Canine kidney cell strain suitable for serum-free full suspension culture and application thereof

Abstract

The invention discloses a canine kidney cell strain suitable for serum-free full suspension culture and an application of the canine kidney cell strain. The name of the canine kidney cell strain is MDCK-JX, the canine kidney cell strain is classified and named as MDCK canine kidney cells, and the preservation number of the canine kidney cell strain in the China General Microbiological Culture Collection Center is CGMCC No.46059. The canine parvovirus can be harvested after being cultured in full-suspension serum-free cultured MDCK-JX cells for 48 hours, the titer of the obtained virus liquid is greater than 107.5 TCID50 / mL, the titer of the virus is far higher than that of the virus cultured in a spinner bottle, the harvested canine parvovirus suspension virus can be used as a suspension seed virus for subculture, and the canine parvovirus suspension virus has the advantages of small inter-batch difference, good immune effect and high safety, and has a good application prospect. Meanwhile, the antigen production method is simple to operate, low in labor intensity and easy to realize large-scale culture, achieves the purposes of increasing the yield and reducing the cost, and has a good market prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary biological products, and particularly relates to a canine kidney cell line MDCK-JX suitable for serum-free full suspension culture and an application thereof. Background Art

[0002] Canine parvovirus disease (CPD) is an acute, highly contagious disease caused by canine parvovirus (CPV). It has a high mortality rate and is one of the most serious infectious diseases affecting dogs. The disease is primarily preventable through vaccination, and there is no specific treatment.

[0003] Currently, domestic veterinary biological product companies produce canine parvovirus using the rotary bottle culture process, as shown in patents CN 1876181 A and CN 101905021 A. This process suffers from low production efficiency, high labor intensity, and poor product uniformity. The application of suspension culture can effectively address these issues. Suspension culture for virus cultivation offers advantages such as high automation, low labor intensity, and minimal batch-to-batch variability. Consequently, an increasing number of vaccine manufacturers are developing and utilizing suspension culture. Summary of the Invention

[0004] The present invention utilizes a suspension culture acclimation method to acclimate MDCK adherent cells (canine kidney cells) into serum-free full-suspension cultured MDCK-JX cells. The successfully acclimated cells can be applied to serum-free full-suspension culture of canine parvovirus, thereby improving antigen quality and yield, reducing production costs, and enabling large-scale culture and application in actual production.

[0005] Based on this, the present invention aims to overcome the shortcomings of the prior art and provide an MDCK-JX cell line adapted to serum-free full suspension culture and the use of the cell in virus culture. The MDCK-JX cell line is deposited in the General Microbiology Center of the China Culture Collection of Microorganisms, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO.46059. The present invention directly inoculates canine parvovirus into serum-free full suspension cultured MDCK-JX cells. The harvested canine parvovirus suspension can be used as a suspension seed virus for production and subcultured, thereby solving the problems of large batch differences and easy contamination in canine parvovirus transfer bottle culture, and can be used for large-scale production.

[0006] The present invention designs a method for acclimating the above-mentioned MDCK-JX cell line adapted to serum-free full suspension culture, and the acclimation method comprises the following steps:

[0007] (1) Passaging and culture of MDCK adherent cells

[0008] The revived MDCK cells were cultured in MEM medium containing 8% fetal bovine serum (FBS). When the MDCK cells grew into a full monolayer, the culture medium was discarded and washed three times with 1xPBS. After washing, 0.25% trypsin was added for digestion at 37°C. When the cells became like quicksand, MEM cell culture medium containing 8% fetal bovine serum was added to terminate the digestion. The attached cells were blown into a suspension with a pipette and divided into culture flasks at a ratio of 1:3 for subculture and culture for 2 to 3 generations.

[0009] (2) Adherent cells adapt to low serum suspension culture environment

[0010] Take a confluent monolayer of MDCK adherent cells and digest them with 0.25% trypsin at 37°C. After the cells shrink and fall off, add MEM cell culture medium containing 8% fetal bovine serum to terminate the digestion. Centrifuge the cells and discard the supernatant. Resuspend the cells in MDCK suspension medium containing 8% fetal bovine serum and transfer them to a shake flask. Incubate the cells in a shaker at 37°C and 5% CO2 in an incubator. Observe and count the cells every 24 hours. During the continuous passage process, gradually reduce the serum concentration and increase the shaker speed. When the serum concentration is reduced to 2%, the shaker speed is increased to 130 r / min, and the cell density is >3×10 6 cells / ml, and when the viability is greater than 90%, the cells are considered to have adapted to the low serum suspension culture environment.

[0011] (3) Acclimating cells to serum-free suspension culture environment

[0012] MDCK cells adapted to low-serum suspension culture were centrifuged, the supernatant discarded, and the cells resuspended in serum-free suspension medium. The cells were cultured in a shaking incubator at 37°C and 5% CO2. The cells were observed and counted every 24 hours. After continuous passage, the cells grew stably and the cell density was >3×10 6 When the viability was >90%, the MDCK cells were considered to have adapted to the serum-free medium suspension culture, and the MDCK cells obtained by this culture were named MDCK-JX cells.

[0013] In addition, the present invention also provides the use of the above-mentioned MDCK-JX cell strain adapted to serum-free full suspension culture in virus culture.

[0014] Furthermore, the above-mentioned MDCK-JX cell line adapted to serum-free full suspension culture is used in canine parvovirus culture.

[0015] The invention directly inoculates canine parvovirus into serum-free full suspension cultured MDCK-JX cells, and the harvested canine parvovirus suspension virus can be used as suspension seed virus for production for subculture, thereby solving the problems of large batch differences and easy contamination in canine parvovirus transfer bottle culture, and can be used for large-scale production.

[0016] The present invention also provides a method for culturing the canine kidney cell line, which is a canine kidney cell line MDCK-JX using an MDCK suspension culture medium; the MDCK suspension culture medium includes MDCK SFM 1415 culture medium, MDCK MS01A culture medium, MDCKSF003 culture medium, Any one or more of MDCK SFM-S medium.

[0017] The culture conditions are culturing in an incubator at 37° C. and 5% CO 2 , with a shaking speed of 130 r / min.

[0018] Finally, the present invention provides a method for culturing canine parvovirus, comprising the following steps:

[0019] 1) Culture the canine kidney cell line MDCK-JX according to the above method; 2) Add appropriate amount of fresh MDCK suspension culture medium to dilute the cell concentration to 3.0×10 6 cells / ml, and inoculated with canine parvovirus suspension seed virus for production according to the optimal inoculation dose MOI=0.05, and cultured at 36-37.5℃.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] The present invention provides a method for acclimating MDCK adherent cells into serum-free full suspension cultured MDCK-JX cells.

[0022] The present invention provides an application of using acclimated serum-free full suspension cultured MDCK-JX cells to culture canine parvovirus. The method can use the harvested canine parvovirus suspension virus as a production suspension seed virus to perform virus subculture to produce canine parvovirus, thereby reducing the restriction of the canine parvovirus adherent seed virus on virus culture and simplifying the process flow.

[0023] The serum-free fully suspended MDCK-JX cells obtained by the invention have a fast growth rate, a short doubling time, a high cell viability, and are suitable for large-scale factory production.

[0024] The present invention utilizes a method for culturing canine parvovirus using serum-free fully suspended MDCK-JX cells. The canine parvovirus can be harvested after being cultured in serum-free fully suspended MDCK-JX cells for 48 hours. The titer of the obtained virus solution is greater than 10 7.5 TCID 50 / mL, the virus titer is much higher than that of roller bottle culture. The harvested canine parvovirus suspension virus can be used as a suspension seed virus for production for subculture, which has the advantages of small batch difference, good immune effect and high safety. Compared with the existing roller bottle culture and microcarrier culture methods, it is simple to operate, has low labor intensity, is easy to realize large-scale culture, and achieves the purpose of increasing output and reducing costs, and has good market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a diagram of MDCK-JX cells adapted to serum-free full suspension culture in Example 1.

[0026] Figure 2 This is a graph showing the growth kinetics of MDCK-JX cell suspension culture in Example 2.

[0027] Figure 3 These are indirect immunofluorescence images of canine parvovirus cultured in suspension MDCK-JX cells in Example 3; A represents the fluorescence image of cells in the virus-infected well, and B represents the fluorescence image of cells in the uninfected control well.

[0028] Figure 4 This is the optimal inoculation dose of canine parvovirus in serum-free full suspension culture of MDCK-JX cells in Example 4.

[0029] Figure 5 The optimal cell density results for serum-free culture of canine parvovirus in full suspension MDCK-JX cells in Example 4 are shown.

[0030] Biomaterial Deposit

[0031] Accession number: CG MCC No.46059

[0032] Name: MDCK-JX

[0033] Classification and nomenclature: MDCK canine kidney cells

[0034] Deposit date: October 24, 2024

[0035] Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0036] Survival: Yes DETAILED DESCRIPTION

[0037] The present invention is described in more detail below through specific implementation methods to facilitate understanding of the technical solution of the present invention, but is not intended to limit the scope of protection of the present invention.

[0038] The MDCK suspension culture medium mentioned in the present invention includes MDCK SFM 1415 culture medium (Gansu Jianshun Biotechnology Co., Ltd.), MDCK MS01A culture medium (Suzhou Womei Biological Co., Ltd.), Any one or more of MDCK SFM-S medium (Suzhou Tianxinhe Biotechnology Co., Ltd.) and MDCK SF003 medium (Shanghai Beianji Biotechnology Co., Ltd.).

[0039] Example 1: Acquisition of MDCK-JX cell line adapted to serum-free full suspension culture

[0040] The method for acclimating MDCK adherent cells to suspension culture to obtain MDCK-JX cells adapted to serum-free full suspension culture specifically comprises the following steps:

[0041] (1) Cell recovery

[0042] Remove the frozen MDCK adherent cells from the liquid nitrogen tank, shake them clockwise in a 37°C water bath to thaw them quickly, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add an appropriate amount of MEM medium containing 8% fetal bovine serum, resuspend and disperse the cells, transfer them to a cell culture flask, and culture them in a 37°C, 5% CO2 incubator until the cells are covered with a monolayer.

[0043] (2) Cell passage

[0044] After the MDCK adherent cells have grown into a full monolayer, the culture medium was discarded and 1xPBS was added to rinse three times. After rinsing, an appropriate amount of 0.25% trypsin was added and the cells were digested at 37°C. When the adherent cells began to fall off, MEM cell culture medium containing 8% fetal bovine serum was added to terminate the digestion. The digested adherent cells were blown into a single cell suspension with a pipette and divided into culture flasks at a ratio of 1:3 for subculture and culture for 2 to 3 generations.

[0045] (3) Adherent cells adapt to low serum suspension culture environment

[0046] Take the MDCK adherent cells that have grown a monolayer, digest them with 0.25% trypsin at 37°C, wait for the cells to shrink and fall off, add MEM cell culture medium containing 8% fetal bovine serum to stop the digestion, transfer the digested cells to a 50ml sterile centrifuge tube, centrifuge at 1000r / min for 5min, and discard the supernatant; use 4 kinds of MDCK suspension culture medium containing 8% fetal bovine serum M1 (MDCK SFM1415 culture medium), M2 (MDCK MS01A culture medium), M3 ( Resuspend the cells in MDCK SFM-S medium) and M4 (MDCK SF003 medium), disperse the cells into a single cell suspension with a pipette and count the cells. Transfer the cell suspension to a shake flask with an initial density of 2×10 6 cells / ml, and cultured on a shaker in a 37°C, 5% CO2 incubator. The initial shaker speed was set at 80 r / min, and the cells were observed and counted every 24 h. During the continuous passage, the serum concentration was gradually reduced and the shaker speed was increased. The suspension acclimation effect of the four suspension media was observed. When the serum concentration was reduced to 2%, the shaker speed was increased to 130 r / min, and the cell density was greater than 3×10 6 cells / ml, and when the cell viability was >90%, the cells were considered to have adapted to the low serum suspension culture environment. The results showed that both M2 and M3 medium were suitable for the acclimation of the MDCK cells. The acclimated cells were labeled MDCK-2 and MDCK-3, respectively. After acclimation, the cell densities of the two cell lines could reach 8 to 10×10 cells / ml after 72 hours of culture. 6 cells / ml, 6~7×10 6 cells / ml.

[0047] Table 1 Results of acclimating MDCK adherent cells with 4 kinds of MDCK suspension medium and low serum

[0048]

[0049] Note: √ indicates that MDCK adherent cells can be successfully acclimated under these conditions; × indicates that MDCK adherent cells cannot be successfully acclimated under these conditions.

[0050] (4) Acclimating cells to serum-free suspension culture environment

[0051] Take two MDCK cell lines that have been adapted to low-serum suspension culture, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Add serum-free M2 and M3 suspension medium respectively to resuspend the cells, use a pipette to disperse the cells into a single cell suspension and count the cells. Adjust the initial cell density to 1.5×10 6 cells / ml, cultured in a 37°C, 5% CO2 incubator for 72 h with a shaker speed of 130 r / min, and the cells were observed and counted every 24 h. After continuous passage, MDCK-2 cells can grow stably in M2 suspension medium without serum supplementation, with a cell density of >3×10 6 cells / ml, with a viability of >90%; after three generations of MDCK-3 cells grown in serum-free M3 suspension medium, the viability gradually decreased, and the fourth generation was completely dead. Therefore, it was believed that MDCK-2 cells had adapted to the serum-free M2 suspension culture medium, and the MDCK-2 cells obtained by this culture were named MDCK-JX cells ( Figure 1).

[0052] MDCK-JX cells have been deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO.46059.

[0053] Example 2: Draw the growth kinetics curve of MDCK-JX cell suspension culture.

[0054] The 4th generation MDCK-JX cells with good growth were taken and cultured at 1.5×10 6 The initial cell density was cells / mL, and the cells were placed in a 250 mL shake flask with a culture volume of 50 mL. The culture conditions were 130 r / min, 37°C, and 5% CO2.

[0055] Samples were taken every 24 hours and cell counts were performed. The growth kinetics curve of suspension cultured MDCK-JX cells was plotted, as shown in Figure 2. Figure 2 As shown in the figure, the growth curve of MDCK-JX cells is "S" shaped. The cells enter the logarithmic growth phase from 24 hours and enter the plateau phase after 96 hours. The cell density increases slowly and the cell viability continues to decline. The cell doubling time is 26 hours, and the maximum cell proliferation density is 14.3×10 6 cells / mL.

[0056] Example 3: Cultivation of Canine Parvovirus Using MDCK-JX Suspension Cells

[0057] 1. According to the MDCK-JX cell suspension culture conditions, different inoculation conditions of canine parvovirus were studied.

[0058] (1) Optimal inoculation dose: MDCK-JX cells cultured according to the acclimation method described in Example 1 were inoculated with 2×10 6 The initial cell density was cells / mL, and canine parvovirus (virus titer: 10 6.8 TCID 50 / ml), and the virus liquid was harvested at 24h, 48h, 72h, and 96h after infection, and the virus titer was determined. The canine parvovirus was the CR86106 strain (Xia Xianzhu, Ye Junhua, Fan Quanshui, et al. Isolation, identification and immunological study of a natural attenuated canine parvovirus strain [J]. Chinese Journal of Livestock and Poultry Infectious Diseases, 1989, (06): 6-9+5.), which can be obtained from China Animal Husbandry Industry Co., Ltd. 。

[0059] (2) Optimal cell density: 1×10 6 cells / mL, 2×10 6cells / mL、3×10 6 cells / mL、4×10 6 cells / mL initial cell density, according to the optimal virus inoculation MOI = 0.05, inoculate canine parvovirus (virus titer: 10 6.8 TCID 50 / ml), and the virus liquid was harvested at 24h, 48h, 72h, and 96h after infection, and the virus titer was determined.

[0060] 2. TCID of canine parvovirus using indirect immunofluorescence 50 Determination:

[0061] 1. The canine parvovirus solution cultured in MDCK-JX suspension cells was diluted 10-fold in a gradient from 10 -1 Dilute to 10 -7 , set aside; take a monolayer of F81 adherent cells, digest them with trypsin, and prepare a single cell suspension with a cell density of 0.5×10 6 cells / ml were plated in 96-well plates, 100 μl was added to each well, and 10 -4 to 10 -7 100 μl of the diluted virus solution was cultured in a 37°C, 5% CO2 incubator for 96 h.

[0062] 2. After 96 hours of culture, fix with 80% acetone at 4°C for 30 minutes; add 200 μl of PBS to each well of a 96-well cell culture plate and wash three times; add 60 μl of a 1:100 diluted canine parvovirus monoclonal antibody (purchased from Beijing Century Yuanheng Animal Epidemic Prevention Technology Co., Ltd.) to each well and incubate in a 37°C incubator for 1 hour.

[0063] 3. Add 200 μl of PBS to each well of a 96-well cell culture plate and wash three times; add a 1:100 diluted fluorescent secondary antibody (Anti-Mouse IgG (whole molecule) FITC produced in goat, purchased from Company) 50 μl, incubated in a light-proof 37°C incubator for 1 h.

[0064] 4. Add 200 μl of PBS to each well of the 96-well cell culture plate and wash three times; observe under a fluorescence microscope. Figure 3 TCID was calculated according to the Reed-Muench method. 50 result.

[0065] The results of the optimal inoculation dose of canine parvovirus in serum-free culture of MDCK-JX cells are as follows Figure 4The results showed that when the MOI was 0.05, the highest titer of the virus liquid harvested at 48 h was 10 7.5 TCID 50 / mL, while when MOI was 0.01, 0.1, and 0.2, the virus titers harvested at 96h, 48h, and 48h were the highest, respectively. 7.0 TCID 50 / mL, 10 6.67 TCID 50 / mL, 10 6.67 TCID 50 Therefore, the optimal MOI was determined to be 0.05.

[0066] The optimal cell density of serum-free culture of canine parvovirus in MDCK-JX cells is as follows Figure 5 The results showed that the titer of the virus harvested from the culture was not positively correlated with the cell density. 6 cells / mL, the virus titer harvested at 48h was the highest, reaching 10 8.0 TCID 50 / mL. When the initial cell density is 1×10 6 cells / mL, the highest titer of the virus liquid harvested at 72 hours was 10 7.33 TCID 50 / mL; cell density was 2×10 6 cells / mL、4×10 6 cells / mL, the virus titer was the highest at 48h, which were 10 7.5 TCID 50 / mL, 10 7.67 TCID 50 / mL. Therefore, it was determined that the optimal initial cell density was 3×10 6 cells / mL.

[0067] The data show that canine parvovirus can be cultured in serum-free suspension MDCK-JX cells. Virus proliferation is affected by the inoculation dose, cell density, and culture time. When the inoculation dose and initial cell density are too low or too high, the optimal culture effect cannot be achieved. When the initial cell density is 3×10 6 cells / mL, the virus inoculation amount was 0.05MOI, and the titer of the virus liquid obtained after culturing for 48h was the highest, indicating that the full suspension culture of MDCK-JX cells can culture canine parvovirus and can achieve a higher virus titer.

[0068] Example 4: Cultivation of Canine Parvovirus Suspension Seed Virus

[0069] 1. Preparation of Canine Parvovirus Adherent Seed Virus

[0070] The virus solution containing 1% (v / v) canine parvovirus seed virus was inoculated into a confluent monolayer of F81 adherent cells and incubated for 1 hour. The virus solution was discarded and supplemented with an appropriate volume of MEM maintenance solution containing 2% (v / v) serum and cultured at 37°C. When the cell lesion rate reached 60% to 80%, the virus solution was harvested and repeatedly frozen and thawed at -80°C for three times. The virus titer was detected by sampling. When the virus content was ≥10 6.5 TCID 50 / mL and save the virus solution for later use.

[0071] 2. Cultivation of canine parvovirus suspension virus

[0072] According to the optimal inoculation cell density and optimal inoculation dose of canine parvovirus cultured in MDCK-JX suspension cells explored in Example 3, the culture of canine parvovirus suspension seed virus was explored using a shake flask. The specific method was as follows: MDCK-JX suspension cells were cultured, and when the initial cell density was 3×10 6 cells / mL, the canine parvovirus adherent seed virus prepared in the above step 1 was inoculated at the optimal inoculation dose MOI=0.05, and the virus liquid was harvested 48 hours after inoculation. This is the canine parvovirus suspension F1 generation virus. The canine parvovirus suspension virus F2 generation, F3 generation, F4 generation, and F5 generation were further cultured according to the above method. The harvested virus liquid was subjected to toxicity titer determination. The experimental results are shown in Table 2.

[0073] Table 2. Results of canine parvovirus suspension inoculation cultured in MDCK-JX suspension cells

[0074]

[0075] The above results show that the canine parvovirus suspension seed virus cultured using MDCK-JX suspension cells can be used as a seed virus for canine parvovirus production to cultivate canine parvovirus.

[0076] Example 5: Cultivation of MDCK-JX suspension cells in a bioreactor

[0077] MDCK-JX suspension cells were cultured in shake flasks for 1 to 3 generations, expanded to the cell number required for inoculation in a 10 L bioreactor, transferred to a 10 L bioreactor, and supplemented with MDCKMS01A medium to a final cell concentration of 0.8 to 1.0 × 10 6 cells / ml. Culture for 48h to 72h at 36.5±0.5℃, pH 7.2±0.1, DO 30% to 60%, stirring speed 70rpm to 90rpm (stirring speed varies with cell density). When the cell density in the reactor reaches 8 to 10×10 6cells / ml, it is used for further amplification culture or inoculation.

[0078] Example 6: Production of canine parvovirus antigen using a bioreactor

[0079] 1. Cultivation of suspension seed virus for canine parvovirus production

[0080] According to the culture method of canine parvovirus suspension seed virus in Example 4, a batch of suspension seed virus for production was cultured, and the titer of the suspension seed virus was 10 7.67 TCID 50 / ml.

[0081] 2. Production of canine parvovirus antigen using bioreactors

[0082] Take MDCK-JX suspension cells cultured in shake flasks or bioreactors for 48 h to 72 h, count the cells, and transfer them to a new bioreactor. The reactor culture parameters are: temperature 36.5 ± 0.5 °C, pH 7.2 ± 0.1, DO value 50% to 60%, and stirring speed 70 rpm to 90 rpm. Dilute the cells to a concentration of 3.0 × 10 6 cells / ml, and inoculated with the canine parvovirus suspension virus prepared in step 1 above at the optimal inoculation dose of MOI = 0.05. After inoculation, the cell viability was monitored and cultured for 48h to 72h. When the cell viability was around 60%, the virus solution was harvested and stored at -80°C. The virus content of the harvested canine parvovirus solution was determined, and the virus content was 10 7.8 TCID 50 / mL.

[0083] Example 7: Application of canine parvovirus antigen

[0084] 1. According to the method of Example 6, 3 batches of canine parvovirus antigen were prepared, with virus contents of 10 7.8 TCID 50 / ml, 10 7.67 TCID 50 / ml, 10 8.0 TCID 50 / ml. The three batches of antigens were used for purity test. After passing the test, the three batches of antigens were diluted respectively and the virus content of the three batches of antigens was adjusted to 10 7.0 TCID 50 / ml, and freeze-dried at 1ml / head to prepare three batches of canine parvovirus live vaccine.

[0085] 2. Safety Test: Select five healthy beagle dogs aged 6-8 weeks with negative canine parvovirus antibodies and observe them in isolation for 5-7 days. Dilute the three batches of vaccine described above and inject them intramuscularly into five test dogs, giving each dog 10 doses. Observe them in isolation for 21 days. The dogs should be normal in spirit, appetite, body temperature, and stool.

[0086] 3. Efficacy test: 25 healthy beagle dogs aged 6-8 weeks with negative canine parvovirus antibodies were selected and divided into 5 groups, I, II, III, IV, and V, with 5 dogs in each group. Groups I, II, and III were immunized with the three batches of vaccine prepared in step 1, and the vaccine was diluted to 2 ml / head. Each dog was injected intramuscularly with 1 dose. Group IV was immunized with a commercialized canine distemper, canine parainfluenza, canine adenovirus, and canine parvovirus disease quadruple live vaccine (China Animal Husbandry Co., Ltd. Jiangxi Biological Pharmaceutical Factory, batch number: 2306061) at 2.0 ml / head intramuscular injection. Group V was a non-immunized negative control group. Blood and serum were collected 21 days later for canine parvovirus neutralizing antibody titer determination.

[0087] The results showed that in safety tests, the spirit, appetite, body temperature and feces of the five test dogs were normal after 21 days of isolation observation; in efficacy tests, the neutralizing antibody titers of groups I, II and III were 1:501, 1:501 and 1:501 respectively, the neutralizing antibody of group IV was 1:256, and the neutralizing antibody of group V was ≤1:2. After the dogs were immunized with canine parvovirus antigen prepared by MDCK-JX suspension cells, the neutralizing antibody titer was much higher than that of the commercialized canine distemper, canine parainfluenza, canine adenovirus and canine parvovirus disease quadruple live vaccine, indicating that the canine parvovirus prepared by MDCK-JX suspension cells has the advantages of high safety and good immune effect.

[0088] In summary, the present invention provides a strain of MDCK-JX full-suspension cells obtained through serum-free suspension acclimation. Using MDCK-JX suspension cells for full-suspension culture of canine parvovirus, the vaccine prepared from the resulting canine parvovirus antigen exhibits high potency, minimal batch-to-batch variability, excellent immune efficacy, and high safety. Furthermore, this antigen production method is simple to operate, requires minimal labor, and is easily scalable, achieving the goals of increasing yield and reducing costs, thus promising promising market prospects.

[0089] The above contents are merely preferred embodiments of the present invention and are not intended to limit the scope of implementation of the present invention. That is, any simple equivalent changes and modifications made in accordance with the claims and description of the present invention are still within the scope of the present invention.

Claims

1. A canine kidney cell line adapted to serum-free full suspension culture, characterized in that: The canine kidney cell line is named MDCK-JX, classified as MDCK canine kidney cell, and its deposit number in the General Microbiology Center of China Culture Collection Administration of Microorganisms is CGMCC No.46059.

2. Use of the canine kidney cells adapted to serum-free full suspension culture according to claim 1 in virus culture.

3. The use according to claim 2, characterized in that Such viruses include canine parvovirus.

4. The method for culturing the canine kidney cell line according to claim 1, wherein the canine kidney cell line MDCK-JX according to claim 1 is cultured using MDCK suspension medium; the MDCK suspension medium includes MDCKSFM 1415 medium, MDCK MS01A medium, MDCK SF003 medium, Any one or more of MDCKSFM-S culture medium; the MDCK suspension culture medium is preferably MDCK MS01A culture medium.

5. The method according to claim 4, characterized in that The culture conditions are culturing in an incubator at 37° C. and 5% CO 2 , with a shaking speed of 130 r / min.

6. A method for culturing canine parvovirus, characterized in that: The following steps are involved: 1) Culturing the canine kidney cell line MDCK-JX according to the method of claim 4 or 5; 2) Add fresh MDCK suspension medium to dilute the cell concentration to 3.0×10 6 cells / ml, and inoculated with canine parvovirus suspension seed virus for production according to the optimal inoculation dose MOI=0.05, and cultured at 36-37.5℃.

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