Method for suspension culture of rabies virus

Abstract

The invention belongs to the technical field of vaccine preparation, and particularly relates to a method for suspension culture of rabies virus. MDCK-siat7e-C is used as a cell matrix for suspension culture of the rabies virus, experiments prove that the rabies virus can be subcultured in the cell, the virus titer can reach 7-81gFFU / ml, and through mouse immunogenicity inspection, the protection index is greater than 100, and the antibody titer reaches 67IU / ml after mice are immunized. The method for suspension culture of the rabies virus by the cell strain provides reference for suspension culture of the rabies virus.

Description

Technical Field

[0001] The present invention belongs to the technical field of vaccine preparation, and particularly relates to a method for culturing rabies virus in suspension. Background Art

[0002] At present, the human rabies vaccines approved for marketing in China are mainly inactivated vaccines produced by cell culture, and the cell matrices used include primary cells, human diploid cells, and Vero cells. For the vaccine produced by primary cells, its side effects are relatively mild, and it has good safety and effectiveness. However, its disadvantages are high labor intensity and low yield. For the human diploid cell rabies vaccine, the incidence of adverse reactions after vaccination is low, the symptoms are mild, and there is no risk of tumorigenesis. However, human diploid cells have slow proliferation, low virus yield, and high vaccine cost, and have not been widely used. At present, the production of human rabies vaccines mostly uses Vero cells for adherent culture to produce rabies virus, with high virus titer, large vaccine yield, high safety and potency. It is difficult to domesticate Vero cells as a cell matrix for suspension, and most adopt the microcarrier adherent culture method. Compared with suspension culture, its production cost is higher, the cell passage operation process is more complex, and the scale-up is limited.

[0003] Common large-scale culture techniques for adherent cells include methods such as roller bottle culture, cell factory culture, and microcarrier culture. The roller bottle culture technique is simple to operate and has low cost. However, due to problems such as low cell density, large space occupation, high labor intensity, low production efficiency, and difficulty in controlling bottle-to-bottle differences, it has been gradually replaced. Cell factories can effectively utilize space compared with roller bottle culture, have low pollution risk, high productivity, and small bottle-to-bottle differences. However, there are also situations such as large space occupation and low production efficiency in large-scale amplification. Microcarrier bioreactor culture can effectively monitor and adjust the growth environment of cells and viruses, has a high level of automation, and good batch-to-batch consistency. However, the microcarrier culture technique is not conducive to large-scale amplification, and the cost of microcarriers is relatively high, and they usually cannot be recycled after use.

[0004] The suspension culture process is easy to operate, has a small pollution risk, and a high cell density. Especially when performing large-scale culture in a bioreactor, it can be easily amplified to the ten-thousand-liter level. The suspension culture process is easy to control, and the culture conditions are stable and uniform, which is beneficial to ensuring product yield and batch-to-batch consistency.

[0005] At present, the production of human rabies vaccines mostly uses serum-containing media, which contain animal-derived substances and are likely to cause significant differences in vaccine quality and batch-to-batch. The use of serum-free and chemically defined media avoids the problems of pollution risks such as animal viruses or TSEs that serum may contain and batch-to-batch differences, further improves the vaccine safety, and at the same time reduces the burden of downstream purification. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides a method for culturing rabies virus in suspension using a serum-free and chemically defined medium for the full suspension culture of canine kidney epithelial cell line MDCK-siat7e-C cells, providing a reference for the suspension culture of rabies virus.

[0007] The present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a serum-free and chemically defined medium for the full suspension culture of canine kidney epithelial cell line MDCK-siat7e-C cells, which is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: C2024292.

[0009] In the second aspect, the present invention provides a serum-free and chemically defined medium for the full suspension culture of canine kidney epithelial cell line MDCK-siat7e-C cell bank, which is constructed by using the aforementioned cell line.

[0010] In the third aspect, the present invention provides the use of the aforementioned cell line or cell bank for culturing viruses or preparing virus vaccines.

[0011] In some embodiments, the virus is rabies virus.

[0012] In the fourth aspect, the present invention provides a method for culturing rabies virus in suspension, including: inoculating rabies virus into the cell suspension of the cell line in the aforementioned cell line or cell bank, culturing in suspension, and harvesting rabies virus from the culture product.

[0013] In some embodiments, the cell line in the aforementioned cell line or cell bank is inoculated into a serum-free cell growth medium, cultured in suspension until it proliferates to a certain cell density to obtain a cell suspension; the serum-free cell growth medium in the cell suspension is replaced with a serum-free cell maintenance medium, and then rabies virus is inoculated to allow sufficient adsorption to obtain an infected virus cell suspension; the serum-free cell maintenance medium in the infected virus cell suspension is replaced with a new serum-free cell maintenance medium, cultured in suspension, and rabies virus is harvested from the culture product.

[0014] In some embodiments, the cell line in the aforementioned cell line or cell bank is inoculated into CD MDCK 244 medium, and cultured in suspension in a shaker at 37 °C, 5% CO2, and 125 rpm. When the cell density proliferates to 3×10 6 ~4×10 6cells / ml to obtain a cell suspension; centrifuge the cell suspension to discard the supernatant, resuspend the cell pellet with CD MDCK 296 medium, inoculate rabies virus at a ratio of MOI = 0.1 - 0.5, and place it in a shaker at 35 - 37 °C, 5% CO2, and 125 rpm for adsorption for 1 - 2 h to obtain a cell suspension infected with the virus; centrifuge the cell suspension infected with the virus to discard the supernatant, resuspend the cell pellet with CD MDCK 296 medium, and then place it in a shaker at 35 °C, 5% CO2, and 125 rpm for suspension culture. Every 2 - 3 days, centrifuge the culture product and harvest 2 / 3 volume of the supernatant, and then resuspend the remaining culture product with CD MDCK 296 medium to make up the culture volume. Culture until the 11th - 15th day, and harvest rabies virus from the culture product.

[0015] In a fifth aspect, the present invention provides a method for preparing a rabies virus vaccine, comprising: inoculating rabies virus into a cell suspension of the aforementioned cell line or the cell line in the cell bank, performing suspension culture, and harvesting rabies virus from the culture product; and concentrating, purifying, and inactivating the harvested rabies virus to obtain a rabies virus vaccine.

[0016] In some embodiments, the cell line in the aforementioned cell line or the cell bank is inoculated into a serum-free cell growth medium, and suspension culture is performed to proliferate to a certain cell density to obtain a cell suspension; the serum-free cell growth medium in the cell suspension is replaced with a serum-free cell maintenance medium, and then rabies virus is inoculated to allow sufficient adsorption to obtain a cell suspension infected with the virus; the serum-free cell maintenance medium in the cell suspension infected with the virus is replaced with a new serum-free cell maintenance medium, suspension culture is performed, and rabies virus is harvested from the culture product; and the harvested rabies virus is concentrated, purified, and inactivated to obtain a rabies virus vaccine.

[0017] In some embodiments, the cell line in the aforementioned cell line or the cell bank is inoculated into CD MDCK 244 medium and placed in a shaker at 37 °C, 5% CO2, and 125 rpm for suspension culture. When the cell density proliferates to 3×10 6 ~4×10 6cells / ml to obtain a cell suspension; centrifuge the cell suspension to discard the supernatant, resuspend the cell pellet with CD MDCK 296 medium, inoculate rabies virus at a ratio of MOI = 0.1 - 0.5, and place it in a shaker at 35 - 37°C, 5% CO2, and 125 rpm for adsorption for 1 - 2 h to obtain an infected virus cell suspension; centrifuge the infected virus cell suspension to discard the supernatant, resuspend the cell pellet with CD MDCK 296 medium, and then place it in a shaker at 35°C, 5% CO2, and 125 rpm for suspension culture. Every 2 - 3 days, centrifuge the culture product and harvest 2 / 3 volume of the supernatant, and then resuspend the remaining culture product with CD MDCK 296 medium to make up the culture volume. Culture until the 11th - 15th day, and harvest rabies virus from the culture product; and, concentrate, purify, and inactivate the harvested rabies virus to obtain a rabies virus vaccine.

[0018] The beneficial effects of the present invention are as follows:

[0019] 1. The present invention obtains a serum-free and fully suspended culture type canine kidney epithelial cell line MDCK-siat7e-C for the first time. This cell line uses the MDCK-siat7e cell line as the parent, and is successively domesticated through serum-free BD MDCK 001 medium and serum-free chemically defined medium CD MDCK 244. After domestication, the cell state is good, the cell viability is high, and the medium used for culturing this cell does not contain serum and animal-derived components, and the components are clear, which is conducive to the quality control of biological products.

[0020] 2. The method for culturing rabies virus provided by the present invention has the following characteristics:

[0021] 1) For the first time, rabies virus is suspended and cultured using MDCK-siat7e-C as the cell matrix, and it is experimentally confirmed that rabies virus can be passaged and cultured in this cell, and the virus titer can reach 7 - 8 lgFFU / ml. After immunogenicity examination in mice, the protection index > 100, and the antibody titer reaches 67 IU / ml after immunizing mice.

[0022] 2) The present invention provides a new culture mode for rabies virus - suspension culture. Compared with the traditional adherent culture of Vero cells, the suspension culture process is easy to operate, has a small pollution risk, and a higher cell density. Especially when culturing on a large scale in a bioreactor, it can be easily scaled up to the ten-thousand-liter level. Moreover, the suspension culture process is easy to control, and the culture conditions are stable and uniform, which is conducive to ensuring the product yield and batch-to-batch consistency. The use of serum-free and chemically defined medium avoids the pollution risks and batch-to-batch differences problems that serum may contain animal viruses or TSE, further improves the vaccine safety, and at the same time reduces the burden of downstream purification. Description of the Drawings

[0023] Figure 1 Curves of viable cell density (A) and cell viability (B) of MDCK-siat7e cells in different growth media;

[0024] Figure 2 Metabolic conditions of MDCK-siat7e cells in different growth media. Among them, A: specific glucose consumption rate; B: specific ammonia production rate;

[0025] Figure 3 Microscopic photo (x200) of MDCK-siat7e-C cells cultured in CD MDCK 244 medium for 120 hours;

[0026] Figure 4 Growth conditions of MDCK-siat7e cells in BD MDCK 001 / CD MDCK 244 medium. Among them, A: cell proliferation curve; B: cell viability; C: specific growth rate of cells;

[0027] Figure 5 Comparison of differences in culture for 3 days in different maintenance fluids after virus inoculation. Among them, A: virus titer at 3 days; B: viable cell density at 3 days;

[0028] Figure 6 Cell glucose metabolism conditions when cultured for 3 days in different maintenance fluids after virus inoculation. Among them, A: initial glucose content in different maintenance fluids; B: final glucose content in different maintenance fluids; C: glucose metabolism rate in different maintenance fluids;

[0029] Figure 7 Comparison of virus and cell culture differences in different maintenance fluids 7 days after virus inoculation. Among them, A: virus proliferation curve; B: cell growth curve; C: peak virus titer; D: virus antigen content;

[0030] Figure 8 Virus proliferation under different MOI conditions. Among them, A: virus titer curve; B: virus titer curve;

[0031] Figure 9 Virus titers and titers curves of generations 1 to 5. Among them, A: virus titer curve; B: virus titer curve;

[0032] Figure 10 Cell photos 13 days after virus inoculation. Among them, A: photo of MDCK-siat7e-C cells infected with CTN-1M strain 13 days later (x200); B: photo of control MDCK-siat7e-C cells cultured for 13 days (x200);

[0033] Figure 11Results of SDS-PAGE of rabies virus CTN-1M strain and WB experiments of CTN-1M strain and CTN-1V strain. Among them, A: SDS-PAGE (CTN-1M strain); B: WB (results of G protein transfection, results of N protein transfection).

[0034] Figure 12 It is the neutralizing antibody titer curve of CTN-1M virus harvested multiple times for 4 generations. Specific implementation manners

[0035] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0036] The experimental materials in the following embodiments are as follows:

[0037] 1. Cell lines, rabies virus strains

[0038]

[0039]

[0040] 2. Main reagents

[0041]

[0042] Example 1 Construction of serum-free fully suspended cultured canine kidney epithelial cell MDCK-siat7e-C cell line

[0043] 1. Acclimation culture in serum-free BD MDCK 001 medium: After the MDCK-siat7e cell line was resuscitated, it was continuously passaged and cultured for 7 generations at 37 °C, 5% CO2, and 125 rpm through the serum-free medium BD MDCK 001 to make it adapt to suspension growth in the serum-free medium BD MDCK 001. The state of the acclimated cells was stable, the growth was good, the cell viability reached more than 98%, the cells grew in single suspension, were uniform in size, and had regular morphology. The cells acclimated with serum-free were used to establish a master cell bank and stored in liquid nitrogen.

[0044] After inspection, the viability of the cells in the master cell bank after resuscitation was above 92%. After 1 to 2 passages, the cell viability increased to 98%. The cell growth state was stable and the dispersibility was good. The master cell bank and the ultra-high passage (40 passages) of the master cell bank were sent to the National Institutes for Food and Drug Control. According to the requirements of the Pharmacopoeia of the People's Republic of China (Volume III, 2015 Edition), a comprehensive verification of the master cell bank (sample number: SH0417201501586) was carried out, including cell identification test, bacteria and fungi inspection, mycobacterium inspection, mycoplasma inspection, intracellular and exogenous virus factor inspection, tumorigenicity inspection, etc. Partial verification (tumorigenicity and tumorigenicity inspection) was carried out on the ultra-high passage (sample number: SH041720161025). The verification report showed that the reverse transcriptase activity test report of the exogenous virus inspection of the master cell bank was negative, and no virus-like particles were seen in the transmission electron microscopy inspection report. The other verification results of the master cell bank met the requirements of the Pharmacopoeia of the People's Republic of China (Volume III, 2015 Edition); for the tumorigenicity inspection, the median tumorigenic dose (TPD 50 ) for nude mice was 10 5.3 viable cells per mouse, not higher than the median tumorigenic dose of the original cells for nude mice (10 5.0 viable cells per mouse). The results of the tumorigenicity inspection of the ultra-high passage cells showed that the tumor formation rate of nude mice was 0 / 15, and no nodules were seen in the main organs such as the heart, liver, spleen, lungs, kidneys, and lymph nodes of the nude mice. Compared with the negative control, there were no obvious differences in the histopathological changes of the heart, liver, spleen, lungs, kidneys, and lymph nodes; for the tumorigenicity inspection, the TPD 50 was 10 4.5 viable cells per mouse. The verification report number of the master cell bank was SH201702389, and the verification report number of the ultra-high passage was SH201702390.

[0045] 2. Further optimization of the serum-free cell growth medium: The development of serum-free media has gone through several stages, including serum-free source, animal-free source (BD), protein-free, and chemically defined (CD). Since the composition of chemically defined medium (CDM) is clear, it is more conducive to the preparation, optimization of the medium, and the removal of impurities during subsequent vaccine preparation, and it is often used as the first choice for cell culture. In recent years, the technology for developing serum-free media for MDCK cells has been relatively mature, and several R & D companies have developed commercially available serum-free media for culturing MDCK cells.

[0046] Therefore, in this experiment, five commercially available serum-free media for MDCK cells (including BD and CD grades) were selected for suspension culture of MDCK-siat7e cells to compare the culture effects and screen out a higher-level serum-free medium suitable for suspension culture of MDCK-siat7e cells. And the growth of MDCK-siat7e cells in it was compared with the growth in the original BD MDCK 001 medium to compare the differences between the two.

[0047] 1) Comparison of the growth of MDCK-siat7e cells in serum-free media CD MDCK 244 (Gansu Jianshun Biotechnology Co., Ltd.), OPM-AM198V2 (Shanghai OPMI Biotechnology Co., Ltd.), Xeno-S001S (Shanghai Baygene Biotechnology Co., Ltd.), Xeno-C001S (Shanghai Baygene Biotechnology Co., Ltd.), and SFM-MDCK (Lanzhou Minhai Bioengineering Co., Ltd.).

[0048] Five commercially available serum-free growth media for culturing MDCK cells were selected for the experiment, namely CD MDCK244, OPM-AM198V, Xeno-S001S, Xeno-C001S, and SFM-MDCK. MDCK-siat7e cells adapted to the serum-free medium BD MDCK001 were resuscitated into the five new media and cultured in suspension under the same conditions. After the growth was stable, cell growth curves and cell metabolism curves were plotted to observe and compare the cell growth conditions. The experimental results are as Figure 1 、 2 shown below:

[0049] As can be seen from Figure 1 A and 1B, the MDCK-siat7e cells reached the highest cell density of 9.31×10 6 cells / ml in Xeno-S001S, and the cell viability could still be maintained above 90% at 10 days. Followed by the CD MDCK 244 medium, with a peak cell density of 7.64×10 6 cells / ml at 8 days, and the cell viability was maintained above 90% within 8 days. According to the cell growth conditions in Figure 1 A and 1B, the preliminary adaptability results of MDCK-siat7e cells in the media were Xeno-S001S > CD MDCK 244

[0050] > OPM-AM198V2 > Xeno-C001S > SFM-MDCK.

[0051] In Figure 2 A, the specific glucose consumption rates of the cells in different media were close. Previous studies have shown that MDCK-siat7e cells are sensitive to ammonia concentration. In Figure 2 B, the specific ammonia production rate of the cells in the CD MDCK 244 medium was low.

[0052] Overall, Xeno-S001S and CD MDCK 244 culture media performed well. Taking all factors into consideration, Xeno-S001S, as a serum-free culture medium, has a more complex composition than the chemically defined culture medium CD MDCK 244, and the ammonia production rate of cells in CD MDCK244 is lower. Therefore, CD MDCK 244 was selected as the growth medium for MDCK-siat7e cells in this experiment.

[0053] 2) Acclimation culture in serum-free medium CD MDCK 244: The master cell bank cells adapted to serum-free medium BD MDCK 001 were revived and serially passaged in 100% chemically defined medium CD MDCK 244 at 37°C, 5% CO2, and 125 rpm. After 72 to 96 hours of resuscitation, the cell density reached 5×10 6 During the subculture, the cells were cultured by adjusting the cell inoculation density and using 200g centrifugal force, centrifuging for 5 minutes and then replacing with fresh medium CD MDCK 244. After the cells recovered, they were cultured to the fourth generation. The cells were in good condition and full in morphology. The specific growth rate was stable at 0.010h -1 ~0.017h -1 The cells were frozen with CD MDCK 244 culture medium + 10% dimethyl sulfoxide, named: Canine kidney epithelial cells MDCK-siat7e-C, and preserved in China Center for Type Culture Collection, deposit address: Wuhan University, Wuhan, China, Postal Code: 430072; deposit date: September 12, 2024, deposit number: CCTCC NO: C2024292. Figure 3 The MDCK-siat7e-C cells were shown to grow in single cell suspension under a 200x microscope, with uniform cell size, regular morphology, and good growth.

[0054] 3) Comparison of the growth of MDCK-siat7e cells in serum-free medium CD MDCK 244 and BD MDCK 001.

[0055] The growth curve of MDCK-siat7e-C cells was compared with the data of BD MDCK 001 cultured MDCK-siat7e cells. Figure 4 shown.

[0056] The growth curves of MDCK-siat7e cells in suspension culture in two culture media (BD MDCK 001 and CD MDCK 244) are shown in Figure 4As shown in A, the cell curve is S-shaped. Compared with the original culture medium BD MDCK 001, the cell density in CD MDCK 244 has been higher than that in BD MDCK 001 since 6 days, and the highest viable cell density in CD MDCK 244 is higher than that in BD MDCK 001 by 5.8×10 6 cells / ml.

[0057] Figure 4 As shown in B, the cell viability of the two is close.

[0058] Figure 4 As shown in C, the specific growth rates of the cells in the two culture media are close. The MDCK-siat7e cells reach the maximum specific growth rate of 0.021 h -1 after being cultured in CD MDCK244 for 72 hours, and the cell doubling time is about 30 - 50 hours.

[0059] In Example 2, the MDCK-siat7e-C cell line was used for serum-free suspension culture of rabies virus

[0060] 1) The MDCK-siat7e-C cell line was inoculated into the growth medium (CDMDCK 244) at a cell density of 0.5×10 6 cells / ml and cultured in a carbon dioxide shaking incubator at 37°C, 5% CO2, and 125 rpm;

[0061] 2) When the cell density of the cell suspension reached 4×10 6 cells / ml, the supernatant was discarded by centrifugation, and the cell pellet was resuspended with the maintenance medium (CDMDCK 296). The rabies virus CTN-1V strain was inoculated at MOI = 0.1 - 0.5 and adsorbed in a shaker at 35°C, 5% CO2, and 125 rpm for 2 h to obtain a cell suspension infected with the virus;

[0062] 3) The cell suspension was centrifuged to discard the supernatant, and after the cell pellet was resuspended with the maintenance medium (CD MDCK 296), it was cultured in a carbon dioxide shaking incubator at 35°C, 5% CO2, and 125 rpm;

[0063] 4) When cultured to 3 / 5 / 7 / 9 / 11 / 13 d, about 2 / 3 volume of the supernatant was harvested by centrifugation, and the remaining culture product was resuspended with the maintenance medium (CDMDCK 296) and the culture volume was made up;

[0064] 5) The rabies virus solution harvested in step 4) was designated as the CTN-1M-1 generation.

[0065] 6) Take the harvest fluid on the 13th day of CTN-1M passage 1 and passage the rabies virus according to steps 2), 3), and 4). The harvested virus fluid is designated as CTN-1M passage 2. Take the harvest fluid on the 13th day of CTN-1M passage 2 and passage it to CTN-1M passage 3 according to steps 2), 3), and 4). Take the harvest fluid on the 13th day of CTN-1M passage 3 and passage it to CTN-1M passage 4 according to steps 2), 3), and 4). Take the harvest fluid on the 13th day of CTN-1M passage 4 and passage CTN-1M to passage 5 according to steps 2), 3), and 4). Harvest the rabies virus fluid from CTN-1M passage 5 according to step 4).

[0066] 7) When the culture of CTN-1M passage 4 ends on the 15th day, take the harvest fluid on the 13th day and centrifuge it at 4000 g for 10 min, then discard the sediment. Filter the supernatant with a 0.65 μm polyethersulfone filter membrane, concentrate the filtered liquid 30-fold using a 100 kD ultrafiltration centrifuge tube, and purify it with a molecular sieve to obtain the purified rabies virus CTN-1M.

[0067] Example 3 Screening Test Study on Serum-Free Suspension Culture Conditions of Rabies Virus

[0068] 1. Screening of Maintenance Medium

[0069] Six commercially available serum-free maintenance media for culturing MDCK cells were selected in the experiment. The nutrient composition ratios of each medium are different, and there may also be different effects on the proliferation and culture of rabies virus in cells. Detect the indicators related to cell status and virus replication, and preliminarily screen out the maintenance medium suitable for rabies virus culture based on this.

[0070] (1) The initial conditions of the six maintenance media are shown in Table 1.

[0071] Table 1 Initial Conditions of Maintenance Medium

[0072]

[0073] (2) Proliferation and Glucose Metabolism of Rabies Virus after Culturing in Different Maintenance Media for 3 Days

[0074] Inoculate the rabies virus CTN-1V strain into MDCK-siat7e-C cells at the same MOI (MOI = 0.1) and culture it in six maintenance media respectively. Detect (calculate) the virus proliferation level, cell growth situation, and cell glucose metabolism in different maintenance media at 3 days after inoculating the cells with the virus. The experimental results are shown in Table 2.

[0075] Table 2 Basic Conditions at 3 Days after Inoculation and Culturing in Different Maintenance Media

[0076]

[0077] ① Virus proliferation situation is asFigure 5 as shown

[0078] In the preliminary comparative experiment, the virus titer was detected 3 days after inoculation with rabies virus as shown Figure 5 A. Except that the virus titer in the SFM-MDCK maintenance medium was <6 lg FFU / ml, the other titer levels were all above 6 lg FFU / ml. The virus titers in the CD MDCK 296, OPM-AM 198V2, and Xeno C001S maintenance media were close to 7 lg FFU / ml.

[0079] Figure 5 B shows the viable cell density of MDCK-siat7e-C cells 3 days after inoculation with rabies virus. The viable cell density was between 5 - 10×10 6 cells / ml, which was higher than 4×10 6 cells / ml at the time of virus inoculation. The SFM-MDCK was the lowest and the Xeno C001S was the highest; the cell viability was above 95% 3 days after virus inoculation.

[0080] Generally speaking, according to the experimental results at 3 days, except for the SFM-MDCK maintenance medium, there was little difference in the cultivation of rabies virus with other different maintenance media.

[0081] ②Cell metabolism - Glucose consumption as shown Figure 6 as shown

[0082] The glucose content in the maintenance medium 3 days after inoculation with rabies virus was detected, and the specific glucose consumption rate was calculated, as shown Figure 6 The results showed that the specific glucose consumption rates were basically the same in the six maintenance media after the cells were inoculated with the virus. Since there was a large difference in the initial glucose content in different maintenance media, the glucose content in all maintenance media was supplemented to the same level of 8.5 g / L in advance in the subsequent 7-day virus inoculation culture experiment (the cultivation effect of SFM-MDCK was not good, and no subsequent experiment was carried out).

[0083] (3) Rabies virus proliferation at 7 days of culture in different maintenance media

[0084] The further virus inoculation operation was as described above. After continuous culture for 7 days, the cell density, virus titer, virus antigen content, and cell metabolism detection indexes were detected during the culture process. The virus proliferation curve was drawn to evaluate the virus proliferation in different maintenance media, and the suitable maintenance medium for culturing rabies virus was comprehensively selected in combination with the above indexes. The results are as shown Figure 7 as shown

[0085] Table 3 Peak virus titers of CTN-1V strain in different maintenance media

[0086]

[0087] From Figure 7 As shown in A, 7C and Table 3, in the experiment of virus proliferation for 7 days, the peak virus titers and the times to reach the peaks were different when using different maintenance media. The peaks appeared approximately 3 to 4 days after virus inoculation. The peak of Xeno C001S appeared earlier, at 2 days. The peaks of CD MDCK 296 and OPM-AM 198V2 both appeared at 4 days, around 7 lg FFU / ml. The virus titer levels of K19 and Xeno S001S were relatively lower.

[0088] Figure 7 B shows that during the virus culture process, the viable cell density levels in the 5 maintenance media were close.

[0089] The rabies virus antigen content in the 5 maintenance media after culture was detected by enzyme-linked immunosorbent assay. The results are as Figure 7 shown in D. The rabies virus antigen content in the culture of CD MDCK 296 was relatively the highest, appearing at the 6th day. The preliminary results showed that it was more suitable for culturing rabies virus.

[0090] Based on the above results, CD MDCK 296 was preferably selected as the maintenance liquid medium for culturing rabies virus in the experiment.

[0091] 2. Experiment on the effect of different MOIs on rabies virus proliferation

[0092] Four experimental groups with MOI = 1; MOI = 0.5; MOI = 0.1; MOI = 0.01 were set up respectively. The CTN-1M rabies virus strain was also infected with MDCK-siat7e-C cells at different MOIs by the method of adsorption and inoculation of virus. Half of the culture medium was changed every 2 to 3 days, and the culture time ended at 15 days. Samples were taken during the medium change to detect the virus titer and potency, and the virus proliferation curve and virus potency curve were plotted. The results are as Figure 8 shown below:

[0093] From Figure 8 A, it can be seen that when the virus was inoculated at 0.1 - 1 MOI, the virus titer proliferation curves were close. The virus titer reached the peak of about 7 - 8 lg FFU / ml 3 days after virus inoculation and remained until the 15th day.

[0094] Figure 8The results of B titer showed that with the extension of virus culture time, the titer of rabies virus gradually increased, and the trend was similar to the previous experimental results. When MOI = 1 / 0.5, the virus titer curve was close, and reached the peak value of about 2 IU / ml at the 11th day. The peak titer of the 3rd group with MOI = 0.1 was 1.89 IU / ml, which was close to that of the 1st and 2nd groups. When MOI = 0.01 in the 4th group, the virus titer curve continued to show an upward trend after 7 days, but the titer was significantly lower than that of other MOI groups. This experiment suggested that when MOI = 0.1 - 1, the CTN-1M strain of rabies virus could obtain higher virus titers and potencies in MDCK-siat7e-C cells. Considering that the inoculation amount of the virus solution should not be too high and the limited dilution method was beneficial to screening dominant viruses, the subsequent experiment used MOI = 0.1 - 0.5 for virus inoculation operation.

[0095] Example 4 Research on the virus characteristics of the rabies virus cultured by the present invention

[0096] I. Research methods

[0097] 1. Virus titer detection (FAT)

[0098] (1) Virus dilution: Pre-add 120 μl of DMEM culture medium to each well of a 96-well dilution plate. Take 30 μl of the sample to be tested and add it to the first well of each row (each sample is detected in duplicate), mix well, and aspirate 30 μl for 1 / 5 serial dilution; use the frozen sample with known virus titer as the positive control and perform the same dilution operation as the test sample; use DMEM culture medium as the negative control.

[0099] (2) Infecting cells: Take a 96-well cell culture plate as the detection plate, add 50 μl of DMEM culture medium to each well, and aspirate 50 μl of the diluted virus mixture prepared above and add it to the corresponding wells of the detection plate. Select well-grown BSR cells and prepare a cell suspension with a concentration of 1×10 6 cells / ml, and add 50 μl of the cell suspension to each well. After mixing, place it in an incubator at 37°C and 5% CO2 for 24 h.

[0100] (3) Acetone fixation: After the culture is completed, take out the culture plate, carefully aspirate the liquid in it and discard it into the waste liquid tank (for disinfection treatment), add 50 μl of pre-cooled 80% acetone at -20°C to each well, and fix it at -20°C for 15 min. Take out the culture plate, discard the acetone, and let it dry naturally by volatilization.

[0101] (4) Binding of anti-rabies virus fluorescent antibody: Dilute the anti-rabies virus nucleoprotein fluorescent antibody (FITC) 1:200 to the working concentration. After the acetone has dried by volatilization, add 50 μl of the working concentration of anti-rabies virus nucleoprotein fluorescent antibody to each well, and incubate it at 37°C for 1 h.

[0102] (5) Washing: Take out the 96-well culture plate, discard the liquid in the plate, add about 50 μl of PBS to each well, wash away the unbound fluorescent antibody in the well, discard the liquid, and repeat 3 - 4 times. After discarding the PBS liquid for the last time, centrifuge to dry, add 50 μl of 5% glycerol to each well, and observe under a fluorescence microscope.

[0103] (6) Result determination: Observe each well under a fluorescence microscope, and record the number of fluorescent foci in 4 wells of the last two dilutions where fluorescent foci appear (the number of fluorescent foci in the well with a lower dilution factor of adjacent wells is recorded as N1, N1', and the number of fluorescent foci in the well with the highest dilution factor is recorded as N2, N2'). Virus titer = (average number of fluorescent foci in the last 2 wells where fluorescent foci appear × dilution factor × 1000) / volume of virus supernatant added per well (FFU / ml); where, average number of fluorescent foci in the last 2 dilutions where fluorescent foci appear = (N1 + N2 × 5 + N1' + N2' × 5) / 2 (5 is the virus dilution multiple, 2 is the number of wells); the dilution factor is the virus dilution multiple corresponding to wells N1 and N1'; the volume of virus added per well is 30 μl.

[0104] 2. Detection of the relative potency of rabies virus by improved antibody binding test (M-ABT)

[0105] (1) Dilution preparation: a. Dilution of the test sample: Pre-add 100 μl of DMEM culture medium to each well of a 96-well plate, take 100 μl of the test sample, standard product, and internal control product and add them to the first well of each row, mix well, take out 100 μl for subsequent 1:2 serial doubling dilutions, and discard 100 μl of the mixed solution from the last well; b. Dilution of the rabies immunoglobulin standard for human use: Dilute the rabies immunoglobulin standard for human use to 0.2 IU / ml with DMEM culture medium for subsequent experiments; c. Dilution of the CVS-11 indicator virus: Dilute CVS-11 with DMEM culture medium according to the determination result of the 80% cell infection amount of the CVS-11 virus to prepare the indicator virus solution.

[0106] (2) Neutralization of the test sample antigen and rabies immunoglobulin for human use: Add 100 μl of rabies immunoglobulin for human use with a concentration of 0.2 IU / ml to each well of the 96-well plate containing 100 μl of the diluted test sample; at the same time, set up an antibody negative control well: the lowest dilution of the test vaccine and standard product wells + 100 μl of normal human serum; mix well and place at 37°C for 1 h for neutralization.

[0107] (3) Neutralization of CVS-11 virus with the remaining rabies immunoglobulin for human use: Take out the neutralized 96-well plate, and transfer 100 μl of the mixed solution from each well to another new 96-well plate. Add 50 μl of the CVS-11 indicator virus solution diluted according to 80% of the cell infection dose to the new 96-well plate. At the same time, set up a vaccine negative control: 50 μl of CVS-11 indicator virus solution + 50 μl of rabies immunoglobulin for human use (concentration 0.2 IU / ml) + 50 μl of DMEM culture medium; an indicator virus control: 50 μl of CVS-11 indicator virus solution + 100 μl of DMEM culture medium. Mix well and incubate at 37°C for 1 h for neutralization.

[0108] (4) Infecting cells: After the neutralization of CVS-11 virus with the remaining rabies immunoglobulin for human use, take out the culture plate and add 50 μl of BSR cell suspension at a concentration of 1×10 6 cells / ml to each well. At the same time, set up a normal cell control well: 100 μl of DMEM culture medium + 50 μl of cell suspension. After mixing, incubate at 37°C in a 5% CO2 incubator for 24 h.

[0109] (5) After the culture is completed, perform acetone fixation, binding with anti-rabies virus fluorescent antibody, washing, and addition of glycerol according to the operations in steps (3), (4), and (5) in the FAT experiment. Finally, observe the results under a fluorescence microscope.

[0110] (6) Result determination: Observe each well under a fluorescence microscope, and record the percentage of virus-infected cells in the two wells before and after the 50% infection rate demarcation point of the cells in the standard product and test sample columns. Use the following formula to calculate the relative virus titer results:

[0111]

[0112] In the formula: A is the dilution factor of the standard product with a fluorescence focus ratio lower than 50%; B is the fluorescence focus percentage of the well of the standard product with a fluorescence focus ratio lower than 50%; C is the fluorescence focus percentage of the well of the standard product with a fluorescence focus ratio higher than 50%; n1 is the dilution multiple of the standard product.

[0113]

[0114] In the formula: E is the dilution factor of the test sample with a fluorescence focus ratio lower than 50%; F is the fluorescence focus percentage of the well of the test sample with a fluorescence focus ratio lower than 50%; G is the fluorescence focus percentage of the well of the test sample with a fluorescence focus ratio higher than 50%; n2 is the dilution multiple of the test sample.

[0115] Potency of the test sample (IU / ml) = 10 (J-K) ×L (Equation 2-3),

[0116] In the formula: J is the lgED of the standard product 50 ; K is the lgED of the test sample50 ; L is the titer of the reference standard, IU / ml.

[0117] (7) The experiment can be considered valid only when the following conditions are met: a. Normal cell control wells: There should be no fluorescence; b. Antibody negative control wells: 80% of the cells should be observed to be fluorescently stained; c. Vaccine negative control wells: There should be no fluorescence; d. Indicator virus control wells: 80% of the cells should be observed to be fluorescently stained; e. The titer of CVS-11 indicator virus should be ≥ 10 6 FFU / ml.

[0118] 3. SDS-PAGE detection: (1) Prepare the sample: Add the sample, reducing agent, and Loading Buffer to the EP tube in a ratio of 13:2:5. After thorough mixing, denature at 100 °C in a metal bath for 10 min; (2) Place a 12% precast gel in the electrophoresis tank, pour in running buffer to submerge it, and load 10 μL per well; (3) Electrophorese at 90 V until the bromophenol blue moves to the bottom of the lower gel; (4) Stain the gel with Coomassie Brilliant Blue staining solution; (5) Rinse the gel with decolorizing solution; (6) After the gel is completely decolorized, take a photo for analysis.

[0119] 4. Western Blot detection: (1) The processes of gel preparation, sample loading, and electrophoresis are the same as those in the SDS-PAGE detection method; (2) Soak the PVDF membrane to be transferred in absolute ethanol, and then equilibrate it in the transfer buffer for at least 5 min; (3) Take out the gel, cut off the upper gel part, soak it in the transfer buffer together, and transfer the membrane using a transfer apparatus in the order of filter paper - PVDF membrane - gel - filter paper; (4) After the transfer is completed, take out the PVDF membrane and soak it in a 10% skim milk solution, and block it at room temperature on a shaker for 2 h; (5) Discard the blocking solution, add the primary antibody diluted with a 5% skim milk solution, and incubate at room temperature for 2 h; (6) Discard the primary antibody, wash the membrane three times with PBST buffer, 5 min each time; (7) Add the secondary antibody diluted with a 5% skim milk solution, and incubate at room temperature for 2 h; (8) Discard the secondary antibody incubation solution, wash the membrane 3 times with PBST buffer, 5 min each time, evenly add the developing solution, and then take a photo for analysis.

[0120] 5. Detection of the neutralizing antibody titer of rabies virus: After inactivating the virus harvest, intraperitoneally inject mice weighing 12 - 14 g, 0.5 ml per mouse, immunize 2 times with a 7-day interval, as the experimental group. Use the same batch of non-immunized mice as the control group. On the 14th day after the primary immunization, collect blood from the experimental group and the control group respectively, and detect the antibody titer in the mouse serum using the RFFIT test. The RFFIT test method is described in Article 3512 of the Third Volume of the Chinese Pharmacopoeia (2020 Edition).

[0121] 6. Immunogenicity test: Refer to the immunogenicity test method of "Rabies Vaccine for Human Use (Vero Cell), Freeze-dried" in the third part of "Chinese Pharmacopoeia" (2020 edition). After inactivating the virus harvest fluid, inject it intraperitoneally into mice weighing 12 - 14 g, 0.5 ml per mouse, immunize twice with a 7-day interval, which is the test group. Use the same batch of non-immunized mice as the control group. On the 14th day after the primary immunization, the test group and the control group are respectively challenged intracerebrally with a 10-fold serial dilution of the CVS virus, with dilution factors from 10 -2 to 10 -6 . Inject 0.03 ml per mouse, inject 10 mice for each dilution factor, observe daily, those that die within 3 days are not counted (the number of dead animals should not exceed 20% of the total number of test animals), observe for 14 days, and the protection index should not be less than 100.

[0122] Calculation method: LD of the control group 50 - LD of the experimental group 50 = difference, and the antilogarithm of the difference is the protection index.

[0123] II. Calculation method

[0124] 1. Specific growth rate of cells: μ = (lnN t - lnN o ) / t (Equation 2 - 4)

[0125] where μ is the specific growth rate (h -1 ), N t is the viable cell density at time t (cells / ml), N0 is the initial viable cell density (cells / ml), and t is the growth time (h);

[0126] 2. Specific consumption (production) rate:

[0127] where Q n is the specific consumption (or production) rate of the substance from time t to t + 1 [mmol / (10 9 cells.h) or g / (10 9 cells.h)], VCD is the viable cell density at time t or t + 1 (10 6 cells / ml), n is the nutrient or metabolite, Δn is the amount of substance n consumed or produced per unit volume from time t to t + 1 (mmol / L or g / L), and Δt is the time value from time t to t + 1 (h).

[0128] III. Research results

[0129] 1. Virus titer and potency: Continuously passage the CTN-1V virus under the above conditions, and detect the proliferation curves of the virus titer and potency of CTN-1M virus in the 1st to 5th generations during the passage process. The results are as Figure 9As shown

[0130] As can be seen from Figure 9 During the virus passage process, the proliferation curve of the CTN-1M virus from generations 1 to 5 was relatively stable. The virus titer reached a relatively high level within 3 days, with the peak virus titer at 7 - 8 lgFFU / ml. The virus titer was still greater than 7 lgFFU / ml on the 13th day, and the overall trend of the titer curve was relatively flat. The virus potency curve showed an upward trend with the extension of the culture time. The virus potency began to gradually increase after 7 days of culture and reached the peak at 13 - 15 days, approximately 2 IU / ml.

[0131] 2. Observation of the cell morphology infected by the virus: As Figure 10 shown in A, 13 days after the CTN-1M strain infected MDCK-siat7e-C cells, the cell morphology became irregular and a large number of cells lysed. After culturing the MDCK-siat7e-C control cells for 13 days, there was no obvious change in the cell morphology, as Figure 10 shown in B.

[0132] 3. SDS-PAGE and WB detection: As Figure 11 shown in A, the SDS-PAGE results of the purified virus showed that the antigen components of the virus were complex, and clear staining bands were visible at the predicted relative molecular weights of the rabies virus G protein (58 - 67 kD) and N protein (50 - 55 kD). The WB result analysis showed that specific reaction bands appeared at the expected main antigen relative molecular weights for the purified virus, indicating that the CTN-1M virus contained the G and N proteins of the rabies virus, as shown in Figure 11 B.

[0133] 4. Detection of the neutralizing antibody titer of the rabies virus: The results are as Figure 12 shown. The neutralizing antibody titers were between 30 - 68 IU / ml, all > 0.5 IU / ml, and showed an overall upward trend with the increase in the culture days. The CTN-1M strain reached the highest neutralizing antibody titer of 67 IU / ml on the 15th day of culture, which was close to that of the CTN-1V virus seed (64 IU / ml).

[0134] 5. Immunogenicity examination: The rabies virus CTN-1M strains harvested in the 2nd and 4th generations were respectively selected for immunogenicity examination, and the protection indices were 324 and 575 respectively, both meeting the requirement of the protection index of ≥ 100 for the immunogenicity examination of the rabies virus specified in the Chinese Pharmacopoeia (2020 Edition).

[0135] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A serum-free full suspension culture type canine kidney epithelial cell MDCK-siat7e-C cell strain, characterized in that: The cell line is deposited in China Center for Type Culture Collection with the deposit number of CCTCC NO: C2024292.

2. A serum-free full suspension culture type canine kidney epithelial cell MDCK-siat7e-C cell bank, characterized in that: The cell bank is constructed using the cell line described in claim 1.

3. Use of the cell line according to claim 1 or the cell bank according to claim 2 for culturing viruses or preparing viral vaccines.

4. The use according to claim 3, characterized in that: The virus is rabies virus.

5. A method for suspension culture of rabies virus, characterized in that: The method comprises: inoculating rabies virus into a cell suspension of the cell line according to claim 1 or the cell line in the cell bank according to claim 2, performing suspension culture, and harvesting rabies virus from the culture product.

6. The method according to claim 5, characterized in that The method comprises: Inoculating the cell line according to claim 1 or the cell line in the cell bank according to claim 2 into a serum-free cell growth medium, and performing suspension culture to allow the cells to proliferate to a certain cell density, thereby obtaining a cell suspension; The serum-free cell growth medium in the cell suspension is replaced with a serum-free cell maintenance medium, and then the rabies virus is added to allow for full adsorption to obtain a cell suspension infected with the virus; The serum-free cell maintenance medium in the virus-infected cell suspension was replaced with a new serum-free cell maintenance medium, suspension culture was performed, and rabies virus was harvested from the culture product.

7. The method according to claim 5 or 6, characterized in that: The method comprises: The cell line described in claim 1 or the cell line in the cell bank described in claim 2 was inoculated into CD MDCK 244 culture medium and placed in a shaking incubator at 37°C, 5% CO2, and 125 rpm for suspension culture. When the cell density reached 3×10 6 ~4×10 6 / ml, and obtain cell suspension; The cell suspension was centrifuged and the supernatant was discarded. The cell pellet was resuspended in CD MDCK 296 medium, and rabies virus was inoculated at a ratio of MOI = 0.1-0.5, and the cell suspension was obtained by adsorption for 1-2 hours in a shaking incubator at 35-37°C, 5% CO2, and 125 rpm; The virus-infected cell suspension was centrifuged and the supernatant discarded. The cell pellet was resuspended in CD MDCK 296 medium and placed in a shaker at 35°C, 5% CO2, and 125rpm for suspension culture. The culture product was centrifuged every 2 to 3 days and 2 / 3 of the supernatant was harvested. The remaining culture product was resuspended in CD MDCK 296 medium and the culture volume was supplemented. The culture was continued until the 11th to 15th day, and the rabies virus was harvested from the culture product.

8. A method for preparing a rabies virus vaccine, characterized in that: The method comprises: inoculating rabies virus into a cell suspension of the cell line of claim 1 or the cell line in the cell bank of claim 2, performing suspension culture, and harvesting rabies virus from the culture product; and concentrating, purifying, and inactivating the harvested rabies virus to obtain a rabies virus vaccine.

9. The method according to claim 8, characterized in that The method comprises: Inoculating the cell line according to claim 1 or the cell line in the cell bank according to claim 2 into a serum-free cell growth medium, and performing suspension culture to allow the cells to proliferate to a certain cell density, thereby obtaining a cell suspension; The serum-free cell growth medium in the cell suspension is replaced with a serum-free cell maintenance medium, and then the rabies virus is added to allow for full adsorption to obtain a cell suspension infected with the virus; The serum-free cell maintenance medium in the virus-infected cell suspension is replaced with a new serum-free cell maintenance medium, suspension culture is performed, and rabies virus is harvested from the culture product; and the harvested rabies virus is concentrated, purified, and inactivated to obtain a rabies virus vaccine.

10. The method according to claim 8 or 9, characterized in that: The method comprises: The cell line described in claim 1 or the cell line in the cell bank described in claim 2 was inoculated into CD MDCK 244 culture medium and placed in a shaking incubator at 37°C, 5% CO2, and 125 rpm for suspension culture. When the cell density reached 3×10 6 ~4×10 6 / ml, and obtain cell suspension; The cell suspension was centrifuged and the supernatant was discarded. The cell pellet was resuspended in CD MDCK 296 medium, and rabies virus was inoculated at a ratio of MOI = 0.1-0.5, and the cell suspension was obtained by adsorption for 1-2 hours in a shaking incubator at 35-37°C, 5% CO2, and 125 rpm; The virus-infected cell suspension is centrifuged to discard the supernatant, the cell pellet is resuspended with CD MDCK 296 culture medium, and then placed in a shaking incubator at 35°C, 5% CO2, and 125rpm for suspension culture, the culture product is centrifuged every 2 to 3 days to harvest 2 / 3 volume of the supernatant, the remaining culture product is resuspended with CD MDCK 296 culture medium to make up the culture volume, and the culture is cultured to the 11th to 15th day, and rabies virus is harvested from the culture product; and the harvested rabies virus is concentrated, purified, and inactivated to obtain a rabies virus vaccine.

Images