CN-122012378-A - Suspension serum-free PK cell based on composite synergist and method for culturing porcine circovirus by using suspension serum-free PK cell

CN122012378ACN 122012378 ACN122012378 ACN 122012378ACN-122012378-A

Abstract

The invention relates to the technical field of veterinary biological product production, and discloses a suspension serum-free PK cell based on a composite synergist and a method for culturing porcine circovirus by using the same, wherein a high-quality suspension cell strain is obtained by constructing an adherent PK cell suspension technology system of gradient domestication-monoclonal screening-performance verification, a composite synergist consisting of IGF1R, L-selenomethionine, polyvinylpyrrolidone (PVP) and a Cu < 2+ >/Mn < 2+ > compound is innovatively introduced, and the special proliferation parameters of PCV2 and PCV3 strains are combined for optimization, so that the efficient and stable culture of PCV2/PCV3 is realized, and the virus titer and the production efficiency are remarkably improved.

Inventors

LI JUNFENG
LI WENTAO
WANG SHUO
HOU YE
LIU XIFENG
SONG CHUNYU
YU HONGMEI

Assignees

北京鼎持生物技术有限公司
浙江壹生科生物技术有限公司
辽宁壹生科生物技术有限公司
浙江鼎持生物制品有限公司

Dates

Publication Date: 20260512
Application Date: 20260228

Claims (9)

1. A suspension serum-free PK cell based on a composite synergistic agent is characterized in that the PK cell is PK-S2 strain, is suggested to be classified and named as pig kidney epithelial cells, and is preserved in China general microbiological culture collection center (CGMCC) No.46750 at the year 2025 and the month 11 and 18.
2. The method for constructing the suspension serum-free PK cells based on the composite synergists as claimed in claim 1, comprising the following specific steps: (1) In the cell pretreatment stage, the adherent PK cells frozen in liquid nitrogen are taken out, and PK cells with stable proliferation rate are obtained through the processes of water bath thawing, resuspension culture and subculture; (2) In the serum gradient reduction domestication stage, four-dimensional collaborative domestication strategies of gradual decrease of serum concentration, gradual increase of serum-free culture medium gradient, gradual increase of shaking strength and concentration gradient adjustment of compound synergistic agent are adopted to perform domestication culture on the obtained PK cells with stable proliferation, and PK cell strains which are completely suitable for serum-free full suspension culture are obtained through screening; (3) And in the suspension adaptability strengthening stage, the screened PK cell strains are respectively transferred to a triangular shake flask for expansion culture, and then a limited dilution method is adopted for monoclonal screening to obtain monoclonal suspension PK-S2 cell strains.
3. The method for constructing suspended serum-free PK cells based on a composite synergist according to claim 2, wherein the composite synergist mother liquor contains IGF1R, L-selenomethionine, polyvinylpyrrolidone and Cu2 + /Mn2+ compound, and the four substances are dissolved in a serum-free special culture medium according to a mass ratio of 3:2:1:0.5 to prepare 10% mother liquor.
4. The construction method of the suspension serum-free PK cells based on the composite synergist according to claim 3, wherein the specific process of the cell pretreatment stage of the step (1) is that frozen adherent PK cells are taken out from liquid nitrogen, rapidly placed in a 37 ℃ water bath for 1-2 min to be melted, so as to avoid damage of the cells due to long-time low temperature, 10mL of DMEM culture medium containing 5% fetal bovine serum and 0.05% composite synergist is added, centrifugation is carried out at 1000rpm for 5min to remove frozen solution, the toxic influence of the frozen protective agent on the cells is reduced, the cells are resuspended and inoculated in T75 culture flasks, 10mL of DMEM culture medium containing 5% fetal bovine serum and 0.05% composite synergist is added into each flask, and the mixture is placed in a 37 ℃ and 5% CO 2 culture box for static culture; After the cell fusion degree reaches 80% -90%, discarding the supernatant, gently flushing for 2 times by using PBS buffer solution with pH of 7.2-7.4, removing residual culture medium and metabolic waste, adding 2mL of 0.25% pancreatin-EDTA digestive solution, incubating for 1-2 min at 37 ℃, immediately adding 5mL DMEM culture medium containing 5% fetal bovine serum and 0.05% composite synergist to terminate digestion after observing cell gap increase and cell rounding under a microscope, and lightly blowing to single cell suspension to avoid cell breakage caused by excessive blowing; Inoculating the cells to a new T75 culture flask according to a 1:3 passage ratio, continuously passaging for 5 times, detecting the cell activity rate by adopting a trypan blue staining method after each passage, stabilizing the cell activity rate to 92% -95%, and simultaneously detecting the cell proliferation activity by adopting a CCK-8 method, so as to ensure the stable cell proliferation rate.
5. The method for constructing a composite potentiator-based suspension serum-free PK cell of claim 3, wherein the stage of serum gradient decreasing domestication in step (2) comprises, The low serum adaptation period is that the stage is completed in the 6 th to 10 th generation of cells, the formula of a culture medium is 2% of fetal bovine serum+30% of serum-free special culture medium+67% of DMEM to remove double-resistant basic culture medium+0.08% of composite synergist, 10mL of the mixed culture medium is added into each bottle of T75 culture flask, the cell inoculation density is adjusted to be 2X 10 5 cells/mL during passage, static culture is still adopted, but the culture flask is manually and gently shaken for 1 to 2 times every day, each time for 5 seconds, the cells are promoted to be fully contacted with the culture medium, the uneven growth of the cells caused by partial nutrition deficiency is avoided, each generation of culture is carried out for 48 hours, the cell morphology is observed under a microscope, the typical epithelial-like fusiform is required to be maintained, no obvious shrinkage or particle increase is required, the trypan blue staining method is adopted to detect the activity rate is required to be more than 90%, the cell counting plate count adherence rate is required to be more than 90% or less than 85%, the serum reduction is required to be suspended when the adherence rate is less than 85%, the serum concentration and the composite synergist concentration is maintained for 1 to 2 times, the next stage is ensured after the cell adaptation; The semi-serum-free vibration induction period is completed in the 11 th to 15 th generation of cells, the formula of the culture medium is adjusted to 1% fetal calf serum, 60% serum-free special culture medium, 39% DMEM basic culture medium and 0.1% composite synergist, the serum dependence is further reduced, the serum-free culture medium proportion is further increased, meanwhile, the concentration of the composite synergist is increased to enhance the cell suspension adaptability, the cells are transferred to 125mL triangular shake flasks, 20mL of cell suspension is inoculated to each flask, the inoculation density is increased to 3X 10 5 cells/mL, and the culture is carried out on shaking flasks at 37 ℃ and 5% CO 2 . The rotation speed of the shaking table is increased by 10rpm from 30rpm for every 2 generations, the shaking strength is finally stabilized at 50rpm, the shaking strength is standardized by that cells are not adhered to and slightly suspended, so that the shearing damage of cells caused by overhigh rotation speed is avoided, part of cells are separated from the bottle wall under a microscope at the stage and are in a single or small-cluster suspension state, the suspension rate is detected by a blood cell counting plate after each generation of passage and is gradually increased from the initial 10% to more than 80%, the activity rate is detected by a trypan blue staining method and is more than or equal to 88%, if the suspension rate is slowly increased, the rotation speed of the shaking table is required to be increased, the lifting amplitude is increased by 15rpm for each generation, and meanwhile, the concentration of the compound synergist is kept unchanged; 203. The serum-free complete adaptation stage is completed in the 16 th to 20 th generation of cells, serum dependence and adherence dependence are completely removed, 100% of serum-free special culture medium is added with 0.12% of compound synergist, 0.1% of Pluronic F-68 is added into the compound synergist to further reduce cell shear force damage, the concentration of the compound synergist is improved to ensure the activity and proliferation capacity of the cells in a serum-free environment, the rotation speed of a shaking table is started from 60rpm, 20rpm is finally stabilized at 140rpm, the culture temperature is maintained at 37 ℃, the carbon dioxide concentration is 5% for passage 1 time every 3 days, pancreatin digestion is not needed in passage, suspension cell suspension is directly taken, centrifugation is carried out for 5min at 1000rpm, fresh serum-free special culture medium containing 0.12% of compound synergist is used for resuspension after supernatant is discarded, the inoculation density is adjusted to be 1×10 6 cells/mL, the cell population which still depends on adherence growth is strictly required in the stage, only the cell population which is kept in a round shape or short form and uniform in size is retained, the cell population is detected by a trypan blue dyeing method, the activity of the cell population is detected by adopting a trypan blue dyeing method to be more than or equal to 90%, the cell population is required to be detected for the activity of the cell population is required to be detected for the next stage to be high in the total cell strain to be 26h, the suspension quality is required to be obtained, and the cell strain is required to be high in the following stage to be subjected to detection to the suspension quality to be multiplied for the cell strain is required to be 5 h to be in a stable, and the following stage is required to be subjected to the quality to the method to a selection stage to be stable to be 5 to be subjected to a stable.
6. The method for constructing the suspension serum-free PK cells based on the composite synergist according to claim 5, wherein the shake flask expansion culture process in the step (3) is characterized in that the screened candidate cell groups are respectively transferred to 125mL triangular shake flasks, 30mL of a serum-free special culture medium containing 0.12% of the composite synergist is added into each flask, the inoculation density is 1X 10 6 cells/mL, the shaking table rotation speed is 140rpm, the culture is 37 ℃ and the culture is 5% CO2, the culture is carried out for 1 time every 3 days, sampling and detection are carried out every day, and the cell density is counted by adopting a cell counting plate, so that the cell density can reach 6X 10 6 ~7×10 6 cells/mL every 3 days; the method comprises the steps of detecting the activity rate by trypan blue staining method, ensuring the activity rate to be more than or equal to 90%, detecting the apoptosis rate by flow cytometry, ensuring the apoptosis rate to be less than or equal to 5%, verifying the stable proliferation capability and activity of cells in a suspension environment by the detection, eliminating candidate cell groups with slow cell density growth or apoptosis rate of more than 5%, retaining 2-3 candidate cell groups with excellent performance to enter a monoclonal screening stage, specifically, taking candidate suspension cells in logarithmic growth phase, diluting the candidate suspension cells to 1cell/100 mu L by a serum-free special culture medium containing 0.12% of compound synergistic agent, ensuring that each cell contains only 1cell on average, avoiding influencing the uniformity of cell lines by multicellular cloning, adding 100 mu L of diluted cell suspension into each cell culture plate in 96-well, placing the cell suspension in a 37 ℃ and 5% CO 2 incubator for static culture, observing and marking holes containing single cells under a microscope after 72h, eliminating multicellular holes and cell-free holes, slightly blowing the single cells by a pipette after the single clone cells grow to a 96-well plate to cover the bottom 50%, transferring to 24 hole plate, adding 1mL serum-free special culture medium containing 0.12% compound synergist into each hole, continuously culturing, transferring to T25 culture flask when cell density of 24 hole plate reaches 1×10 6 cells/hole, adding 5mL serum-free special culture medium containing 0.12% compound synergist, culturing, and finally amplifying to 125mL shake flask when cell density of T25 culture flask reaches 5×10 6 cells/mL to obtain monoclonal suspension PK-S2 cell strain.
7. A method for culturing porcine circovirus by using suspension serum-free PK cells based on a composite synergist is characterized by comprising the following steps of: (1) Preparing virus seeds, screening porcine circovirus PCV2 and PCV3 from diseased porcine lymph node disease, inoculating, culturing and screening the obtained PCV2 seed viruses and PCV3 seed viruses by using the PK-S2 cells of claim 1 of a serum-free culture medium according to an access amount of MOI=0.01, adding 0.12% of a compound synergist into the culture medium of PCV2, adding 0.15% of the compound synergist into the culture medium of PCV2, (2) Performing virus scale culture, firstly performing amplification culture on PK-S2 cells according to claim 1, gradually completing the amplification of PK-S2 cells through 125mL shake flask primary amplification, 500mL shake flask middle-level amplification and 5L shake flask high-level amplification culture stages, adding 0.12% composite synergist into each amplification stage, then respectively introducing 5L shake flask amplified seed cells into a 50L bioreactor according to 1X 10 6 cells/mL, culturing by using a serum-free special culture medium, respectively introducing PCV2 and PCV3 viruses according to MOI=0.01, wherein PCV2 adopts 37 ℃ culture in the whole course, PCV2 DNA polymerase activity is highest at the temperature, the virus replication efficiency is optimal, PCV3 adopts a sectional temperature control strategy, the former 24h adopts 37 ℃ culture, promoting virus adsorption and invasion, PCV3 capsid protein and cell receptor binding efficiency are carried out in the stage, then 48h is converted into 35 ℃ culture, low temperature induction of virus particle assembly, the virus capsid protein is lifted to be stable, PCV2 and PCV3 is carried out at the temperature of 20% -0.80% by controlling the concentration of PCV2 to be 0.80-30% by means of a paddle concentration of composite oxygen concentration of the PCV3 in the stage of 0.80-3 rpm, and maintaining the concentration of PCV2 at the composite culture paddle concentration of 0.80-3 rpm in the culture process to be controlled to be 30%; (3) And (3) harvesting venom, namely performing multi-generation virus harvesting by adopting a continuous harvesting-feed culture strategy.
8. The method for culturing porcine circovirus in suspension serum-free PK cells based on a composite synergist according to claim 7, wherein the amplification culture in the step (2) is specifically performed by, Primary expansion, namely inoculating PK-S2 cell strains into a new 125mL shake flask according to 1X 10 6 cells/mL, culturing for 72 hours at 37 ℃ and 140rpm by using a serum-free special culture medium containing 0.12% of a composite synergistic agent, wherein the target cell density is 6X 10 6 cells/mL, the activity rate is more than or equal to 95%, and detecting the apoptosis rate by adopting a flow cytometry method, wherein the apoptosis rate is less than or equal to 3%, so that the influence of apoptotic cells on subsequent expansion is avoided; Medium-grade expansion, namely transferring the cells after primary expansion to a 500mL shake flask, inoculating a serum-free special culture medium with the density of 1 multiplied by 10 6 cells/mL, culturing for 72 hours at 37 ℃ and 140rpm, wherein the density of target cells is 6.5X10 6 cells/mL, the activity rate is more than or equal to 95%, detecting the pH value of the culture medium by a pH meter, and if the pH value deviates, adjusting by adding 1mol/L hydrochloric acid or 1mol/L sodium hydroxide solution, thereby ensuring the stable growth environment of the cells; High-grade amplification, namely transferring the cells after medium-grade amplification to a 5L shaking bottle, inoculating a serum-free special culture medium with the density of 1 multiplied by 10 6 cells/mL, culturing for 72 hours at 37 ℃ and 140rpm by using a serum-free special culture medium containing 0.12% of a compound synergist, wherein the target cell density is 7 multiplied by 10 6 cells/mL, the activity rate is more than or equal to 95%, detecting the dissolved oxygen concentration of the culture medium by adopting an oxygen dissolving instrument, keeping the dissolved oxygen concentration at 30% -50%, regulating the dissolved oxygen by regulating the rotating speed of the shaking bottle, ensuring that the respiratory requirement of the cells is met, and gradually expanding the cell scale by step-like amplification.
9. The method for culturing porcine circovirus in suspension serum-free PK cells based on a composite synergist according to claim 8, wherein the specific process of harvesting the viruses for multiple generations in the step (3) is as follows, The first generation of harvest, namely 72 hours after virus inoculation, when the virus titer reaches a peak value, harvesting 75% of culture, and rapidly harvesting high-concentration virus, wherein in the harvest process, the stirring rotation speed is kept at 50rpm, so that the turbidity of harvest liquid caused by cell precipitation is avoided; The second generation of harvest, namely, supplementing fresh serum-free special culture medium containing the compound synergist with the corresponding concentration to the rest 25% of culture to 50L, and continuously culturing for 72h, wherein the virus titer is still stable at the moment, and harvesting all the culture; And harvesting in the third generation, namely supplementing fresh serum-free special culture medium containing the compound synergist with corresponding concentration to 50L again, continuing to culture for 72h, wherein the virus titer is slightly reduced and still meets the production requirement, harvesting all cultures, detecting the cell viability after harvesting, stopping harvesting if the viability is less than 80%, avoiding the subsequent harvesting liquid titer to be too low, continuing to feed culture and harvesting in the fourth generation if the viability is more than or equal to 80%, and determining whether to continue harvesting according to the production requirement if the fourth-generation virus titer is possibly further reduced.

Description

Suspension serum-free PK cell based on composite synergist and method for culturing porcine circovirus by using suspension serum-free PK cell Technical Field The invention relates to the technical field of laser processing, in particular to a suspension serum-free PK cell based on a composite synergist and a method for culturing porcine circovirus by using the suspension serum-free PK cell. Background In the intensive and large-scale development process of pig industry, porcine Circovirus (PCVAD) induced porcine circovirus-related diseases (PCVAD) have become key factors for restricting the healthy development of industry. PCV mainly comprises PCV2 (containing main flow subtypes such as 2a, 2b, 2d and the like) and PCV3, wherein pathogenicity of the PCV2 and PCV3 is extremely strong and the hazard range is wide, namely, after 2-10 weeks of piglet infection, the PCV is prone to burst a post-weaning multisystem failure syndrome (PMWS), which is represented by typical symptoms such as mental depression, progressive emaciation, dyspnea and the like, the death rate is as high as 10% -30%, and when adult sow infection is infected, abortion, stillbirth, weaning and the like are caused, so that the reproductive efficiency of the sow is reduced by more than 30%, and more seriously, PCV damages the immune system of the pig body, induces immunosuppression, so that the susceptibility of the pig group to epidemic diseases such as swine fever and blue ear disease is greatly improved, and the mixed infection rate can reach 40% -60%. According to industry data statistics, annual economic loss of a large-scale pig farm caused by PCVAD can reach 15% -20% of total revenue, and economic loss exceeding billions dollars per year is caused for the global pig raising industry. At present, PCV related vaccine production mainly depends on the traditional adherent PK cell culture technology, but the technology has a plurality of inherent defects, and is difficult to meet the industrial production requirement. The serum dependence and the potential safety hazard are prominent, 8% -10% of fetal bovine serum is needed to be added in the traditional process, the purchase cost is high (market average price is 500-800 yuan/L), and the vaccine is extremely easy to pollute due to carrying mycoplasma (detection rate is about 5% -8%), bovine viral diarrhea virus (BVDV, detection rate is about 2% -3%), and other exogenous factors (the last page), and meanwhile, the piglet allergic reaction is caused by serum residues, the occurrence rate is about 5%, and the use safety and the immune effect of the vaccine are seriously affected. The production efficiency is low, the wall-attached PK cells are limited by a growth space, the maximum density is only 5 multiplied by 10 6 cells/mL, the PCV proliferation potency (TCID 50) is less than or equal to 5.5/mL, and the wall-attached PK cells are limited by the wall-attached growth characteristics, only 'single harvest' can be realized, and only 1L of virus liquid is actually harvested by taking a single batch of 10L revolving bottles as an example, so that the yield can not meet the vaccine requirement of a large-scale pig farm, and the contradiction between vaccine supply and demand is caused. The process is complicated and has poor stability, the production process needs manual operations such as pancreatin digestion passage, multi-step liquid exchange and the like, the production period is as long as 45-50 days, and more importantly, the component difference (such as fluctuation of 15 percent of growth factor content) among serum batches can cause great fluctuation of cell proliferation efficiency and virus titer, the inter-batch variation coefficient is more than or equal to 20 percent, the quality control difficulty is obviously increased, and the quality uniformity of vaccine is difficult to ensure. The strain has weak suitability, the PCV genotype is easy to generate recombination variation, especially PCV3 is used as an emerging strain discovered in 2016, the traditional adherent cells need 6-8 months to carry out cell adaptation and process optimization, the optimal prevention and control time is often missed, the vaccine strain is not matched with the epidemic strain, the prevention and control effect is greatly reduced, and the epidemic spread of PCV3 cannot be effectively restrained. In order to solve the problems, the suspension serum-free cell culture technology becomes an industry innovation direction, but the prior art still has two key bottlenecks that on one hand, the lack of a domestication process of mature adherent PK cells to suspension cells, most domestication schemes can only realize semi-suspension growth, the cell serum-free tolerance capacity is weak, the activity decay after passage is fast, the survival rate after passage is reduced to below 85 percent after 20 times, stable suspension cell strains with high proliferation activity and virus sensitivity are difficult to obtain,