CN106237324B - Method for producing transmissible gastroenteritis of swine vaccine by using full suspension technology - Google Patents
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Abstract
The invention relates to a method for producing a swine transmissible gastroenteritis vaccine by using a full suspension technology. The ST cell full suspension culture process has the key technology that after the ST cell full suspension domestication is successful, the large-scale culture of the transmissible gastroenteritis virus can be carried out in a bioreactor, has the advantages of simple operation, strong controllability, high culture efficiency and automation degree, no need of a carrier and the like, and provides a new path for the large-scale production of cells or vaccines.
Description
Technical Field
The invention relates to a method for producing a swine transmissible gastroenteritis vaccine by using a full suspension technology, belonging to the field of biological products for livestock.
Background
Porcine Transmissible Gastroenteritis virus (TGEV) is a member of the genus coronavirus of the family Coronaviridae, and can cause porcine Transmissible Gastroenteritis (TGE). The disease is an acute, highly contagious disease in pigs (saifectal, 1992) and is clinically characterized by diarrhea, severe vomiting and dehydration. Pigs of different ages and breeds are susceptible to the disease, have short incubation period and rapid spread, and can reach swinery within 2-3 days. The disease has high lethality to newborn piglets, the morbidity and the lethality of piglets within two weeks of age can reach 100 percent, and the disease is a worldwide disease of pigs. TGE is reported in China from the 60 s, in recent years, TGE is frequently generated and popular in partial areas of China, and in areas with dense swinery, TGE is one of the main causes of piglet morbidity and mortality, and brings serious harm to the pig industry. Once a pig farm is infected with TGEV, the TGEV is difficult to eradicate, and at present, the pig farm can only depend on vaccine prevention and control.
ST cells, namely pig testicular cells, belong to fibroblasts, can be continuously subcultured adherent to in vitro, and are sensitive to various viruses, such as: porcine parvovirus, porcine pseudorabies virus, classical swine fever virus, porcine transmissible gastroenteritis virus and the like are one of main raw and auxiliary materials for propagating viruses in vaccine production. ST cell culture is a key process link of virus vaccine production, directly influences the quality of the vaccine, and how to stably obtain sufficient cells is particularly important for ensuring the production capacity and the product quality.
The ST cell culture technology by means of spinner flasks is the most common cell culture method used in the production of the virus vaccines for livestock at present, and has the advantages of simple process and low cost, but the problems of low production efficiency, high labor intensity, poor product uniformity and the like are difficult to solve fundamentally (Chenwenqing, Wangjian super, Liuhuajie and the like, comparative analysis of a suspension culture process and a spinner flask culture process, Chinese veterinary medicine, 2010,44(10): 37-41).
Based on the disadvantages of spinner flask culture, suspension culture is beginning to be applied in production practice. The ST cell suspension culture process can control a series of cell culture parameters, has the advantages of high virus culture content, no need of mixing, small batch difference, automatic culture mode, no need of mass production personnel, small culture required area, small pollution, low cost and the like, and is a preferred cell culture technology in vaccine preparation.
At present, the application of suspension culture is mainly microcarrier suspension culture. In the aspect of veterinary vaccines, full suspension culture is firstly applied to the production of foot-and-mouth disease vaccines, and then a suspension culture process causes certain effects in the biopharmaceutical industry, so that vaccine production enterprises pay more and more attention to new processes.
The invention relates to a process for producing a transmissible gastroenteritis of swine vaccine by whole suspension of passage cells. The key technology is that after ST cells are successfully domesticated in a full suspension manner, the ST cells can be used for culturing the transmissible gastroenteritis virus of swine. Compared with other microcarrier suspension culture systems, the system has the advantages of simple operation, strong controllability, high culture efficiency and automation degree, no need of carriers and the like, and provides a new path for the large-scale production of cells or vaccines.
Disclosure of Invention
The invention aims to provide a process for producing a swine transmissible gastroenteritis vaccine by ST cell full suspension, so that the production cost is reduced, and large-scale and uniform production is carried out to meet the current domestic epidemic situation of swine transmissible gastroenteritis.
Technical scheme of the invention
1. A process for preparing the vaccine of transmissible gastroenteritis of pig by full suspension technique includes such steps as culturing cells, exchanging culture liquid of cells, inoculating the cells to bioreactor, suspension culturing, exchanging part of culture liquid of cells, inoculating the seed liquid of virus, culturing in bioreactor, adding freeze-drying protecting agent, mixing, loading in containers and vacuum freeze-drying.
2. The method for producing transmissible gastroenteritis of swine vaccine using full suspension technology as claimed in claim 1, wherein said step of replacing cell culture fluid and inoculating is to replace 1/3 of cell culture fluid with new cell culture fluid and inoculating transmissible gastroenteritis virus when ST cells are suspension cultured in bioreactor to be able to be inoculated.
3. The method for producing the swine transmissible gastroenteritis vaccine by using the full suspension technology as claimed in claim 1, wherein the method is characterized by comprising the following steps:
(1) suspending and culturing ST cells for 48-72 h by using a shake flask, centrifuging, resuspending the centrifuged cells by using a cell culture solution, and then performing density control on the cells by using 3 × 105～3×106Putting the solution/ml into a bioreactor for ST cell full suspension culture;
(2) culturing ST cells in full suspension to reach the density of 6 × 105/ml～6×106The solution/ml is settled in a bioreactor for 2 hours at 15 ℃, the original culture solution of the upper layer 1/3 is pumped out and injected into new cell culture solution, and 0.1 to 0.5 percent (V/V) of porcine transmissible gastroenteritis virus seed virus is inoculated;
(3) continuously culturing suspension cells in a bioreactor, when about 80% of cells have typical CPE, harvesting virus liquid, adding appropriate heat-resistant freeze-drying protective agent, mixing uniformly, packaging, and freeze-drying under vacuum to obtain the vaccine.
4. The method for producing the transmissible gastroenteritis virus vaccine of swine as claimed in claims 1-3, wherein the virus is transmissible gastroenteritis virus SD/L strain with preservation number of CGMCC No. 6001.
5. The method of claim 1, wherein the ST cell line is established by subjecting a porcine testis continuous cell line (ST cell line) to adherent culture and low serum culture suspension acclimation, wherein the cell line is named as a porcine testis cell line (SwineTests) suspension adapted strain (ST-1 strain), and the cell line is delivered to China general microbiological culture Collection center (CGMCC No. 12698) with the collection number of CGMCC No. 2016 at 23 days 2016, 23 days.
The cell culture solution of the present invention is a low serum cell culture solution, and the serum content (V/V) of the cell culture solution is: 0.5 to 2 percent.
The bioreactor used was a APP L IKON 30L-2000L cell bioreactor.
The process for full-suspension culture of the transmissible gastroenteritis of swine vaccine by the ST cells, disclosed by the invention, has the advantages of simplicity in operation, strong controllability, high culture efficiency and automation degree, no need of a carrier, capability of suspension culture in a low-serum culture solution and capability of providing a new path for large-scale production of the cells or the vaccine.
Detailed Description
1. Process for establishing and domesticating ST cell strain in full suspension culture
(1) Acclimatization of ST cell adherent culture stage (serum reduction)
ST (CVCC, No. C L27, catalog of Chinese veterinary drug strains (second edition), p171) adherent cell seeds are taken from a liquid nitrogen tank, 10% cell growth liquid of newborn bovine serum is added into a T75 culture bottle, ST cell recovery and adherent culture are carried out, subculture domestication is carried out by using adherent culture liquid which gradually reduces serum, and the serum content is reduced from 10%, 8%, 5%, 3%, 2% and 1% to 0.5%.
(2) Suspension acclimation of ST cells (Low serum culture fluid)
A portion of successfully acclimated ST cells F39 was harvested and cryopreserved as ST-1F 0.
ST cell acclimatization process shows that the serum content of the cell is reduced from 10 percent to low serum level (0.5 percent to 2.0 percent) in the adherent stage, the number and the activity of the cell can meet the experimental requirements, and from the adherent stage to the suspension stage, the cell can adapt to the suspension culture state through 39 generations of acclimatization culture and can be stably added to 1.2 × 106The cell activity reaches more than 90 percent per ml. Through the process optimization of the full suspension culture conditions (pH and rotating speed), the pH value of 7.0 +/-0.1 and the rotating speed of 120r/min are determined, the expanded culture of ST cells from a triangular culture bottle to a bioreactor is realized, the dissolved oxygen in the bioreactor is improved, the whole domestication process of the ST cells from wall attachment to full suspension is completed, the successfully domesticated suspension cells are named as a Swine Testis cell line (Swine Testis) suspension adapted strain, called ST-1 strain for short, and the strain is delivered to the general microorganism center of China Committee for microorganism management of institute of microbiological sciences, China institute of Microbiol, North West Lu No.1, Inward, Beijing, 23 days in 2016, for collection, with the collection number of CGMCC No. 12698. (details are shown in example 1)
2. Preparation of Whole suspension cells
(1) The cells are cultured in a shake flask for expanding, and 5 × 10 is added after the cells are recovered5The cells were inoculated into 125ml triangular flasks at 120r/min in a 37 ℃ 5% CO2 incubator until the cells grew to 1.2 × 106At the time of the culture, the cells were passaged and supplemented with fresh medium in such a proportion that the initial cell density was 5 × 105/ml。
(2) Cell inoculation: before cell inoculation, the dissolved oxygen electrode is corrected and CO is introduced2Adjusting pH to 7.0 + -0.1, and collecting required amount of shake flask cells (density of about 1.2 × 10)6/ml) was centrifuged, and 200ml (200ml/1000ml) of the culture solution was resuspended and then transferred to a bioreactor.
3. Preparation of venom for preparing vaccine and preparing vaccine
(1) Inoculation, the ST-1 cells are cultured in a full suspension way to reach the density of about 6 × 105/ml～6×106And/ml, settling in a bioreactor at 15 ℃ for 2 hours, pumping out the original culture solution of the upper layer 1/3, injecting a new cell culture solution, and inoculating 0.2% (V/V) porcine transmissible gastroenteritis virus strain SD/L (CGMCC No. 6001).
(2) Harvesting virus liquid, and preparing seedlings: continuously culturing suspended cells in a bioreactor, when about 80% of the cells have typical CPE, harvesting virus liquid, adding a proper heat-resistant freeze-drying protective agent, adding a proper freeze-drying protective agent, uniformly mixing, subpackaging, and freezing and vacuum drying to obtain the vaccine.
4. Inspection of transmissible gastroenteritis vaccines for swine
(1) The character is faint yellow spongy loose block which is easy to be separated from the bottle wall, and the loose block is quickly dissolved after being added with diluent.
(2) The sterility test was carried out according to the appendix of the current "Chinese veterinary pharmacopoeia" (Committee of Chinese veterinary medical science, pharmacopoeia of people's republic of China, good quality, good year edition, China agricultural publishing agency, 2011, hereinafter referred to as "Chinese veterinary pharmacopoeia"), and the growth was carried out aseptically.
(3) The mycoplasma test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and no mycoplasma grows.
(4) Exogenous virus inspection after neutralizing vaccine and swine transmissible gastroenteritis specific positive serum, inoculating Vero cell, MDBK cell and ST cell monolayer, and inspecting according to the appendix of the existing Chinese veterinary pharmacopoeia without exogenous virus pollution.
(5) Diluting the vaccine into 1 head part/ml according to the head part of the bottle label, mixing well, and diluting to 200TCID500.1ml, mixed with an equal amount of TGEV-specific positive serum, and neutralized at 37 ℃ for 30 minutes, and then seeded into 24-well cell culture plates of ST cells that have grown into monolayers, each of which was seeded into 12 wells, 0.5ml per well. Simultaneously, a non-neutralizing control group is set (the vaccine is diluted into 1 head part/ml according to the head part marked by a bottle label, mixed evenly and then diluted to 200TCID500.1ml, mixed with an equal volume of culture medium) and a control of normal cells, each inoculated into 6 wells at 0.5ml per well. Each well was supplemented with 0.5ml of cell culture medium containing 2% newborn calf serum. The cell culture plate was placed at 37 ℃ and 5% CO2Culturing in an incubator, and observing for 96-120 hour. The neutralization and cell control groups should exhibit no CPE, and the neutralization control groups should exhibit CPE in their entirety.
(6) 5 healthy susceptible piglets of 28-35 days old are used for safety inspection, 10 vaccines are inoculated to each muscle, and the piglets are all healthy and alive after observation for 14 days.
(7) Efficacy test any of the following methods was selected.
① TGEV virus content determination vaccine is diluted to 1 part/ml with culture solution containing 2% newborn calf serum, and 10 times serial dilution is carried out to obtain 10-3、10-4、10-5、10-6And 4 dilutions are respectively inoculated to a 96-well micro-cell culture plate of the ST cells which have grown into a monolayer, 8 dilutions are inoculated to each well, 100 mu l of each well, and 8 wells of a non-toxic control are arranged. Each well was supplemented with 100. mu.l of cell culture medium containing 2% newborn bovine serum. Standing at 37 deg.C and containing 5% CO2Culturing in an incubator, observing for 96-120 hours, and recording the number of CPE holes. TCID calculation by Reed-Muench method50The virus content of each vaccine is more than or equal to 105.0TCID50。
② piglet immunization method comprises immunizing 5 healthy susceptible piglets of 3-5 days old with 1 part of vaccine, injecting the vaccine into muscle, immunizing for 14 days later, and orally administering 4ml of TGEV/Q strain (containing 1000 ID) with 5 immunized piglets and 5 control piglets under the same conditions50) Continuously observing for 7 days, controlling the whole disease of piglets, and protecting immune piglets for at least 4 heads; or 4 piglets are attacked and the immunized piglets are completely protected.
(8) The vacuum degree is determined according to the appendix of Chinese animal pharmacopoeia, and the vacuum degree is in accordance with the regulations.
(9) The residual water content is determined according to the appendix of Chinese animal pharmacopoeia, and the determination is in accordance with the regulations.
The invention relates to biological material resource information
The Swine Transmissible gastroenteritis virus SD/L strain is obtained by separating a disease material of piglets suspected to suffer from Swine Transmissible Gastroenteritis (TGE) by using a cell passage method to obtain a TGE virus SD strain, wherein the virus is weakened and has good immunogenicity, can be used as a vaccine strain and is named as a Swine Transmissible gastroenteritis virus (Transmissible gastroenteritic virus) SD/L strain, and the strain is delivered to China Committee for microbiological culture Collection of microorganisms of Ministry of China, West institute of south China, No.1, of south China institute of sciences, No. 3, of south China institute of south America, at 4 months and 13 days in 2012, and is deposited at China center for microbiological culture Collection, China CCC No.6001, a Swine Testis cell ST strain, with the deposition number of CVCC, No. C L, purchased from China veterinary medicine, China center for microbiological culture management center catalog of China center for culture Collection of China (China culture Collection of China, China publication of culture Collection of Swine), Swiss strain 2002, Swiss strain, Beijing institute of south China center for culture Collection of China, China center for culture Collection of south China, China center, China center for culture Collection of south China, China.
Positive significance of the invention
The invention relates to a method for producing a swine transmissible gastroenteritis vaccine by using a full suspension technology. The ST cell full suspension culture process has the key technology that after the ST cell full suspension domestication is successful, the large-scale culture of the transmissible gastroenteritis virus can be carried out in a bioreactor, and the ST cell full suspension domestication process has the advantages of simple operation, strong controllability, high culture efficiency and automation degree, no need of a carrier and the like, and provides a new path for the large-scale production of cells or vaccines.
Examples
The following are specific embodiments of the invention to further illustrate the invention, but are not to be construed as limiting the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Example 1
Establishment of a full suspension culture of ST cell lines
Acclimatization of ST cell adherent culture stage (Low serum acclimatization Process)
ST adherent cell seeds (porcine testis passage cell ST strain, preservation) were taken from liquid nitrogen tankSerial number CVCC, No. C L27, purchased from China institute of veterinary medicine, China center for culture Collection of microorganisms, catalogues of Chinese veterinary medicine (second edition), China agricultural Press, 2002, p171), adding 10% MEM cell growth solution to a T75 culture bottle, recovering cells, culturing at a seed ratio of 1:1, changing the solution after 24h, digesting with 0.05% pancreatin 2 times when the cells grow into a compact monolayer and have a good growth state, gently blowing with a pipette to obtain a single cell suspension, passaging at a ratio of 1:3(V/V), performing ST cell adherent culture, continuously passaging downward to F3., separating the cells with 0.05% pancreatin, centrifuging at 500-1000 r/min, remixing with the culture solution, and mixing uniformly with 3 × 105～5×105And performing subculture domestication on each ml. The serum content is reduced from 10%, 8%, 5%, 3%, 2%, 1% to 0.5%, and each serum content continues to be generated for three generations after the cell state is stable, and then the next serum content is switched. The growth of the cells was observed. See table 1.
TABLE 1 results of acclimatization test of culture solution for ST cell adherent culture low serum cell
| Serum content | Density of inoculation | Adherence condition | Growth conditions | Number of | Vitality of the body |
| 10％ | 3.2×105 | ++++ | ++++ | 3.4×106 | 98％ |
| 8％ | 2.7×105 | ++++ | ++++ | 3.5×106 | 99％ |
| 5％ | 2.9×105 | +++ | ++++ | 3.0×106 | 96％ |
| 3％ | 3.0×105 | +++ | ++++ | 3.1×106 | 97％ |
| 2％ | 2.8×105 | +++ | ++++ | 2.9×106 | 97％ |
| 1％ | 3.1×105 | +++ | ++++ | 2.8×106 | 96％ |
| 0.5％ | 3.1×105 | +++ | ++++ | 3.0×106 | 98％ |
After slow (37 generations) cell acclimation and subculture, the test result shows that the cells are cultured by using culture solution with 10-5% of serum content, and a compact cell monolayer can be grown after about 48 hours; culturing cells in a culture solution with the serum content reduced from 3% to 0.5%, wherein a little dead cells exist when the cells are cultured in the 1st generation, but the adherence and growth of the cells are not influenced, bottle-dividing passage can be carried out according to a conventional method after the cells are cultured for 3 to 5 generations, and after the cells are stably domesticated, the cells grow into compact cells for about 72 hours and the cell state is good. Considering the production cost, the serum content of the cell growth liquid is set to be 0.5-2%.
Suspension acclimation of ST cells (cell culture fluid containing 0.5% -2% newborn calf serum)
(1) Domestication of different generation sub-cell suspension culture
ST adherent cells grown into a dense monolayer cultured by an acclimated cell culture solution (0.5-2% of a cell culture solution of newborn calf serum) are stably passaged for 3 generations and then are marked as ST F0.
1) When ST adherent cells (F5) grow into a compact cell monolayer by using the domesticated cell culture solution, digesting with 0.05 percent of pancreatin, gently blowing and beating into a single cell suspension by using a pipette, and adding 5 × 105Inoculating into 125ml triangular culture flask, adding low serum culture solution to 40ml, rotating at 120r/min at 37 deg.C and 5% CO2Culturing in an incubator. Centrifuging every 72h to change the solution (containing2% newborn bovine serum) and continuously culturing and subculturing by the method until 50 generations, sampling and counting each generation of cells during the culturing process and observing the cell morphology.
2) When ST adherent cells (F8) grow into a compact cell monolayer by using the domesticated cell culture solution, digesting with 0.05 percent of pancreatin, gently blowing and beating into a single cell suspension by using a pipette, and adding 5 × 105Inoculating into 125ml triangular culture flask, adding low serum culture solution to 40ml, rotating at 120r/min at 37 deg.C and 5% CO2Culturing in an incubator. And (4) centrifuging every 72h to change the culture solution (cell culture solution containing 2% newborn calf serum), continuously culturing and subculturing by the method until 50 generations, sampling and counting cells of each generation in the culture process, and observing cell morphology.
3) When ST adherent cells (F11) grow into a compact cell monolayer by using the domesticated cell culture solution, digesting with 0.05 percent of pancreatin, gently blowing and beating into a single cell suspension by using a pipette, and adding 5 × 105Inoculating into 125ml triangular culture flask, adding low serum culture solution to 40ml, rotating at 120r/min at 37 deg.C and 5% CO2Culturing in an incubator. And (4) centrifuging every 72h to change the culture solution (cell culture solution containing 2% newborn calf serum), continuously culturing and subculturing by the method until 50 generations, sampling and counting cells of each generation in the culture process, and observing cell morphology.
The test result shows that adherent cells are acclimatized into suspension cells from '2)' F8, the number of cell death is large at the beginning, and when the '2)' is continuously centrifuged and liquid-changed for passage and transmitted to F39, the cells are completely cultured in suspension for 72h, and the cell density reaches 2.26 × 106And/ml, the cell activity reaches 94%. 1) And 3) none of them succeeded in acclimatization. A portion of successfully acclimated ST cells F39 was harvested and cryopreserved as ST-1F 0.
(2) Effect of culture solution pH on suspension culture of cells
Under the same conditions as other culture conditions, the pH of the cell culture solution was set to 6.8. + -. 0.1, 7.0. + -. 0.1, 7.2. + -. 0.1 and 7.4. + -. 0.1, respectively, and after culturing at 37 ℃ for 72 hours, the cells were sampled, counted and observed for morphology.
When the pH of the ST cell culture solution is 7.0 +/-0.1, the pH of the culture solution is reduced rapidly, cells grow rapidly, the cell density is higher than that of other test groups in the same period, and the cell activity is better. When the pH is 6.8 +/-0.1 or 7.4 +/-0.1, the cells grow slowly after 48 hours of culture, and the cell density is obviously lower than that of other test groups. Therefore, the ST cell culture solution is desirably pH 7.0. + -. 0.1.
(3) Effect of rotational speed on suspension culture of cells
The culture conditions were the same as "(2)", and suspension culture was performed at different rotation speeds, type 1: 80r/min, 2 nd strain 120r/min, culturing at 37 deg.C for 72h, sampling, counting and observing cell morphology.
ST cells are cultured at the rotating speed of 80r/min, the cells are seriously aggregated, the cells are frequently attached to the wall and the bottom of the tank, and the rotating speed of 120r/min is more favorable for the growth of the ST cells.
3. Amplification of low serum full suspension cultured ST-1 cells in bioreactor
One day before cell inoculation, 800ml (800ml/1000ml) of cell culture solution (containing 2% newborn calf serum) is pumped into a bioreactor, saturated air is introduced for overnight, the ST-1 suspension cells which are well domesticated are recovered from liquid nitrogen by a conventional method, centrifugation is carried out at 500-1000 r/min, the cells are added into a 250ml triangular flask, 0.5-2% newborn calf serum cell culture solution is added, and the cell density is adjusted to 5 × 105The cells were grown to 1.2 × 10, and incubated at 37 ℃ and 120rpm in a shaker6The cell culture volume reaches 200ml, the suspension cell can be transferred to a 3L bioreactor for culture, the cells are transferred to a 10L bioreactor and a 30L bioreactor, the optimal conditions of cell growth are found, the pH is respectively set to be 7.0 +/-0.1, the dissolved oxygen is set to be 40 percent and 80 percent, the proliferation condition of the low serum suspension cell ST-1 in the bioreactor is observed under two conditions, and the highest cell density is more than 2.0 × 10 and 10 when the dissolved oxygen is 40 percent and 80 percent6The activity can reach more than 90 percent per ml, and the difference between the activity and the activity is not great. Therefore, 40% dissolved oxygen was selected as the ventilation for ST cell suspension culture. The test results are shown in Table 2.
TABLE 2ST-1 suspension cell growth test results in each reactor
From ST cell acclimation process, it can be seen that the serum content of the cell is reduced from 10% to low serum level (0.5% -2%) at the stage of adherence, and the number and activity of the cell can meet the experimental requirements, while from adherence to suspension, the cell can adapt to the suspension culture state through 39 generations of acclimation culture and can be stably added to 2 × 106The cell activity reaches more than 90 percent per ml. Through the process optimization of the full suspension culture conditions (pH and rotating speed), the pH value of 7.0 +/-0.1 and the rotating speed of 120r/min are determined, the expanded culture of ST cells from a triangular culture bottle to a bioreactor is realized, the dissolved oxygen in the bioreactor is improved, the whole domestication process of the ST cells from wall attachment to full suspension is completed, the successfully domesticated suspension cells are named as a Swine Testis cell line (Swine Testis) suspension adapted strain, called ST-1 strain for short, and the strain is delivered to the general microorganism center of China Committee for microorganism management of institute of microbiological sciences, China institute of Microbiol, North West Lu No.1, of Beijing city, No. 3 of sunward, in 2016, 23 days, 2016 (year 08 month) for preservation, and the preservation number is CGMCC No. 12698.
Example 2
Amplification culture of ST cell lines adapted to full suspension culture
Reviving and subculturing the pig testicular cell suspension adapted strain ST-1 preserved by liquid nitrogen, and then inoculating the pig testicular cell suspension adapted strain ST-1 into a bioreactor for full suspension amplification culture to obtain an ST-1 cell suspension culture solution. The method comprises the following specific steps:
1. taking an ST-1 cell strain frozen by liquid nitrogen, quickly recovering, adding the cell strain into a shake flask filled with a culture solution, culturing at 36-37 ℃ for 60-72 h, and carrying out subculture amplification culture according to a ratio of 1: 3-1: 5;
2. one day before cell inoculation, 800ml (800ml/1000ml) of culture medium (containing 2% serum) was added to the bioreactor and saturated air was passed overnight.
3. Cell inoculation: before cell inoculation, the dissolved oxygen electrode is corrected and CO is introduced2Adjusting the pH value to 7.0 +/-0.1, culturing at the temperature of 36-37 ℃, and taking required amount of shake flask cells (density)About 1.2 × 106/ml), and then 200ml (200ml/1000ml) of the culture solution is added back to the suspension and inoculated into a bioreactor for suspension culture.
Example 3
Process for producing transmissible gastroenteritis of swine vaccine by full suspension of ST cells
1. Preparing the virus seeds for production, namely taking an ST cell monolayer with good growth, discarding cell growth liquid, replacing with cell maintenance liquid containing 0.1-0.5% of virus seeds of the porcine transmissible gastroenteritis virus SD/L strain, continuously culturing at 36-37 ℃, harvesting when about 80% of cells have typical CPE, quantitatively subpackaging, indicating the harvest date, the virus seed algebra and the like, and freezing and storing.
2. Propagation of venom for vaccine production
(1) Cleaning and sterilizing the bioreactor: the bioreactor was cleaned and sterilized at 121 ℃ for 30 min.
(2) Cell inoculation, ST-1 cells are cultured and expanded in a shake flask in a suspension way to reach 1.2 × 106After/ml, the cells were inoculated into a bioreactor under the following cell culture conditions: the working volume of the reactor is 30 liters, the dissolved oxygen electrode is corrected before cell inoculation, and CO is introduced2Adjusting the pH value to 7.0 +/-0.2, culturing at 36-37 deg.C with dissolved oxygen of 40%, and collecting the required amount of shake flask cells (density about 1.2 × 10)6And/ml), back-suspending with a cell culture solution, inoculating into a bioreactor for suspension culture, and continuously culturing for 2-3 days.
(3) Inoculating and harvesting to obtain ST-1 cells, culturing to density of about 1.2 × 106And/ml, settling for 2 hours at 15 ℃ in a bioreactor, pumping out an upper layer 1/3 original cell culture solution, replacing a cell culture solution of 0.5-2% newborn bovine serum, inoculating a porcine transmissible gastroenteritis virus SD/L strain according to 0.2% of the final volume of the cell culture solution, wherein the virus culture conditions are that the temperature is 33-35 ℃, the pH value is 7.0 +/-0.2, the dissolved oxygen is 40%, harvesting and removing the tank when about 80% of cells have typical CPE, and producing 3 batches of virus solution in total.
3. Inspection of semi-finished product
(1) The sterility test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the bacteria should grow uniformly.
(2) The virus content per ml is not less than106.5TCID50。
4. Preparing vaccine and subpackaging SD/L strain virus liquid which is qualified in inspection, adding a freeze-drying protective agent serving as a stabilizer and a proper antibiotic according to a ratio of 1:1, fully mixing, and quantitatively subpackaging.
5. Freeze-drying, packaging, and vacuum-drying.
Example 4
-inspection of finished products of porcine transmissible gastroenteritis vaccines
1. The yellow spongy loose lumps are easy to separate from the bottle wall and can be quickly dissolved after being added with diluent.
2. The sterility test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the growth is sterile.
3. The mycoplasma test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and no mycoplasma grows.
4. Exogenous virus inspection after neutralizing vaccine and swine transmissible gastroenteritis specific positive serum, inoculating Vero cell, MDBK cell and ST cell monolayer, and inspecting according to the appendix of the existing Chinese veterinary pharmacopoeia, wherein no exogenous virus pollution exists.
5. Diluting the vaccine into 1 head part/ml according to the head part of the bottle label, mixing well, and diluting to 200TCID500.1ml, mixed with equal amount of swine transmissible gastroenteritis specific positive serum, and after being neutralized at 37 ℃ for 30 minutes, 24-well cell culture plates of ST cells grown in a monolayer were inoculated, 12 wells for each cell, 0.5ml per well. Simultaneously, a non-neutralizing control group is set (the vaccine is diluted into 1 head part/ml according to the head part marked by a bottle label, mixed evenly and then diluted to 200TCID500.1ml, mixed with an equal volume of culture medium) and a control of normal cells, each inoculated into 6 wells at 0.5ml per well. Each well was supplemented with 0.5ml of cell culture medium containing 2% newborn calf serum. The cell culture plate was placed at 37 ℃ and 5% CO2Culturing in an incubator, and observing for 96-120 hours. The neutralization group and the cell control group showed no CPE, and the neutralization control group showed CPE in its entirety.
6. 5 healthy susceptible piglets of 28-35 days old are used for safety inspection, 10 vaccines are inoculated to each muscle, and the piglets are all healthy and alive after 14 days of observation.
7. Efficacy test any of the following methods was selected.
(1) TGEV virus content determination vaccine is diluted to 1 part/ml with culture solution containing 2% newborn calf serum, and 10 times serial dilution is carried out to obtain 10-3、10-4、10-5、10-6And 4 dilutions are respectively inoculated to a 96-well micro-cell culture plate of the ST cells which have grown into a monolayer, 8 dilutions are inoculated to each well, 100 mu l of each well, and 8 wells of a non-toxic control are arranged. Each well was supplemented with 100. mu.l of cell culture medium containing 2% newborn bovine serum. Standing at 37 deg.C and containing 5% CO2Culturing in an incubator, observing for 96-120 hours, and recording the number of CPE holes. TCID calculation by Reed-Muench method50The virus content of each vaccine is more than or equal to 105.0TCID50。
(2) The piglet immunization challenge method uses 5 healthy susceptible piglets of 3-5 days old and 1 part of each intramuscular injection vaccine. After 14 days of immunization, 4ml of pig transmissible gastroenteritis virus virulent TGEV/Q strain (containing 1000 ID) is orally administered to 5 immunized piglets and 5 control piglets under the same conditions50) Continuously observing for 7 days, controlling the whole disease of piglets, and protecting immune piglets for at least 4 heads; or 4 control piglets with disease, and immune piglets are all protected
8. The vacuum degree is determined according to the appendix of Chinese animal pharmacopoeia, and the vacuum degree determination conforms to the regulations.
9. The residual water content is determined according to the appendix of Chinese animal pharmacopoeia, and all the measurements accord with the regulations.
Claims (2)
1. A method for using the whole suspension technology to produce the pig transmissible gastroenteritis vaccine, characterized by that its preparation method includes cell culture, change cell culture fluid and then the virus is received and harvested the process, namely carry on the suspension culture amplification to ST-1 strain pig testis cell line cell with shaking flask first, insert the bioreactor to carry on the suspension culture, change a part of cell culture fluid and insert SD/L strain pig transmissible gastroenteritis virus seed liquid after the cell grows to the inoculation density of the virus, continue culturing in the bioreactor until finishing, receive the virus liquid, add appropriate freeze-drying protective agent to mix, split charging, vacuum freeze-drying to get final product;
the method specifically comprises the following steps:
(1) suspending and culturing ST-1 strain cells for 48-72 h by using a shake flask, centrifuging, resuspending the centrifuged cells by using a cell culture solution, and then performing density control on the cells by using 3 × 105～3×106Putting the solution/ml into a bioreactor for ST-1 cell full suspension culture;
(2) the ST-1 cells are cultured in a full suspension way to reach the density of 6 × 105/ml～6×106The solution/ml is settled in a bioreactor for 2 hours at 15 ℃, original culture solution of the upper layer 1/3 is pumped out and injected into new cell culture solution, and 0.1 to 0.5 percent (V/V) SD/L strain porcine transmissible gastroenteritis virus seed virus is inoculated;
(3) continuously culturing suspension cells in a bioreactor, when about 80% of the cells have typical CPE, harvesting virus liquid, adding a proper freeze-drying protective agent, uniformly mixing, subpackaging, and freezing and vacuum drying to obtain a vaccine;
the ST-1 cell strain is obtained by carrying out a low serum domestication process and a low serum culture solution suspension domestication process in an adherent culture stage on ST strain cells of a pig testicle passage cell line, wherein the ST strain cells are named as a pig testicle cell line (Swine Testis) suspension adaptive strain, ST-1 strain for short, and the ST-1 strain cells are preserved in the common microorganism center of China Committee for culture Collection of microorganisms of institute of microbiological, China academy of sciences, Ministry of microbiology, No.1, North West No. 3, Inward areas, Beijing, 23 days 2016, and have the preservation number of CGMCC No. 12698;
the low serum culture solution refers to a low serum cell culture solution, and the serum content (V/V) of the low serum cell culture solution is as follows: 0.5 to 2 percent.
2. The method for producing the transmissible gastroenteritis virus vaccine of swine as claimed in claim 1, wherein the virus is transmissible gastroenteritis virus SD/L strain with preservation number of CGMCC No. 6001.
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| CN103550771A (en) * | 2013-10-31 | 2014-02-05 | 成都天邦生物制品有限公司 | Production method of transmissible gastroenteritis virus vaccine |
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| CN103550771A (en) * | 2013-10-31 | 2014-02-05 | 成都天邦生物制品有限公司 | Production method of transmissible gastroenteritis virus vaccine |
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