TW201040257A - Vaccine produced with suspension microcarrier cell culturing system and method thereof - Google Patents

Abstract

This invention relates to a vaccine produced with a suspension microcarrier cell culturing system and its method comprising the following steps: (1) inoculating cells for producing vaccine in a culture pot containing culture medium and microcarriers; (2) uniformly mixing the cells with microcarriers to attach the cells on the microcarriers; (3) providing adequate nutrients and gas for the foregoing cells at a proper temperature so that the cells grow on the microcarriers continuously; (4) preparing the viruses for producing the vaccine as a virus suspension that is inoculated on the aforementioned cells to be continuously cultured; harvesting the virus liquid or cells containing the viruses every one to three days and changing the culture medium; and (5) purifying the above harvested virus liquid, inactivating the virus liquid upon demands after purifying the harvested virus liquid, adding a proper adjuvant to the inactivated virus liquid, adding a proper lyophilization protection agent to the virus liquid without undergoing inactivation process, mixing thoroughly, and quantitatively packing after adequately mixing to obtain a desire vaccine.

Description

201040257 - VI. Description of the invention: [Technical field to which the invention pertains] The present invention relates to the field of biological products, and more particularly to a vaccine produced by a suspension microcarrier cell culture system and a method thereof. [Prior Art] The currently known method of producing a whole virus vaccine 'produces a virus by chicken embryo egg, animal tissue or cell culture method; however, 'the egg embryo quality produced by the chicken embryo egg has an egg source quality and the source is unstable Ο °, etc. The problem is that the whole virus vaccine produced by animal tissues contains too much foreign protein, which is easy to cause allergic reactions and neurological complications to the target; therefore, the preferred way is to find the cells that are sensitive to the virus. The cell culture method is used to produce a large amount of virus. • Producing a virus by attaching cell culture to produce a whole virus vaccine (including live and inactivated vaccines), in which a large number of cells are cultured, which is a seedling cell line (attached type) The culture is first subjected to EDTA-pancreatin cell dispersion digestion and passage, and cultured in a culture medium or culture medium at a suitable temperature in a cell culture medium to form a good monolayer for further passage or virus inoculation. At present, the laboratory or enterprise f big _ bottle automatic transfer (check bottle System) ‘allows cells to get sufficient gas and nutrient exchange. However, the disadvantages of this conventional cell culture process are that the preparation time is complicated and the cells cannot be cultured in large quantities at the same time, and only a single layer of cells can be cultured per culture or culture flask. In order to cultivate a large amount of money in a short period of time, the cost of the company is huge, which increases the cost burden of human and material resources. It can be seen that 1 soul loses ___ as recorded points, and needs 3 201040257 - to improve. In view of the above-mentioned cell culture method, the inventor of the present invention produced a variety of vaccines derived from the county's vaccines, and he was able to improve the mines, and after years of painstaking research, he finally succeeded in research and development of this article_浮iweilongcheng culture Qiansi's vaccine and its methods. SUMMARY OF THE INVENTION The object of the present invention is to overcome the deficiencies of the prior art and to provide a method for culturing a secret silk vaccine with a suspension microcarrier. The method has a red-green effect and is stable, easy to age, high in virus content, and small in batch-to-batch quality control, which can significantly improve vaccine yield and quality. The vaccine produced by this method has good safety and high immunity, and has complete immune protection effect against the county. In order to achieve the above object, the present invention adopts the following technical solution: The method for producing a vaccine by using a suspension microcarrier cell culture system comprises the following steps: L inoculation of seedling cells into a culture tank containing a medium and a microcarrier; 2. Mixing the above cells with the microcarriers uniformly, and attaching the cells to the microcarriers; 〇3. Providing the above-mentioned cell foot nutrients and gases at a temperature to allow the cells to continue to grow on the microcarriers; The seedling virus is made into a virus suspension, inoculated on the above cells, and the culture is continued; the virus liquid is harvested every few days, and the medium is changed; 5. The above-mentioned harvested virus liquid is purified, and according to whether it needs to be inactivated. Add the inactivating agent or not add the inactivating agent, add the appropriate adjuvant to the inactivated virus solution, add the appropriate lyoprotectant without the inactivated virus solution, mix well and quantify the package to get inactivated. Or a live vaccine. The microcarriers in the step (1) are glass beads, polyester fiber discs, and polyester fiber plates. 4 201040257 * The step (3) is to obtain gas exchange by stirring or by raising and lowering the medium. The cell culture temperature described in the steps (2), (3), and (4) is 30 to 37. <:, and contains 2. 55% carbon dioxide. The cell in the step (1) is dog kidney cells (MDCK), and the virus in the step (4) is avian influenza virus (Η5Ν1). The cells in step (1) are hamster rat kidney cells (ΒΗΚ), rabbit kidney cells (Pku) or non-Pascia green monkey kidney cells (VER0), and the virus in step (4) is porcine pseudorabies virus (PRV). . The cells in the step (1) are porcine kidney cells (PK15), hamster rat kidney cells (BHK) or African green monkey kidney cells (VERO), and the virus in the step (4) is Japanese encephalitis virus (JEV). • The cells in step (1) are hamster pups kidney cells (BHK), and the virus in step (4) is bovine-popular fever virus (BEFV). The cell in the step (1) is an African green monkey kidney cell (VER〇), and the virus in the step (4) is a rabies virus. [Embodiment] The following description and drawings are intended to be illustrative and not restrictive. Other embodiments of the invention will be apparent to those skilled in the art from reading this description. Example 1 Production of Live Avian Influenza Vaccine 1.1 Virus and Cell Line The virus used to make the avian influenza vaccine is a reassortant of the H5N1 reconstituted vaccine, and the 5 201040257 virus strain is isolated from the highly pathogenic avian influenza virus strain. Attenuated strains genetically recombined by the World Health Organization (WHO) (eg A/Hong Kong/213/2003 or A/Viet Nam/1194/2004, or NIBRG-14) have been tested for pathogenicity' results showing H5N1 The reconstituted vaccine prototype strain virus can not be used to make H5N1 avian influenza vaccine. Furthermore, a cell line having good sensitivity to the virus strain was used (in this example, the dog kidney cell line Madin Darby Canine was used.

Kldney, MDCK) 'As a seedling cell line, to infect and multiply H5N1 avian influenza virus 0 〇 1.2 method (1) Inoculate seedlings with dog kidney cell line (MDCK cells) cells containing DMEM liquid culture medium (GIBC0 company) (containing secret (v/v) calf also clear) and microcarrier culture tank; • (2) at 37 ° C, 5% C 〇 2 culture environment, using mild Lin, or pumping, vacuum In a manner, the liquid medium level is raised and lowered in the culture tank for a small period of time, so that the cells are uniformly mixed with the microcarriers, and the cells are attached to the microcarriers; 〇(3) at 37 ° C, 5% c 〇 2 In the culture environment, the above-mentioned cell foot_nutrients are also provided by means of mixing or pumping or vacuuming, and the cells are allowed to grow on the above-mentioned micro-health for about 4 to 7 days until the cells are full of the microcarriers. DMEM medium was added with 2% calf serum to prepare for inoculation of the virus; (4) The virus-coated vaccine strain virus was made into a virus suspension (inoculation concentration of WM.O丄) in DMEM New Zealand medium, and inoculated into the above cells. On the top, in the 34〇c, ji c〇2 culture environment to continue the cultivation, the same use of the mixing Or pumping and vacuuming to provide sufficient nutrients and gas for the cells; after 2 to 3 days, the virus solution is harvested and stored in w; the harvested virus solution is fed into 6 201040257 • Toxic species identification to confirm compliance with various standards; Identification (8) Viral content: The virus content was calculated by the plaque forming unit (pfii) method, and the virus content per l.OmL was required to be 1 x 106 PFU. (b) Sterility test: no bacteria, mold, mycoplasma and foreign virus contamination. (5) The tested virus content = lxl 〇 6PFU / mL H5Nl reconstituted vaccine prototype virus antigen stock solution after centrifugation purification, the virus is destroyed by formaldehyde or binary 〇ethyleneimine (ΒΕΙ) After the operation, the oily vaccine is prepared by aluminum hydroxide adjuvant or by emulsification (oil-in-water, w/0) or biphasic emulsification (water-in-water, w/o/w), and quantitatively divided and added. Store the lid at 2~8 after sealing. (: Get the finished product. The three batches of vaccines will be numbered H5N1-T001, • H5N1-T002, H5N1-T003. U results virus content: H5N1 reconstituted vaccine prototype strain virus inoculated with MDCK cells, harvested virus sputum, a total of In three batches, the virus content was determined to be 109〇2ΡΡυ, 109.15 PFU, and 1〇9 06 PFU per lmL of virus. Sterility test: Three batches of products were grown aseptically. Compare the "suspended microcarrier cell culture system" of the present invention with The transgenic flask culture system was used to culture the MDCK cell line to proliferate the avian influenza virus Η5Ν1. The virus yield of the two-system cell culture is shown in Table 1-1. 7 201040257 Table 1-1 Comparison of the yield of the avian influenza virus H5N1 in different culture systems Culture method Suspension culture Rotary flask culture (85〇cm2) Cell inoculation number 3χ108 ΙχΙΟ7 toxic cells number 1.3χ109 1χ108 toxic concentration (ΜΟΙ) ΙΟ"4 ίο·4 Virus yield 500ml (=lxl09TCID5〇/ml) 200ml (1χ108 TCID5〇/ml) Ο The results of the above comparative test show that the yield of avian influenza vaccine produced by the suspension microcarrier cell culture system of the present invention is much larger than that of the Xizhi bottle culture system. Production of the production. Example 2 2.1 Preparation of pseudorabies live vaccine 2.1.1 Virus and cell strain The virus used to make the pseudorabies virus is a pseudorabies virulent strain (4) on the lion 8 G1 glycoprotein gene on PRV) (gene sequences such as Genbank No. M14336, AB440295, AJ271966, AY683134, AY683135, AY683136, AJ581561, AY368490, AF3〇6511, etc.) and thymidine kinase (τκ) gene (gene sequence) Such as

Genbank No. AF199413, AF080571, AY217095, AY171242, AY173125 # Genetically engineered gene-deficient attenuated strains. After causing the cockroach, the results showed that the gl vinegar protein and the thymocyte kinase (TK) double gene defect attenuated strain virus were not pathogenic, and could be used to make a live pseudorabies vaccine. And using a cell line which has good sensitivity to the virus strain (in this embodiment, the hamster rat „_ ΒΙ1Κ _ : other cells such as rabbit kidney fine 8 201040257 cells (PK13) or African green monkey kidney cells (VERO) also Porcine pseudorabies virus has good susceptibility. It is used as a cell line for seedlings to infect and multiply the pseudorabies virus. 2.1.2 Method (1) Inoculate BHK cells into the field containing ι〇% (ν/ν) ) calf serum DMEM liquid medium (GIBCO) and microcarrier culture tank; (2) at 36~37 ° C, 5% C 〇 2 culture environment, paste temperature and mixing, or pumping, vacuuming 0 (4) The liquid level of the medium (4) is raised and lowered in a small amount in the culture, and the side is about 3 trees, so that the above cells are uniformly mixed with the microcarriers, and the cells are attached to the microcarriers; (3) at 36 to 37 ° C, 5% C 〇 2 In the culture environment, the same method of mixing or pumping and vacuuming provides the above-mentioned cell foot nutrient and gas, so that the cells grow on the above-mentioned micro-health for about 2~5 days. Until the cells are full of microcarriers. Replace the new ❹ MEM medium and add 2% (v / v) calf serum 'prepared to inoculate the virus; (4) the pig mad Disease gI7TK-double gene defect strain virus w DMEM liquid medium (containing ❹2% shaking serum) was made into virus cake liquid (_ concentration is i tcid ^ cells), inoculated on the above cells 'placed at 36 ~ 37 ° C, 5% Continue to culture under the C〇2 culture environment, and also use the method of mixing or pumping and releasing the true two to provide sufficient nutrients and gas per cell for every 18 to 24 hours (when cytopathic (more than 90%) The liquid is stored in the next generation; the harvested virus solution is sterilized to confirm compliance with various standards; Toxic species identification (8) Viral content: The harvested virus solution is diluted with DMEM liquid medium to obtain 10, 10, 10, 10 3 dilutions, each dilution was inoculated with cells 201040257 and 4 bottles of virus, cultured in 36~37 ° C, 5% C〇1 incubator for 16 to 120 hours, each recorded cytopathic lesion (CPE) Calculate TCID50 according to Reed-Muench, and the content per 〇吼 须 = ΐχΐ 5 τ τ τ τ 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 无菌 无菌 5 无菌 无菌Specificity: Diluted harvested virus solution into 2 3 TCI in DMEM liquid medium Dso/mL, mixed with the same amount of anti-PRV rabbit immune serum, after 37 τ - 〇 hours, inoculated in pig kidney (ΡΚ), rabbit kidney (RK) and other strains of cells for 5 days, no The cell degeneration effect, and subcultured by sub-generational homologous cells for 7 days, also had no cell degeneration effect, and the adsorption test was carried out with erythrocytes of guinea pig, goose and chicken, respectively, and all of them must be negative. (5) Tested Qualified virus content=1χ1〇5 TCIDso/mL pig pseudorabies gI7TK-double gene defect strain virus antigen solution after centrifugation purification, 5 〇% (volume ratio) lactose as lyoprotectant, shake well, freeze vacuum drying And quantitatively dispensed, sealed and stored in

2~ 8 °C 〇 get aa. The three batches of vaccines produced were numbered PR_T1, PR_T2, and PR-T3. Finished product inspection: According to the "Inspection Standard for Animal Drugs", the inspection shall be in accordance with the regulations, and there shall be no side effects on the safety of the animal. The vaccine virus content is small and you are busy taking it. Sterility test: According to the "medical medication aa test; ^ quasi" regulations ^ _ test, there should be no live bacteria that may be detected. 1 丄3 result 2 disease glyce content. Porcine pseudorabies gI7TK-double gene defect strain virus inoculated with sputum cells, 3 harvested virus solution 'total 2 batches, respectively determine the virus content, each ImL virus content is 201040257 107 15tcid50, io7 20tcid50 , io735tcid50. The results of the vaccine trait test are shown in Table 2-1. Table 2-1 Three batches of vaccine trait test results Integrity: rf mesh results PR-T1 PR-T2 PR-T3 vaccine appearance light yellow solid, no foreign matter light yellow solid, no foreign matter light yellow solid, no red matter dissolved after foreign matter, The concentration is uniform, no odor, red liquid, uniform concentration, no odor, red liquid, uniform concentration, no odor, pH 7.15 7.18 7.20 virus content (TCID5〇/ml) 1〇7.15 1〇7.20 1〇7.35 The sterility test is negative, the results are shown in Table 2_2. Table 2-2 Three batches of vaccine sterility test results BHI Broth Friis Broth PPLO Agar PR-T1 negative negative negative PR-T2 negative negative negative PR-T3 negative negative negative £. Control positive _ moldy bacteria » positive positive each batch of vaccine 10 Test only after the sample is dissolved

BHI. The extract medium (Brajn Heart was inoculated as (10) Broth) and cultured at 37 ° C for 1 week. PPLO Agar: Mycoplasma solid medium. The samples were first inoculated into Friis liquid medium, and the culture medium was transferred to ppl 〇 Agar for 1 week, and further cultured for 1 week under 5% C 〇 2 conditions, and then microscopic examination for the presence of mycoplasma colonies. Specificity test: no other virus contamination, the results are shown in Table 2-3. 11 201040257 Table 2-3 Three batches of vaccine specific test results Test item vaccine + immune seropositive PR-T1 PR-T2 PR-T3 Control cell strain test PK CPE (1) CPE (1) CPE (-) CPE (+) CPE (-) RK CPE(1) CPE(1) CPE(1) CPE(+) CPE(-) Follow-up culture 7曰PK CPE(1) CPE(1) CPE(1) CPE(1) CPE(1) RK CPE(1) CPE(1) CPE(1) CPE(+) CPE(-) Blood cell adsorption test guinea pig blood cell negative negative negative positive negative chicken blood cell negative negative negative positive Negative Hematopoietic Negative Negative Negative Positive Negative PK: Porcine Kidney Cell Line RK: Rabbit Kidney Cell Line 〇CPE: Cytopathic 2.2 Comparative Experiment of Porcine Pseudorabies Vaccine Vaccine 本Comparative Products Made by the Invention 2.2.1 Materials (1) Vaccine: Porcine pseudorabies live vaccine (made by suspension microcarrier cell culture system) 3 batches, batch number: PR-T1, PR-T2, PR_T3. Porcine pseudorabies live vaccine (made by rotary bottle automatic culture system) 1 batch, batch number: PR-RB ° (2) Experimental animals: 1 month old weaned piglets, susceptible gilts' PR disease antibodies are Yin 12 201040257 - Gender, Qing Zhong and price = 1: 2). (3) Virus: Porcine pseudorabies virus (PRV) virulent strain. 2.2.2 Method (1) Safety test a. Safety test for piglets: 16 piglets weaned with pR virus antibody negative (serum neutralizing antibody = 1 · · 2) were randomly divided into 4 groups, each The group was injected with PR-T1, PR-T2, pR_T3 and PR-RB vaccines respectively, and 4 ml of each vaccine was intramuscularly injected for 21 days. At the same time, temperature was measured and food intake and respiration were observed. b. Safety test of gilts: 8 gilts were taken, 4 times before the breeding, and the muscles were seeded with PRV-gI'/TIC vaccine (batch number: PR-T1) and 4 samples of PR-RB vaccine, 8 ml each. /head. After immunization, the temperature was measured daily for routine clinical observation. After breeding, observe whether there are reproductive disorders such as premature birth, miscarriage, stillbirth, mummified fetus and weak babies, and observe until delivery. (2) Antibody detection and challenge protection test: Take 1 month old PR virus antibody negative (Jinqing Zhonghe ❹ antibody-1 · 2) susceptible piglets 20 'randomly divided into 5 groups, 4 in each group, 1, 2, 3 groups were injected with PR-T1, PR-T2, PR-T3 three batches of PRV-gI7TK-vaccine 2ml/head, 4 groups of intramuscular PR-RB vaccine 2mV head, 5 groups as negative control, blood collection after 21 days The serum was neutralized and the antibody titer was determined by separating the serum. At the same time, each pig was given 5 C 5 OTCID 5 〇 2 ml of the pig pseudorabies virulent virus, and 14 连续 was observed continuously. The clinical manifestations of the piglets were measured and observed. 2.2.3 Results (1) Safety test 13 201040257 Table 2-4 Vaccine safety test results Ο 〇 1 month old weaned pigs were injected with 4 ml of vaccine (pr_t1, pr_T2, PR-T3) and PR RB field respectively. Piglets have no increase in body temperature, and their feeding, mental status and growth and development are normal, and all piglets are tested, 3⁄4•. After the gilts were immunized with 8 ml of vaccine (pR Ti) and pR_RB vaccine, they were devoid of body/dish 1 normal appetite. After normal breeding, no target symptoms occurred. The results were fine, and the three batches of the experimental vaccine and the PR_RB vaccine were safe for vaccination. Wire is shown in Table 2 4. Vaccine animal immunization quantity immunization route immunization dose result PR-T1 piglet 4 intramuscular injection 4ml/head no body temperature rise, feeding, mental condition and growth and development are normal sow 4 intramuscular injection 8ml / head no body temperature change, normal appetite . No target disease occurred after normal breeding PR-T2 piglets 4 intramuscular injection 4ml / head no body temperature increased, feeding, mental status and growth and development were normal PR-T3 piglets 4 intramuscular injection 4ml / head no body temperature rise south, feeding , mental status and growth and development were normal PR-RB piglets 4 intramuscular injection 4ml / head no body temperature increased, feeding, mental status and growth and development of normal sow 4 intramuscular injection 8ml / head no body temperature changes, normal appetite . When the target did not occur, the target 痣楙^(2) antibody detection and challenge protection test was tested by the neutralization test method to detect serum neutralizing antibodies after vaccination, and the results showed that no 浐β PR-ΊΙ, PR-T2, PR-T3 After the vaccine or the PR-RB vaccine, the antibodies were all above i: ΐ6, 4/4 protection after challenge, and 4/4 control, the results are shown in Table 2-5. Table vaccine immunopotency test surname fruit group immune immunization number if #丨晋 neutralizing antibody titer

Attack protection rate 4/4 14 201040257 Group immune immunization quantity jl 丨景 neutralizing antibody titer PR-T2 PR-T3 PR-RB Negative control 4 4 4 4 2ml/head 2ml/head 2ml/head 2ml/head 1 1 64 64 16 Attack protection rate 1: 32 1 : 64 1 : 32 1 : 32 1 : 64 1 : 16 1 : 64 1 : 128 1 : 32 4/4 4/4 4/4 0/4 Compare this The invention discloses the "suspended micro-system" and (4) raising the BHK age to increase the pseudorabies scale, and the virus yield of the two-line cell culture is shown in Table 2-6. 2表2-6不_养^辑_假狂狂魅A(四)吾—culture method suspension culture spinner flask culture (1700 cm2) cell seeding number 2xl〇8 2.96x10s seed cell number 4.5xl〇9 2.96χ108 toxic concentration (MOI) 0.1 0.1 virus yield 3000ml (= lxl09pfu / ml) 600ml (9.6xl07pfti / ml) The above comparison test results show that the pigs produced by the micro-carrier cell culture of the invention

The vaccine produced by the pseudorabies vaccine and the conventional method has good fullness and immune effect on the susceptible piglets and the gilts, and the yield of the pseudorabies vaccine produced by the suspension microcarrier cell culture system of the present invention is much larger than that of the conventional use. The bottle culture system produces vaccine production. Example 3 3.1 曰 期 } 炎 炎 炎 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 3.1 .JEV-SA14 'its gene sequence information such as Gejjbajjjj; No. U14163), hamster kidney cells (BHK cell) after passage through 220 generations, became attenuated strains. After the pathogenicity test, the results showed that the attenuated strain virus was not pathogenic and could be used to make a Japanese encephalitis live vaccine. Also, a cell line having good sensitivity to the virus strain is used (in this embodiment, hamster kidney cells (BHK) are selected; other cells such as porcine kidney cells (PK) and African green monkey kidney cells (VER〇) are also used for Japanese encephalitis. The virus has good susceptibility. 'As a cell line for seedlings, it infects and multiplies the sputum cerebral inflammatory virus. 3.1.2 Methods • (1) Inoculate seedlings with BHK cells into MEM liquid medium (GIBCO, containing • 10% (cafe) calf serum, 〇·〇1Μ NaHC03, 〇.lmg/mi kanamycin sulfate ( Ka_ycin sulfate), 1〇〇, 〇〇〇ιυ penicillin G sodium salt (peniciiHn g Sodium)) and microcarrier culture tank; 〇 (2) in 37 ° C, 5% C 〇 2 culture environment, using mild Stir, or pumping or vacuuming, so that the liquid medium level rises and falls in the culture tank for a small amount of time, and the cells are uniformly mixed with the microcarriers, and the cells are attached to the microcarriers; (3) at 37 Under the culture environment of °C and 5%C〇2, sufficient nutrients and gases of the above cells are also provided by stirring or pumping and vacuuming, so that the cells grow on the above microcarriers for about 2-5 days until the cells are full of microscopic cells. Vector, replace with new MEM medium and add 1% (v/v) calf serum 'prepared to inoculate virus; (4) Add Japanese encephalitis attenuated strain virus to MEM liquid medium (including ι〇%(ν/ν) Calf serum) 16 201040257 • Prepared as a virus suspension (inoculation concentration of 0.005 Μ.Ο_Ι·), inoculated in the above fine On the cell, place it in a 37〇c, 5%C02 culture environment and continue to culture. Also use the method of stirring or pumping or vacuum to provide sufficient nutrients and gas for the cells; harvest the virus solution every 3~5 days and place it in 4 . (: preservation; harvested virus liquid for virus species identification to confirm compliance with various standards; virus species identification (a) virus content: the harvested virus solution is diluted 10 times in MEM liquid medium, taking 10_6, 10_7, 10_8 3 Each dilution was inoculated with 4 Q bottles of BHK cells, and cultured in 36 37 ° C, 5% c〇2 incubator for 16 to 120 hours. Daily observation and recording of cytopathic effect (CPE), press Reed- Muench calculates TCIDso, and the virus content must be = lxl 〇 5TCID5 每 per 1 〇. • (b) Sterility test: According to the Agricultural Committee's Animal Testing Standards, it must not contain any live bacteria that may be detected. Specificity: The harvested virus solution was diluted to SOC liquid medium to soOpfij/mL, and an equal amount of neutralizing antibody was used at a price of 104 or more (measured by plaque assay) against swine fever virus rabbit ◎ immune serum, 37° c feeling 1.5 hours after inoculation in hamster kidney cells (BHK) for 5 days, must have no cell degeneration effect, and subcultured by sub-generational cells for 7 days, also have no cell degeneration effect 'and scorpion 1% red blood cells of goose and chicken are subjected to adsorption test and must be negative. (5) Qualified virus content = lxl 〇 5 TCID5 〇 / mL Japanese encephalitis attenuated strain virus antigen solution after centrifugation purification, 50% (volume ratio) lactose as a lyoprotectant, and added 5 pg /ml kanamycin (Kanamycin), 5 IU / mL penicillin (Penicillin), shake well, freeze and vacuum dry, and quantitatively dispense, seal and store at 2~8. (: Get the finished product. Will be made 3 17 201040257 ' Batch vaccine number is JE-T01, JE-T02, JE-T03. Finished product inspection: According to the "Animal «Product Inspection Standard" for inspection, it should meet the requirements, and there is no side effect on the safety of the animal. Small 1〇5 redundant (four) site. Sterility test: According to the "Intestinal Drug Testing Standards" for testing, there should be no live bacteria that may be detected. 3.1.3 Results Virus content. Japanese encephalitis attenuated strain virus inoculated with BHK cells The virus solution was harvested for a total of 30 batches, and the virus content and vaccine traits were determined respectively. The test results are shown in Table 3-1. Table 3-1 Three batches of vaccine trait test results Test project results JE-T01 JE-T02 JE-T03 vaccine Normal appearance, no foreign matter, no foreign matter, no normal The concentration of the substance is uniform, the concentration is uniform, the concentration is uniform, no odor after dissolution, no odor, no odor, humidity 2.17% 2.66% 1.77% virus content 1065 1〇6.25 1〇6.25 (TCID50/ml) The sterility test is negative, the results are shown in Table 3-2 Table 3-2 Three batches of vaccine sterility test results BHI Broth Friis Broth PPLO Agar JE-T1 negative negative negative JE-T2 negative negative negative 18 201040257 BHI Broth Friis Broth PPLO Agar JE-T3 negative negative negative E.coli control, positive positive Positive Mycoplasma - Positive positive for each batch of vaccines. Only the sample was dissolved and tested. The sample was inoculated with Brain Heart Infusion Broth and cultured for 1 week at 37 °C. Ο PPLO Agar: Mycoplasma solid medium. The samples were first inoculated into Friis liquid medium, and the culture medium was transferred to ppL 〇耵 under 5% C〇2 for 1 week, and then examined by microscopy for the presence of mycoplasma colony specificity test: none Other virus contamination, the results are shown in Table 3-3. Table 3-3 Three batches of vaccine specific test results test items - vaccine + immune serum ~~~~~---- positive negative---- - JE-T01 ------- "E-T02 JE- T03 Control Cell Line Test HK Subculture 7曰CPE(1) CPE(-) CPE(1)----- CPE(+) -- CPE(-) HK Blood Cell Adsorption Test CPE(1) CPE(1) CPE(1) CPE(+) CPE(-) Scorpion Blood Cell Negative Negative negative positive negative chicken blood cell negative negative negative positive negative hematocrit negative negative negative positive negative 〇19 201040257 - HK: hamster kidney cell CPE: cytopathic 3.2 Comparative experiment of Japanese sputum active vaccine prepared by the present invention and similar products 3.2 .1 Materials (1) Vaccine··曰本脑炎活活 vaccine (made by suspension microcarrier cell culture system) 3 batches, batch number: JE-T (H, JE-T02, JE-T03. Sputum encephalitis Live poison vaccine (made by rotary bottle automatic culture system) 1 batch, batch number: JE-RB01. ❹ (2) Experimental animals: 4 weeks old weaned piglets, weighing 2~3kg, Japanese encephalitis antibodies are negative (serum neutralization) Price = 0.9). 3.2.2 Method (1) Safety test: 4 weeks old Japanese encephalitis virus antibody negative (serum neutralizing antibody) =0.9) 8 weaned pigs were randomly divided into 4 groups. Each group was injected with jE_T01, JE_T〇2, JE-T03 and JE-RB01 vaccines. Each dose was intramuscularly injected with the recommended dose of vaccine (2 mL) for 14 consecutive days. At the same time, weight measurement, observation of feeding, neurological symptoms, etc. (2) Antibody testing test 4 weeks old Japanese encephalitis virus antibody negative (serum neutralizing antibody = 1:10) susceptible piglets 1 hoe, Randomly divided into 5 groups, 2 in each group, the first and third groups of the muscles were injected with i times the dose of the guilty meal, JE-T02, ΙΕ·Τ03 vaccine' group 4 intramuscular injection of the i-dose dose of the vaccine. The first group did not shoot any vaccines as a control; after 14 days of immunization, the serum was removed to determine the neutralization of 20 201040257 - (NT) antibody titer and hemagglutination inhibition (HI) antibody power. 3_23 results (1 Safety test 4 weeks _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Both survived. The results showed that three batches of experimental vaccine and positive sputum vaccine Q were safe for overdose vaccination of target animals. The results are shown in Table 3-4. 4 vaccine safety test results vaccine pig number injection site clinical symptoms weight (ks) appetite neurological symptoms test initial test end JE-T01 1 normal normal no 2.7 4.6 2 normal normal no 2.9 4.9 JE-T02 3 normal normal no 2.3 4.3 4 Normal normal no 2.7 4.5 JE-T03 5 Normal normal no 2.4 4.4 6 Normal normal no 2.6 4.6 JE-RB01 7 Normal normal no 2.5 4.5 8 Normal normal no 2.7 4.8 Negative control 9 Normal normal no 2.5 4.4 (2) Antibody The test used the neutralizing test method to detect the serum neutralizing antibody after the vaccine immunization. The results showed that the antibody titer of JE-T (H, JE-T02, JE-T03 vaccine or JE-RB01 vaccine after immunization was 1:2). 〇21 201040257 Above, the results are shown in Table 3-5. Table 3-5 Vaccine Immune Efficacy Test Results _ Vaccine Pig No. Hemagglutination Inhibiting Antibody Force Preimmunity Immunization 14 days Preimmunization Immunity 14夭1 < 1:10 1:320 < 1:10 1:160 2 < 1:10 1:80 < 1:10 1:20 3 < 1:10 =1:320 < 1:10 =1:1280 4 < 1:10 160 < 1:10 1:160 5 &lt ; 1:10 =1:320 < 1:10 1:320 6 < 1:10 =1:320 < 1:10 =1:1280 7 < 1:10 1:160 < 1:10 1 :80 8 < 1:10 1:160 < 1:10 1:20 9 < 1:10 < 1:10 < 1:10 < 1:10 10 < 1:10 < 1: 10 < 1:10 < 1:10 neutralizing antibody valence JE-T01 JE-T02 JE-T03 JE-RB01 〇 negative control comparing the "suspended microcarrier cell culture system" of the present invention with a conventional spinner flask culture system, The HK cell line was cultured to proliferate Japanese encephalitis virus, and the virus yield of the two-system cell culture is shown in Table 3-6. Table 3-6 Different culture system proliferation Japanese encephalitis virus production comparison culture method suspension culture spinner flask culture (1700 cm2) cell seeding number 2χ108 6.5χ107 virulence cell number 3.5χ109 6.5χ107 toxic concentration (MOI) 0.005 0.005 virus The yield of 4900ml (=1062TCIDWml) 600ml (105 5 TCIDso/ml) The results of the above comparative test show that the vaccine produced by the Japanese inflammatory disease field and the one-use method produced by the suspension microcarrier cell culture system of the present invention has Good safety and immunity 22 201040257 - The effect, and the production of the Japanese encephalitis vaccine produced by the suspension microcarrier cell culture system of the present invention is much larger than the production of the vaccine produced by the conventional rotary bottle culture system. Example 4 4.1 Preparation of bovine epidemic heat inactivated vaccine 4.1.1 Virus and cell strain Bovine epidemic heat inactivated Q epidemic field was prepared with bovine epidemic fever virus virulent strain (for example, Tn73 or ΤΠ88128 strain), and the opposite virus was used. The cell line with good susceptibility (in this embodiment, the hamster rat kidney cell sputum) is used as a seedling cell line to infect and mass-produce the bovine epidemic fever virus. 4丄2 method α) Inoculate seedling cells with ΒΗΚ 液体 meditation medium (GIBC〇, containing U, Nikaki, 0.G1M NaHO) 3, aimg/ml sulphate card anamydn sulfate), ΙΟΟ'ΟΟΟ IU Penicillin G sodium salt (Penicillin G Sodium) and microcarrier culture tank; (2) in 37 ° C, 5% C 〇 2 Pei Wei, using mild mixing, or pumping, vacuum method to make liquid medium The liquid level is in the culture _ Xiao Cai New, gamma for about 2 hours, so that the above cells and microcarriers are evenly mixed, and the cells are attached to the microcarriers; (3) at 37 ° C, 5% C 〇 2 culture environment 'also use The method of mixing or pumping and vacuuming is provided to provide sufficient nutrients and gases of the cells, and the cells are grown on the microcarriers for about 2 to 5 days until the cells are filled with the entire IU-loaded MEM medium and added. 2 Correction, bovine serum, ready to inoculate the virus; (4) The bovine epidemic fever virus virulent strain is made into MEM liquid medium (containing 2% calf serum) 23 201040257 - Virus suspension (vaccination degree is 0.05 MOI ), inoculate on the above cells, and continue to culture in a 37〇c, 5%c〇2 culture environment, also using stirring or The vacuum is provided to provide sufficient nutrients and gas for the cells; the virus solution is harvested every 3 to 5 days and stored at 4 ° C; the virus solution is harvested for identification to confirm compliance with various standards; The virus contains cockroaches: the harvested virus solution is diluted 1 〇 in MEM liquid medium, and 10, 10, and 10 dilutions are taken, and each dilution is inoculated with 4 bottles of βηκ 〇 cells, respectively, at 5% C 〇 2 Incubate for 120 hours in the incubator, observe and record the cytopathic effect (CPE) daily. ' Calculate TCID50 according to Reed-Muench. For every 10 mT, the virus content should be 1x106TCID5. . (b) Sterility test: According to the “Testing Standard for Animal Drugs” of the Council of Agriculture, it shall not contain any live bacteria that may be detected. (3) Qualified virus content = lxl 〇 6 TCID5 〇 / mL bovine epidemic virus antigen ❹ 双 双 双 双 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( C paid. The three batches of vaccines produced were numbered bef-TOI, BEF-T02, BEF-T03. Finished product inspection: According to the "Inspection Standard for Animal Drugs", the inspection shall be in accordance with the regulations, and there shall be no side effects on the safety of the animal. The vaccine virus content is small, you Ding Qingla. Sterility test: According to the provisions of the "Test for the use of drugs for animal use", there should be no live bacteria that may be detected. 4丄3 Results Disease content: After the cattle epidemic fever was inoculated with ΒΗκ cells, the venom was collected, a total of 3 24 201040257 batches were determined for virus content, and the virus content per lmL was lxlO8.2 TCID5Q, ΙχΙΟ8·5 TCID5〇&gt 1x108 35TCID5〇° Sterility test: According to the “Intestinal Standard for Animal Drugs”, all three batches of products are grown aseptically. 4.2 Comparison test of bovine epidemic heat inactivated vaccine prepared by the present invention and similar products 4.2.1 Materials (1) Vaccine: Bovine epidemic heat inactivated vaccine (prepared by suspension microcarrier cell culture system) 3 batches, batch number: BEF -T (H, BEF-T02, BEF-T03. 1 batch of pig warm live vaccine (made by rotary bottle automatic culture system), batch number: BEF-RB01. (2) Laboratory animals: 6 months old cattle, cattle popular The heat virus antibodies were all negative (serum neutralization price = 1:10). 4_2.2 Method Antibody detection and challenge protection test: Take 6-month-old calves 1 taro, bovine epidemic fever virus antibody was negative (serum neutralizing antibody =1:10), randomly divided into 5 groups, 2 in each group, 1st, 2nd, and 3rd groups were injected with 1x doses of BEF-TCU, BEF-T02, BEF-T03 vaccine, and group 4 intramuscularly. One dose of BEF-RB01 vaccine, the fifth group did not inject any vaccine as a negative control; after 8 weeks of immunization, serum was separated to determine the neutralizing antibody titer. 4·2·3 results were detected by neutralization test Serum neutralizing antibody after vaccine immunization, the results show that unselfish is BEF-T01, BEF-T〇2, BEF-T〇3 vaccine or BEF_RB〇1 vaccine immunized antibody Price 岣 25 201040257 Above 1:32, the results are shown in Table 4-1. Table 4-1 Vaccine Immunization Efficacy Test Results Vaccine Neutralizing Antibody Price Price 1000 copies before immunization 8 weeks 1 <1:10 1:256 BEF-T01 2 <1:10 1:320 3 < 1:10 1:512 BEF -T02 4 < 1:10 1:1024 5 < 1:10 1:256 BEF -T03 6 <1: 10 1:320 7 < 1:10 1:160 BEF-RB01 8 < 1:10 1:256 9 < 1:10 < 1:10 Negative control _ 10 < 1:10 < 1:10 The BHK cell line was cultured by the "suspended microcarrier cell culture system" and the conventional spinner flask culture system of the present invention to proliferate the bovine epidemic fever virus, and the virus yields of the two system cell cultures are shown in Table 4-2. Table 4-2 Comparison of the yield of bovine epidemic fever virus in different culture systems. Culture method Suspension culture Rotary flask culture cell inoculation number 2χ108 2.96χ108 Number of cells at the time of poisoning 4.5χ109 2.96x10s Toxic concentration (MOI) 0.05 0.05 Virus yield 3800ml (=1x108TCID5〇) 600ml (1x106TCID5〇) The results of the above comparative test show that the bovine 26 201040257 produced by the suspension microcarrier cell culture system of the present invention is used for the epidemic study. A method of producing a vaccine money calves have good safety and immune effect, to yield a cell culture system and producing bovine ephemeral fever vaccine suspension of microcarriers of the invention, far larger than conventional roller bottle culture production system for producing a vaccine. Example 5 Preparation of a rabies attenuated vaccine 5.1.1 Virus and cell strain A rabies inactivated vaccine was prepared using a rabies virus virulent strain (for example, CW1 or aGV strain), and a cell line having good sensitivity to the virus strain was used. (This example uses African green monkey kidney cell VERO) 'as a seedling rib cell line' to infect and mass-produce rabies virus. 5.1.2 Method (1) Seedlings were seeded with VERO cells in MEM liquid medium (GIBC〇, containing 7% calf serum, 〇._NaHC〇3, 〇lm_sodium sulfate kanamycin such as __) 100,000 IU penicillin G sodium salt (Penicillin G Sodium)) and microcarrier culture tank; 〇 (2) in a 36 ° C, 5% C 〇 2 culture environment, using mild agitation, or pumping, vacuuming The liquid medium level is raised and raised in a small amount, and the side is about 2 hours, so that the above cells are uniformly mixed with the microcarriers, and the cells are attached to the microcarriers; (3) culture environment at 36〇C, 5%C〇2 Next, using the method of stirring or pumping or vacuuming, sufficient nutrients and gases of the cells are provided, and the cells are grown on the microcarriers for about 7 days until the cells are full of the microcarriers, and the new MEM medium is replaced and added. Looking for calf serum, ready to inoculate the virus; 27 201040257 - (4) The rabies anti-virus strain was made into a virus suspension (inoculation concentration of 0.05 MOI·) in MEM pigment medium (containing 2% oblique serum), and inoculated on the above cells. , placed in a 36τ, 5% c〇2 culture environment _ culture, the same cake or pumping, release the true way Provide enough nutrients and gas for the cells. · Harvest the second virus solution on the fourth day of inoculation, add a new mem medium (containing 2% calf serum), continue to culture, and continuously harvest the virus solution every 2 days. The harvested virus solution is placed at 4. (: preservation, and the virus is determined to confirm compliance with various standards; the sterility test is carried out in accordance with the "Testing Standard for Animal Drugs" of the Council of Agriculture, and must not contain any possible detectable activities. Bacteria, and determine the prion titer 'per L0mL virus content must be = 5.5 lg LD5 〇 / rnl; (3) Qualified virus content = 5 · 5 lg of rabies virus antigen stock after centrifugation purification 'to wake up or 2 - taethylamine (BEI) is inactivated by a water-soluble dye or by emulsification (oil-in-water 'W/〇) or two-phase emulsification (water-in-oil, water/w/0/w) Oily vaccine, quantitatively packed, sealed and stored at 2~8 °C to obtain finished products. Finished product inspection. According to the "Intestinal Drug Testing Standards" for inspection, it should meet the requirements and have no side effects on animal safety. Viral content = 5.5 lgLDWml. Sterility test: according to the "test mark for animal drugs" 》Regulations shall be carried out without any live bacteria that may be detected. 5·1·3 Results Viral content: After vaccination with rabies virus, the virus solution is continuously harvested, and the test is repeated twice to determine the virus content per lmL of virus. The contents are shown in Table 54. Table 5-1 Virus harvesting amount --- ______________________ days of virus inoculation - (IgLPso / rid) __ —— ---- - __ production batch 1 production batch 2 7.8 28 4 201040257

Virus inoculation days virus titer (lgLD50/ml, production batch 1 production batch 2 6 7.8 7.7 8 8.0 7.8 10 7.3 8.0 15 7.0 7.1 16 7.3 7.2 Aseptic test: Three batches of products are aseptically grown. Compare with the present invention The "suspended microcarrier cell culture system" and the conventional spinner flask culture system were used to culture the VERO cell line to proliferate rabies virus. The virus yields of the two system cell cultures are shown in the table. Table 5-2 Comparison of the yield of rabies virus in different culture systems Culture medium suspension culture bottle culture (1700 cm2) Cell inoculation number 1.4χ1〇8 1.4χ1〇8 Number of cells 5.6xl〇9 1.4xl〇8 Toxicity (MOI) 0.05 0.05 600ml (6 lgLD5〇/ml The virus yield is 8500ml (=7.0 lgLD5n/ml). The results of the above comparative test show that the yield of the suspension microcarrier cell culture system vaccine of the present invention is much larger than that of the conventional vaccine bottle production system. , virus strains, etc. can be obtained in commercial pipelines, government approved units; for example: the MDCK cells can be purchased from the American Standard Biological Collection Center (AmericanlypeCuiture)

Collection, ATCC) (eg. ATCC No. CCL-34, CRL-2285, CRL-2286, etc.) or consortium law 29 201040257 - Bioresource Conservation and Research Center (BCRC) of the People's Food Industry Development Institute (eg BCRC No.) 60567); The BHK cells are commercially available from ATCC (eg, ATCC No. CCL-10, CRL-1632, CRL-8544, CRL-10314, etc.); the PK13 cells are commercially available from ATCC (eg, ATCC No. CRL-6489) The VERO cells are commercially available from ATCC (eg, CCL-81, CRL-1587, etc.); the PK15 cells are commercially available from ATCC (eg, ATCC No. CCL-33); the BHK cells are commercially available from ATCC (eg, ATCC No. CCL-10, CRL-1632, CRL-8544, CRL-10314, etc.). Although the present invention has been described in terms of specific embodiments and applications, it is intended that the invention may be range. Therefore, the drawings and the description of the present invention are intended to be illustrative of the invention and are not intended to limit the scope. - The method for producing a vaccine by using a suspension microcarrier cell culture system provided by the present invention has the following advantages when compared with other conventional techniques: 1. The cell culture method provided by the present invention can be allowed in a bottle The increase in cell attachment surface can reduce the use of culture flasks and culture media. Its efficiency is dozens of times that of traditional rotary bottle automatic culture systems, which can save a lot of cost and manpower, and reduce the chance of operators coming into contact with viruses. To reduce the chances of operators infecting human-to-animal common viruses (eg, poultry H5N1 virus, Japanese encephalitis virus, rabies virus). 2. The method provided by the invention has low cell inoculation amount, easy control, high virus infection efficiency and high production efficiency. The detailed description above is a detailed description of one of the possible embodiments of the present invention, but the embodiment is not intended to be used in the present invention, and is not equivalent to the spirit of the present invention. , should be included in the scope of the patent in this case. In summary, the 'in this case, but the method is indeed _, and can carry out the above-mentioned multiple functions in comparison with the evaluation model, should fully comply with the statutory invention patent requirements of new and progressive, and love to apply according to law. The bureau approved the application for the invention patent, in order to invent the invention, to the sense of virtue. BRIEF DESCRIPTION OF THE DRAWINGS The technical content of the present invention and its effects will be further understood by referring to the following detailed description of a preferred embodiment of the invention and the accompanying drawings; : Circle 1 is a preparation flow chart of the present invention. [Main component symbol description] Benefits Μ»,

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Claims (1)

201040257 ♦ VII. Patent application scope: 1. A method for transducing floating microcarrier cell culture Yangxian silk vaccine, comprising the following steps: (1) inoculating cells for seedlings into a culture tank containing medium and microcarrier; (2) Mixing the above cells with the microcarriers uniformly to attach the cells to the microcarriers; (3) providing the above-mentioned cell foot nutrients and gas at a suitable temperature to continue to grow on the microcarriers; (4) seedlings (four) diseased silk The diseased silk floating liquid is inoculated on the above cells and continues to be cultured; the virus liquid is continuously harvested at a certain time or for a period of time, and the medium is replaced; (5) after the above-mentioned harvested virus liquid is purified, an appropriate lyoprotectant is added. , fully mixed and quantitatively dispensed to obtain the desired live vaccine. 2. The method for cultivating a nucleus cell culture vaccine according to the ninth item of the invention, wherein the microcarrier in the step (1) is a glass bead, a polyester fiber disc, a polyester fiber slab Various types of microcarriers. 〇 3. The method for producing a vaccine by a suspension microcarrier cell culture system according to claim 1, wherein the cell in the step (1) is dog kidney cells (MDCK), and the virus in the step (4) is avian influenza virus. (H5N1). 4. The method for producing a vaccine by a suspension microcarrier cell culture system according to claim 1, wherein the cell in the step (1) is a hamster rat kidney cell (BHK), a rabbit kidney cell, and a ^ Or African green monkey kidney cells (VERO), the virus in step (4) is Porcine Pseudorabies Virus (prv). 5. The method for producing a vaccine by the suspension microcarrier cell culture system as described in claim 1 of the patent scope 32 201040257 * The method wherein the cells in the step (1) are porcine kidney cells (ΡΚ), hamster kidney cells (HK) Or African green monkey kidney cells (VERO), the virus in step (4) is Japanese encephalitis virus (jev). 6. If you apply for a patent scope! The method for producing a vaccine by a suspension microcarrier cell culture system is described in which the cell in the step (1) is a hamster rat kidney cell (bhk), and the virus in the step (4) is a bovine epidemic fever virus (BEFV). 7. The method for preparing a suspension vaccine for suspension microcarrier cell culture according to the fourth aspect of the patent application, wherein the cell in the step (1) is an African green monkey kidney cell (VERO), and the virus in the step (4) is Rabies virus (RV). 8. The method of producing a vaccine by the Sugao microcarrier cell culture system as described in the patent phantom item, wherein the 3H step (3) is to obtain gas exchange by means of agitation. 9. The method of (4) floating micro-ship cell culture secret vaccine according to the cap (4), wherein the 5 Hai step (3) is to raise the medium by liquid level to provide sufficient nutrients and gas exchange. 〇 10. A method for producing a vaccine by a suspension microcarrier cell culture system as described in claim 1, wherein the step (4) is to take the secondary virus solution in units of 1 to 5 days, and continuously harvest the virus solution. 11_-A vaccine prepared according to the method described in Patent No. 1, Item 1. 12. A method for cultivating a money silk vaccine with a cake micro-resolving cell, comprising the technical steps: (1) cultivating the cell into a culture tank containing a transgenic group; 33 201040257 (2) mixing the above cells with the microcarriers uniformly , attaching the cells to the micro-cuts; (3) providing the above-mentioned _foot_nutrients and gases at appropriate temperatures to allow the cells to continue to grow on the microcarriers; (4) making the virus for seedlings into a virus suspension Inoculate on the above cells, continue to culture; continuously harvest the virus solution during the day or for a period of time, and change the medium; (5) Purify the above-mentioned harvested virus solution, inactivate it with an inactivating agent, and add appropriate The adjuvant is thoroughly mixed and quantitatively dispensed to obtain the desired inactivated vaccine. Ο I3. For example, in the method of the patent item 12 (10), the micro-carrier in the step (1) is a glass bead, a polyester fiber disc, a polyester fiber. Various microcarriers such as flat plates. 14. The method for producing a vaccine by a suspension microcarrier cell culture system according to claim 12, wherein the cell in the step (1) is dog kidney cells (MDCK), and the virus in the step (4) is avian influenza. Virus (H5N1). Ο 15_ The method for producing a vaccine by a suspension microcarrier cell culture system according to the scope of claim 5, wherein the cells in the step (1) are hamster rat kidney cells (BHK), rabbit kidney cells (pKl3) Or African green monkey kidney cells (VERO), the virus in step (4) is Porcine Pseudorabies Virus (pRV). 16. A method for producing a vaccine by a suspension microcarrier cell culture system according to claim 12, wherein the cells in the step (1) are porcine kidney cells (PK), hamster rat kidney cells (BHK). Or African green monkey kidney cells (VERO), the virus in step (4) is sputum encephalitis virus (JEV). 17. The method of producing a vaccine by a suspension microcarrier cell culture system as described in claim 12 of claim 12 201040257 ^ wherein the cell in the step (1) is a hamster rat kidney cell ,, the virus in the step (4) It is a bovine epidemic fever virus (BEFV). 18. A method for producing a vaccine according to the fourth embodiment of the present invention, wherein the cell in the step (1) is an African green monkey kidney cell (VER〇), in the step (4) The virus is rabies virus (rV). The method of producing a vaccine by a suspension microcarrier cell culture system as described in claim 12 of the patent scope is wherein the step (3) is to obtain gas exchange by stirring and aeration. • The vaccine is prepared by a suspension microcarrier cell culture system as described in claim 12 of the Shenqing patent scope, wherein the step (3) is to raise the medium by a liquid level to provide sufficient nutrients and gas exchange of the cells. 21. The method for producing a vaccine by a suspension microcarrier cell culture system as described in claim 12 of the patent scope, wherein the step (4) is to harvest the virus solution once in a period of 1 to 5 days, and continuously. reward Viral fluid. 22 __ · A vaccine prepared according to the method described in claim 12 of the patent application. 35