogenous virus inspection to reach various indexes specified in quality Standard of veterinary biological products of the people's republic of China, wherein the virus titer is not lower than 10⁶TCID₅₀0.1mL, used as attenuated antigen seed virus of porcine epidemic diarrhea and porcine delta coronavirus cell passage, and stored in a refrigerator at-80 ℃ for later use. PEDV attenuated strains are currently deposited in the China center for type culture Collection at the address: wuhan, Wuhan university, post code 430072, preservation number CCTCC NO: V202007, preservation date 2019, 12 months and 19 days. The PDCoV low virulent strain is currently preserved in China center for type culture Collection, and the address: wuhan, Wuhan university, post code 430072, preservation number of V202008, preservation date of 2019, 12 months and 19 days.

II preparation of Virus solution for producing vaccine

Comprises cell amplification culture, virus inoculation, virus liquid harvest, virus titer determination, sterility and exogenous detection.

1. And (3) expanding and culturing cells in a seeding tank: the density of suspended ST cells in a shake flask reaches 7 multiplied by 10⁶～10×10⁶When the number of cells per mL reaches more than 95%, the cells are transferred to a seeding tank. Before transfer, the seed tank Dissolved Oxygen (DO) electrode, pH electrode, and temperature electrode were calibrated, and the tank was autoclaved. Pumping 20% culture solution according to the volume of the seeding tank at 0.5 × 10⁶～1×10⁶Suspension ST cells were seeded at a density of one/mL under culture conditions of 37 ℃, pH7.2, DO 50%, and rotation speed 75 rpm.

2. Virus inoculation: when the density of suspended ST cells in the bioreactor reaches 7X 10⁶～10×10⁶At cell/mL, the cell density was diluted to 2X 10 with the culture medium⁶～3×10⁶Adding pancreatin with the final concentration of 10ug/mL, taking PEDV and PDCoV virus liquid as seed virus, inoculating porcine epidemic diarrhea cell passage attenuated strain or porcine delta coronavirus cell passage attenuated strain according to the culture volume ratio of 1%, continuously culturing at 37 ℃, wherein the pH is 7.2, the DO is 50%, and the rotating speed is 75 rpm.

3. Harvesting virus liquid, measuring virus titer, and detecting sterility and exogenous source: harvesting virus 24-72 h after inoculation when cytopathic effect reaches more than 80%, sucking supernatant of naturally precipitated virus, filtering through a 0.65-micron filter, concentrating through a 300KDa membrane package, sampling, performing sterility test, mycoplasma test and exogenous virus test to reach indexes specified in quality Standard of veterinary biological products of the people's republic of China, wherein the virus titer is not lower than 10⁶ TCID₅₀0.1mL, the virus solution is used as a virus solution for preparing the vaccine.

III vaccine preparation and lyophilization

The prepared virus liquid for preparing attenuated vaccine by porcine epidemic diarrhea and porcine delta coronavirus cell passage with all qualified indexes is prepared according to the volume ratio of 1: 1, and then mixing the mixture with a freeze-drying protective agent in a volume ratio of 7: 1, and freeze-drying in a low-temperature freeze-dryer to obtain the porcine epidemic diarrhea and porcine delta coronavirus bigeminal cell attenuated vaccine, and storing at-20 ℃.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

In the following examples, the lyoprotectant is prepared by adding water to gelatin and sucrose, wherein the gelatin is 8g/L, and the sucrose is 48 g/L.

Example 1 preparation of porcine epidemic diarrhea and porcine delta coronavirus bivalent attenuated vaccine

1. Breeding of seed virus

Discarding original culture solution, inoculating 1% of the original culture solution to the adherent ST cells with good monolayer growth, respectively inoculating the porcine epidemic diarrhea virus cells with attenuated virus due to passage or porcine delta coronavirus cells with attenuated virus due to passage, adsorbing for 2h, replacing with DMEM maintenance solution containing penicillin and streptomycin with 100IU/mL respectively, adding pancreatin with final concentration of 10ug/mL at 37 deg.C and 5% CO₂Culturing in a concentration incubator for 24-72 h, harvesting virus when cytopathic effect reaches more than 80%, and freezing at-80 deg.CThe box is repeatedly frozen and thawed twice, and then aseptic inspection, mycoplasma inspection and exogenous virus inspection are carried out to reach all indexes specified in veterinary biological product quality standard of the people's republic of China, and the virus titer is not lower than 10⁶TCID₅₀0.1mL, used as attenuated antigen seed virus of porcine epidemic diarrhea and porcine delta coronavirus cell passage, and stored in a refrigerator at-80 ℃ for later use.

2. Preparation of virus liquid for preparing vaccine

2.1 cell expansion culture in seeding tank: the density of suspended ST cells in a shake flask reaches 7 multiplied by 10⁶～10×10⁶When the number of cells per mL reaches more than 95%, the cells are transferred to a seeding tank. Before transfer, the seed tank Dissolved Oxygen (DO) electrode, pH electrode, and temperature electrode were calibrated, and the tank was autoclaved. Pumping 20% culture solution according to the volume of the seeding tank at 0.5 × 10⁶～1×10⁶Suspension ST cells were seeded at a density of one/mL under culture conditions of 37 ℃, pH7.2, DO 50%, and rotation speed 75 rpm.

2.2 virus inoculation: when the density of suspended ST cells in the bioreactor reaches 7X 10⁶～10×10⁶At cell/mL, the cell density was diluted to 2X 10 with the culture medium⁶～3×10⁶Adding pancreatin with the final concentration of 10ug/mL, taking virus liquid of low virulent PEDV and PDCoV as seed virus, inoculating porcine epidemic diarrhea cells or porcine delta coronavirus cells to the seed virus according to the culture volume ratio of 1%, continuously culturing at 37 ℃, wherein the pH is 7.2, the DO is 50%, and the rotating speed is 75 rpm.

2.3 harvesting virus liquid, measuring virus titer, and detecting sterility and exogenous source: harvesting virus 24-72 h after inoculation when cytopathic effect reaches more than 80%, sucking supernatant of naturally precipitated virus, filtering with 0.65 μm filter, concentrating with 300KDa membrane, sampling, performing sterility test, mycoplasma test and exogenous virus test to obtain indexes specified in quality Standard of veterinary biological products of people's republic of China, and determining virus titerNot lower than 10⁶TCID₅₀0.1mL, the virus solution is used as a virus solution for preparing the vaccine.

3. Formulation and lyophilization of vaccines

The prepared virus liquid for preparing attenuated vaccine by porcine epidemic diarrhea and porcine delta coronavirus cell passage with all qualified indexes is mixed according to the volume ratio of 1: 1, and then mixed with a freeze-drying protective agent according to the volume ratio of 7: 1, and freeze-drying in a low-temperature freeze-dryer to obtain the porcine epidemic diarrhea and porcine delta coronavirus bigeminal cell attenuated vaccine, and storing at-20 ℃.

EXAMPLE 2 Return to virulence test for attenuated strains of Virus

The test method comprises the following steps: 2mL (10) of each of the porcine epidemic diarrhea virus cell passage attenuated virus strain and the porcine delta coronavirus cell passage attenuated virus strain⁶TCID₅₀and/mL) 3-day-old artificially-fed piglets which are inoculated with non-colostrum orally, respectively taking 2mL orally for 5 piglets in the 1 st group, killing the piglets after 72h of planting, observing whether gastrointestinal tract tissues have visual abnormalities, preparing small intestine tissue filtrate (each individual mixed solution), taking 2mL orally for inoculating the piglets in the 2 nd group and 3 days old piglets, killing the piglets after 72h, observing and inoculating the piglets in the 3 rd group by the same method, and repeating the above steps for 5 generations (5 groups in total).

And (3) test results: after 3-day-old piglets are continuously passaged for 5 generations, no obvious lesion is found in clinical observation of experimental piglets and general caesarean section examination of gastrointestinal tracts, which shows that the vaccine strain has strong avirulent reversion.

Example 3 safety test of bivalent attenuated vaccine

Selecting 21-28 days old piglets in a certain pig farm, detecting main pathogeny and related antibodies of the piglets, and selecting 20 piglets which are negative in porcine circovirus type 2, classical swine fever virus and porcine reproductive and respiratory syndrome virus detection, and porcine epidemic diarrhea and porcine delta coronavirus neutralizing antibody negativity (PEDV and PDCoV neutralizing antibody titer is not higher than 1: 2).

Test pigs were randomly divided into 4 groups of 5 pigs each.

Groups

1 and 2 were each treated with 1 part/head of the bivalent attenuated vaccine prepared in example 1 by intramuscular injection, group 2 was treated with 1 part/head of the bivalent attenuated vaccine prepared in example 1 by intramuscular injection 2 weeks later, group 3 was treated with 2 parts/head of the bivalent attenuated vaccine prepared in example 3 by intramuscular injection, and group 4 was treated with no immunization by intramuscular injection as a control. The observation was carried out for 4 weeks.

The experimental result shows that compared with the control group, the

immune groups

1, 2 and 3 have no abnormality in food intake and drinking, no adverse reaction is seen at the injection part, and 100 percent of pigs are healthy and alive.

Example 4 immunoprotective efficacy assay of bivalent attenuated vaccine

Selecting 10 pregnant sows with 35-42 d prenatal porcine epidemic diarrhea and porcine delta coronavirus neutralizing antibody negativity (PEDV and PDCoV neutralizing antibody titer is not higher than 1: 2), randomly dividing into 2 groups, injecting 5 pregnant sows into each group, injecting the bivalent attenuated vaccine prepared in the embodiment 1 into the group 1 through muscle, injecting the same amount of physiological saline into the group 2 through muscle as a control group, collecting blood respectively at 1, 2, 3, 4 and 5 weeks after immunization, and detecting the level of neutralizing antibodies; after the sows farrowing, respectively and randomly selecting 10 piglets of 3-day-old piglets and 7-day-old piglets in the group 1 and 10 piglets of 3-day-old piglets and 7-day-old piglets in the group 2; 2mL (10 mL) of each of the porcine epidemic diarrhea virus HB201503 (isolation and identification of the porcine epidemic diarrhea virus Hubei strain and analysis of s1 gene evolution, Guirui et al, (Hubei agricultural science, 2016) and the porcine delta coronavirus CHN-HN-2014 (CN 107815440A) were administered orally (10 mL)⁶TCID₅₀mL), and observing the clinical manifestations of the piglets after the challenge.

The experimental results showed that the level of neutralizing antibodies rose rapidly 2 weeks after immunization and that the level of neutralizing antibodies could be maintained until farrowing in sows. After the piglets born by sows immunized with the porcine epidemic diarrhea and porcine delta coronavirus bigeminal cell attenuated vaccine are attacked at 3 days and 7 days, no abnormality is found in the lactation, spirit and feces of 10 immunized piglets, and the piglets are 100% healthy and alive; after 3 days old and 7 days old of piglets born by the control group injected with normal saline are detoxified, all 10 immunized piglets show typical porcine epidemic diarrhea symptoms and die. The experimental data are shown in tables 1 to 3.

TABLE 1 neutralizing antibody levels against porcine epidemic diarrhea Virus after immunization of a bivalent attenuated vaccine against a sow

	Week 1	Week 2	Week 3	Week 4	Week 5
						Neutralizing antibody levels	1：8	1：16	1：32	1：32	1：16

TABLE 2 neutralizing antibody levels against porcine delta coronavirus after immunization of a bivalent attenuated vaccine against a sow

	Week 1	Week 2	Week 3	Week 4	Week 5
						Neutralizing antibody levels	1：16	1：32	1：32	1：32	1：16

TABLE 3 protective rate of challenge for piglets born by immunized sows

The invention provides a safe and reliable porcine epidemic diarrhea and porcine delta coronavirus bigeminal attenuated vaccine for emergency vaccination, which has high immune protection rate, good stability and high yield by adopting a cell continuous passage method and a cell suspension culture technology, and provides an effective means for the prevention and control of PEDV and PDCoV.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. The porcine delta coronavirus attenuated strain is characterized in that the preservation number is CCTCC NO: V202008.

2. Use of the porcine delta coronavirus attenuated strain of claim 1 in the preparation of a vaccine.

3. A composition comprising the porcine epidemic diarrhea virus attenuated strain with the collection number of CCTCC No. V202007 and the porcine delta coronavirus attenuated strain of claim 1 and a pharmaceutically acceptable carrier.

4. The porcine epidemic diarrhea and porcine delta coronavirus bivalent attenuated vaccine is characterized in that the bivalent attenuated vaccine is obtained by respectively inoculating a porcine epidemic diarrhea virus attenuated strain with the preservation number of CCTCC NO: V202007 and the porcine delta coronavirus attenuated strain of claim 1 to susceptible cells for culture, mixing the obtained virus liquid in proportion, adding a freeze-drying protective agent, uniformly mixing, and then freezing and drying.

5. The bivalent attenuated vaccine according to claim 4, wherein the virus titer of the harvested virus fluid of the attenuated strain of porcine epidemic diarrhea virus and the virus fluid of the attenuated strain of porcine Deltacoronavirus is not less than 10⁶TCID₅₀/0.1mL。

6. The bivalent attenuated vaccine according to claim 5, wherein the two viral fluids are mixed in equal volumes.

7. The bivalent attenuated vaccine according to any one of claims 4 to 6, wherein the lyoprotectant is gelatin and sucrose.

8. The bivalent attenuated vaccine according to claim 7, wherein the lyoprotectant is prepared by adding water to gelatin and sucrose, wherein the gelatin is 8g/L and the sucrose is 48 g/L.

9. A process for the preparation of the bivalent attenuated vaccine according to any one of claims 4 to 8, comprising the steps of:

1) suspending and culturing ST cells in a bioreactor, respectively inoculating the cultured ST cells with porcine epidemic diarrhea virus low-virulent strains and porcine delta coronavirus low-virulent strains, continuously culturing, harvesting viruses when cytopathic effect reaches more than 80%, taking supernatant of the naturally precipitated viruses, filtering and concentrating to obtain virus liquid for preparing the vaccine;

10. The method of claim 9, wherein the cell culture conditions are: 37 ℃, pH7.2, DO value 50%, rotation speed 75 rpm.

11. The method as claimed in claim 9 or 10, wherein the medium used for suspension culture of ST cells is MEM medium containing 100IU/mL penicillin and 100IU/mL streptomycin.