Method for culturing and screening of mdck monoclonal cell line

Abstract

The present application relates to the field of cell culture, in particular to a culture and screening method of MDCK monoclonal cell strain, and provides a culture method of MDCK monoclonal cell strain, comprising the following steps: S1, culturing pretreated cells to logarithmic phase; S2, inoculating the cells in logarithmic phase in S1 to the first culture carrier after diluting to a concentration of 1 cell / 100 μL; S3, inoculating the single cell colony in the first culture carrier in S2 to the second culture carrier after digestion, until the cell fusion degree reaches 70-80%, and obtaining the MDCK monoclonal cell strain. The present application realizes early detection without expanding the monoclonal cells to T75 culture flask, avoids the step of gradually expanding and culturing for 7-10 days in the traditional method, greatly shortens the overall screening period from the original 14-21 days, and can quickly match the demand of H3N2 virus variation for highly adaptive cell strains.

Description

Technical Field

[0001] This invention relates to the field of cell culture, and more particularly to a method for culturing and screening the MDCK monoclonal cell line. Background Technology

[0002] Influenza is a highly prevalent acute respiratory infectious disease worldwide. Due to its high transmissibility and frequent mutation rate, the influenza virus frequently causes seasonal influenza epidemics, posing a significant threat to vulnerable populations such as the elderly and children. The development and production of highly effective influenza vaccines is a core means of controlling this disease—and one of the key aspects of vaccine production is obtaining cell lines capable of supporting efficient viral replication.

[0003] MDCK cells (canine kidney epithelial cells), with their advantages of "no dependence on chicken embryos, flexible production, and high antigen stability," have become a core direction for global influenza vaccine technology innovation. Their research and production must meet stringent technical, quality, and regulatory requirements. Currently, a development pattern of "leading the market abroad and tackling clinical trials domestically" has emerged, making them a "golden cell line" in the field of influenza viruses: ① High sensitivity to H3N2 virus, supporting large-scale viral replication; ② Stable adherent or suspension growth, easy for large-scale culture; ③ No risk of exogenous viral contamination, meeting the biosafety requirements for vaccine production. However, MDCK cell populations exhibit heterogeneity; different monoclonal cell lines can vary in "adaptability" (i.e., viral proliferation capacity) to the virus by 3-5 times. Highly adaptable monoclonal cell lines can increase viral yield by more than 2 times. Therefore, screening for highly adaptable MDCK monoclonal cell lines is a core prerequisite for ensuring vaccine production capacity and improving virus isolation success rates. Summary of the Invention

[0004] In view of this, the present invention provides a method for culturing and screening MDCK monoclonal cell lines. This invention achieves early detection without expanding monoclonal cells to T75 culture flasks, avoiding the 7-10 day step-by-step expansion and culture required in traditional methods. This significantly shortens the overall screening cycle from the original 14-21 days, enabling rapid matching of the high adaptability cell lines required by H3N2 virus mutations. The present invention establishes a quantitative detection method unaffected by blood source, overcoming the fluctuations in detection results caused by the differences in sensitivity of different H3N2 strains to chicken or guinea pig blood in the traditional hemagglutination titer method. This allows for precise quantification of virus concentration, ensuring the consistency and reliability of detection results for different strains.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for culturing the MDCK monoclonal cell line, comprising the following steps:

[0007] S1: Culture the pretreated cells to the logarithmic growth phase;

[0008] S2: Dilute the logarithmic phase cells described in S1 to a concentration of 1 cell / 100 μL and then seed them into the first culture medium for culture.

[0009] S3: After digesting the single cell colonies in the first culture vector in S2, they are inoculated into the second culture vector until the cell confluence reaches 70-80%, thus obtaining the MDCK monoclonal cell line.

[0010] In some embodiments of the present invention, the culture time described in the culture method S2 above is 7 to 10 days.

[0011] In some embodiments of the present invention, the culture time described in the culture method S3 above is 3 to 4 days.

[0012] In some embodiments of the present invention, the first culture carrier and the second culture carrier in the above culture method include: microplates.

[0013] In some embodiments of the present invention, the microplate in the above culture method includes: a 24-well plate and / or a 96-well plate.

[0014] The present invention also provides an MDCK monoclonal cell line obtained by the culture method described above.

[0015] This invention also provides a method for screening highly adaptable MDCK monoclonal cell lines, comprising the following steps:

[0016] S1: Infect the above MDCK monoclonal cell line with the virus, culture it, and obtain the virus-infected cells;

[0017] S2: Extract RNA from the virus-infected cells, perform reverse transcription, and q-PCR to obtain the viral RNA copy number;

[0018] S3: Compare the viral RNA copy number obtained in S2 with the threshold. Cells with a copy number higher than the threshold are considered highly adaptable MDCK monoclonal cell lines.

[0019] In some embodiments of the present invention, in the above screening method, the viral MOI at the time of infection is 0.01; the transfection time is 48~72h.

[0020] In some embodiments of the present invention, in the above screening method, when the transfection time is 48 hours, the threshold is ≥5.1×10⁻⁶. 9 copies / μL;

[0021] When the transfection time is 60 hours, the threshold is ≥6×10⁻⁶. 9 copies / μL;

[0022] When the transfection time is 72 hours, the threshold is ≥5.4 × 10⁻⁶. 9 copies / μL.

[0023] In some embodiments of the present invention, in the above screening method, the primer set of the q-PCR has:

[0024] (1) A nucleotide sequence as shown in any of SEQ ID NO:1 to SEQ ID NO:4; or

[0025] (2) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (1), and which has the same or similar function as the nucleotide sequence shown in (1); or

[0026] (3) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (1) or (2).

[0027] In some embodiments of the present invention, in the above screening method, the primer set of the q-PCR includes: primer set 1 and / or primer set 2;

[0028] The sequences of primer set 1 are as shown in SEQ ID NO:1 and SEQ ID NO:2, which are nucleotide sequences.

[0029] The sequence of primer set 2 is as shown in SEQ ID NO:3 and SEQ ID NO:4, which are nucleotide sequences.

[0030] This invention also provides a primer set for screening highly adaptable MDCK monoclonal cell lines, the primer set having:

[0031] (1) A nucleotide sequence as shown in any of SEQ ID NO:1 to SEQ ID NO:4; or

[0032] (2) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (1), and which has the same or similar function as the nucleotide sequence shown in (1); or

[0033] (3) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (1) or (2).

[0034] In some embodiments of the present invention, the primer set includes: primer set 1 and / or primer set 2;

[0035] The sequences of primer set 1 are as shown in SEQ ID NO:1 and SEQ ID NO:2, which are nucleotide sequences.

[0036] The sequence of primer set 2 is as shown in SEQ ID NO:3 and SEQ ID NO:4, which are nucleotide sequences.

[0037] The present invention also provides a detection product comprising: the above-described primer set and acceptable adjuvants.

[0038] The beneficial effects of this invention include:

[0039] (1) Significantly shortened screening cycle: By enabling early detection without expanding monoclonal cells to T75 culture flasks, the traditional method of gradually expanding and culturing for 7 to 10 days is avoided, and the overall screening cycle is significantly shortened from the original 14 to 21 days, which can quickly meet the demand of H3N2 virus mutation for highly adaptable cell lines.

[0040] (2) Improve the accuracy and stability of detection: A quantitative detection method that is not affected by blood source has been established, which overcomes the problem of fluctuation in detection results caused by the difference in sensitivity of different H3N2 strains to chicken blood or guinea pig blood in the traditional hemagglutination titer method. It can realize the accurate quantification of virus concentration and ensure the consistency and reliability of detection results of different strains. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0042] Figure 1 The primer melting curve of q-PCR using fluorescent dye method is shown (horizontal axis: temperature (°C), vertical axis: fluorescence signal value). The curve shows a single peak (peak temperature about 85°C) with no extraneous peaks, which proves the primer specificity.

[0043] Figure 2 The standard curve for q-PCR using fluorescent dyes is shown (x-axis: logarithm of plasmid concentration (lg copies / μL), y-axis: Ct value). The linear regression equation is y = -3.35x + 36.79, R0. 2 =1.000, amplification efficiency of 98.69%, proving the accuracy of quantification;

[0044] Figure 3 The standard curve of q-PCR using nonspecific forward primer F1: 5'-CTATTGGACAATAGTAAAACCGGGRGA-3' (as shown in SEQ ID NO:5) / reverse primer R1: 5'-GTCATTGGGRATGCTTCCATTTGG-3' (as shown in SEQ ID NO:6) was obtained. The curve (x-axis: logarithm of plasmid concentration (lg copies / μL), y-axis: Ct value) was obtained. The linear regression equation was y=-5.31x+64.695, R2 =0.9952, amplification efficiency 54.3%;

[0045] Figure 4 The non-specific forward primer F1: 5'-CTATTGGACAATAGTAAAACCGGGRGA-3' (as shown in SEQ ID NO:5) / reverse primer R1: 5'-GTCATTGGGRATGCTTCCATTTGG-3' (as shown in SEQ ID NO:6) was used. The primer melting curve of the fluorescent dye q-PCR method (x-axis: temperature (°C), y-axis: fluorescence signal value) shows a double peak (peak temperature below 80°C), indicating the presence of non-specific amplification products or primer dimers.

[0046] Figure 5 The scatter plot shows the correlation between virus concentration detected in 24-well plates and T75 vials (x-axis: 24-well plate concentration (copies / μL), y-axis: T75 vial concentration (copies / μL)). The data points of the 10 candidate strains show a linear distribution, R0. 2 =0.94, demonstrating the reliability of early detection; where: A shows the copy number of H3N2 virus in monoclonal strains detected by Q-PCR; B shows the hemagglutination titer of H3N2 virus in monoclonal strains detected by traditional hemagglutination method; C shows the correlation results of virus concentration detected by PCR and hemagglutination methods.

[0047] Figure 6 The flowchart of the screening process for highly adaptable monoclonal cell lines of MDCK is shown (steps: parental cells → 96-well plate monoclonal cloning → 24-well plate culture → q-PCR preliminary screening → T75 bottle verification → stable line), which visually demonstrates the screening process. Detailed Implementation

[0048] This invention discloses a method for culturing and screening MDCK monoclonal cell lines.

[0049] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0050] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0051] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0052] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0053] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0054] The cell line ATCC NBL-2 of this invention was purchased from Beijing Zhongyuan Heju Biotechnology Co., Ltd., ATCC batch number: 70040764.

[0055] In Examples 1 to 6, Comparative Examples, and Effect Examples of the present invention, all raw materials and reagents used can be purchased from the market.

[0056] The present invention will be further illustrated below with reference to the embodiments:

[0057] Example 1: Pretreatment of parental MDCK cells

[0058] Cell source: International standard parental MDCK cell lines were used, mostly ATCC NBL-2 strain (accession number ATCCCRL-2936). Cells had to pass tests in advance for mycoplasma (PCR method), exogenous viruses (such as canine parvovirus, human parainfluenza virus, and 42 other viruses), and cell viability (trypan blue staining) to ensure uncontaminated cells and a viability ≥95%.

[0059] Culture conditions: Use DMEM medium (Opmai) containing 10% fetal bovine serum (FBS, Gibco brand) and culture in a constant temperature incubator at 37℃, 5% CO2, and 95% humidity. Passage the cells every 2-3 days at a ratio of 1:3. Once the cells have grown to the logarithmic growth phase (cells cover 80%-85% of the bottom area of ​​the culture flask), they can be used for subsequent operations.

[0060] Example 2: Construction of MDCK monoclonal cell bank

[0061] Limiting dilution method:

[0062] Cell concentration adjustment: Take logarithmic-phase parental MDCK cells, gently pipette them into DMEM medium containing 10% FBS (avoid cell clumping), count them using a hemocytometer, and adjust the cell concentration to 1 cell / 100μL (to ensure that each well contains at most 1 cell).

[0063] 96-well plate seeding and culture: Add 100 μL of adjusted cell suspension to each well of a 96-well cell culture plate (Corning 3596, V-bottom), and set up 3 blank control wells (culture medium only, no cells). Incubate statically at 37℃ and 5% CO2 for 7-10 days. During this period, observe daily using an inverted microscope (Olympus CKX41, 10× objective lens) and mark the wells "containing only a single cell colony" (colony diameter 0.5-1 mm, morphology typical of epithelial cells, no contamination by other cells).

[0064] Monoclonal cell proliferation: For labeled monoclonal wells, add 100 μL of 0.25% trypsin (containing 0.02% EDTA), incubate at 37°C for 2 min to allow cell detachment, then add 900 μL of DMEM medium containing 10% FBS to terminate digestion. After gentle pipetting and aspiration, transfer all cells to 24-well plates (1 mL per well); culture at 37°C and 5% CO2 for 3-4 days until cell confluence reaches 70%-80%.

[0065] Example 3: H3N2 Influenza Virus Infection and Culture

[0066] Virus preparation: The WHO-recommended H3N2 influenza virus standard strain (Singapore / INFIMH-16-0019 / 2016 strain) was used. It was passaged 1-2 times in SPF chicken embryos (incubated at 35℃ for 72h). Chicken embryo allantoic fluid was collected, and the initial virus titer was determined by hemagglutination titer method (≥1:64). The virus was diluted with serum-free DMEM medium containing 2μg / mL TPCK-trypsin.

[0067] Cell infection procedure: Dilute H3N2 virus to MOI=0.01 in serum-free DMEM medium containing 2 μg / mL trypsin (Sigma T4174), add 200 μL of diluted virus solution to each well of a 24-well plate, and incubate at 37°C for 1 h (gently shake the culture plate once every 15 min to ensure that the virus is evenly contacted with the cells). After incubation, add 800 μL of serum-free DMEM medium and continue culturing for 48-72 h.

[0068] Example 4: RNA extraction and reverse transcription from viral culture supernatant

[0069] RNA extraction from the supernatant was performed using the Super FastPure Cell RNA Isolation Kit (Novizan RC102-01): ① Pipe 200 μL of cell supernatant from a 24-well plate, add 500 μL of lysis buffer RL (provided with the kit), and vortex for 10 s; ② Add 200 μL of chloroform, vortex for 30 s, and centrifuge at 12000 rpm for 15 min at 4 °C; ③ Pipe the upper aqueous phase (approximately 400 μL) into the adsorption column of the kit, centrifuge at 10000 rpm for 30 s at 4 °C, and discard the waste liquid; ④ Add 500 μL of wash buffer RW1 (provided with the kit), centrifuge for 30 s, and discard the waste liquid; ⑤ Add 700 μL of wash buffer RW2 (provided with the kit), centrifuge for 30 s, and discard the waste liquid, repeating once; ⑥ Transfer the adsorption column to a new centrifuge tube, add 30 μL of RNase-free enzyme-free water, incubate at room temperature for 2 min, centrifuge at 12000 rpm for 2 min, and collect the eluent (i.e., total viral RNA).

[0070] RNA reverse transcription was performed using the Novizan Strand cDNA Synthesis Kit (R211-01): ① Prepare a 20 μL reverse transcription system: 4 μL of 5×PrimeScript Buffer, 1 μL of PrimeScript RT Enzyme Mix I, 1 μL of Oligo dTPrimer (50 μM), 1 μL of Random 6 mers (100 μM), 2 μL of viral RNA, and 11 μL of RNase-free water; ② Reaction conditions: incubate at 42℃ for 30 min (reverse transcription to synthesize cDNA) → heat at 85℃ for 5 min (inactivate reverse transcriptase) → store at 4℃ to obtain the cDNA template.

[0071] Example 5: q-PCR quantitative detection

[0072] 1) Specific primer design and synthesis: Primer sequences (designed based on the 200-400bp conserved region of the HA gene) Forward primer (H3N2-2007-F1): 5'-GCACAGGGAATCTAATTGCTCCT-3' (as shown in SEQ ID NO:1), Reverse primer (H3N2-2007-R1): 5'-AYCTCATTAYTGAGCTTTTCCCACTT-3' (as shown in SEQ ID NO:2) (Y=C / T, adapted to slight variations in the HA gene to ensure universality for different H3N2 strains). The above primers were synthesized by Universal Biosystems (Anhui) Co., Ltd., with a purity of HPLC grade (≥99%). They were dissolved in RNase-free enzyme-free water to 10μM, aliquoted, and stored at -20℃ protected from light.

[0073] 2) Preparation of standard curve: A linear plasmid containing the HA gene target fragment was synthesized using general biosynthesis. The plasmid was diluted to 10⁻⁶ with RNase-free enzyme-free water. 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 Eight concentration gradients per copy / μL;

[0074] 3) Detection Procedure: For q-PCR quantitative detection, use Novizan ChamQ Blue Universal SYBR qPCRMaster Mix (Q312-02). In an RNase-free 0.2 mL q-PCR tube, first add 10 μL of 2×ChamQ SYBR qPCRMaster Mix, then add 0.4 μL of 10 μM H3N2 virus forward primer (H3N2-2007-F1) and 0.4 μL of 10 μM reverse primer (H3N2-2007-R1); then add 2 μL of cDNA template obtained from reverse transcription; finally, add 7.2 μL of RNase-free ddH2O to bring the volume to 20 μL. Each sample should have 3 replicate wells, and a standard curve sample and a negative control (using RNase-free ddH2O instead of cDNA template) should also be set up to eliminate contamination interference. After the system is prepared, gently tap the side of the q-PCR tube to mix, and briefly centrifuge to remove air bubbles.

[0075] 4) Concentration conversion: The copy number (copies / μL) of the HA gene in cDNA was calculated using the q-PCR instrument software based on the standard curve. Combined with the sample dilution factor, the viral concentration in the supernatant of the 24-well plate was converted: Virus concentration (copies / μL) = Copy number calculated by software × Dilution factor.

[0076] Example 6: Screening and Stability Verification of Highly Adaptive Monoclonal Cell Lines

[0077] Screening threshold setting: First, detect the viral concentration of MDCK parental cells in the 24-well plate stage (3 replicates, average value 3 × 10⁻⁶). 9 (copies / μL), when the viral infection time is 60 h, the screening threshold is set to "viral concentration ≥ 6 × 10⁻⁶". 9 "copies / μL (more than twice the parental cells)";

[0078] When the viral infection time is 48 hours, the detection concentration is approximately 85% of that at 60 hours, and the screening threshold is set at ≥5.1×10⁻⁶. 9copies / μL;

[0079] When the viral infection time is 72 hours, the detection concentration is approximately 90% of that at 60 hours, and the screening threshold is set at ≥5.4×10⁻⁶. 9 copies / μL.

[0080] Preliminary screening: By comparing the viral concentration of all 24-well plate monoclonal cell lines, those that meet the criteria are selected and identified as "candidate high-fitness strains".

[0081] Comparative Example

[0082] 1. Pretreatment of parental MDCK cells

[0083] Cell source: International standard parental MDCK cell lines were used, mostly ATCC NBL-2 strain (accession number ATCCCRL-2936). Cells had to pass tests in advance for mycoplasma (PCR method), exogenous viruses (such as canine parvovirus, human parainfluenza virus, and 42 other viruses), and cell viability (trypan blue staining) to ensure uncontaminated cells and a viability ≥95%.

[0084] Culture conditions: Use DMEM medium (Opmai) containing 10% fetal bovine serum (FBS, Gibco brand) and culture in a constant temperature incubator at 37℃, 5% CO2, and 95% humidity. Passage the cells every 2-3 days at a ratio of 1:3. Once the cells have grown to the logarithmic growth phase (cells cover 80%-85% of the bottom area of ​​the culture flask), they can be used for subsequent operations.

[0085] Key objective: To ensure that parental cells are in optimal physiological condition and to avoid monoclonal failure due to cell aging or contamination.

[0086] 2. Construction of MDCK monoclonal cell bank using limiting dilution method

[0087] Cell concentration adjustment: Take log-phase parental MDCK cells and gently disperse them by pipetting in DMEM medium containing 10% FBS (avoid cell clumping). Count the cells using a hemocytometer and adjust the cell concentration to 1 cell / 100μL (to ensure that each well contains a high probability of only 1 cell when seeded).

[0088] 96-well plate seeding and culture: Add 100 μL of adjusted cell suspension to each well of a 96-well cell culture plate (Corning 3596, V-bottom), and set up 3 blank control wells (culture medium only, no cells). Incubate statically at 37℃ and 5% CO2 for 7-10 days. During this period, observe daily using an inverted microscope (Olympus CKX41, 10× objective lens) and mark the wells "containing only a single cell colony" (colony diameter 0.5-1 mm, morphology typical of epithelial cells, no contamination by other cells).

[0089] Stepwise expansion of monoclonal cells: For labeled monoclonal wells, add 100 μL of 0.25% trypsin (containing 0.02% EDTA), incubate at 37°C for 2 min to allow cell detachment, then add 900 μL of DMEM medium containing 10% FBS to terminate digestion. Gently mix by pipetting and aspirating, then transfer all cells to 24-well plates (1 mL per well); incubate at 37°C and 5% CO2 for 3-4 days until cell confluence reaches 70%-80%, then transfer to 6-well plates (2 mL per well) and incubate for 4 days. Finally, expand to T75 culture flasks (10 mL per flask) and incubate for 7 days to obtain approximately 1 × 10⁶ cells / well. 8 Single-clonal cells per vial were simultaneously cryopreserved to establish a Research Cell Bank (RCB) (each vial containing 1×10⁶ cells). 6 (1 cell), used for subsequent viral adaptability testing.

[0090] 3. H3N2 Influenza Virus Infection and Culture

[0091] Virus preparation: The WHO-recommended H3N2 influenza virus standard strain (Singapore / INFIMH-16-0019 / 2016 strain) was used. It was passaged 1-2 times in SPF chicken embryos (incubated at 35℃ for 72h). Chicken embryo allantoic fluid was collected, and the initial virus titer was determined by hemagglutination titer method (≥1:64). The virus was diluted with serum-free DMEM medium containing 2μg / mL TPCK-trypsin.

[0092] Cell infection procedure: Wash monoclonal cells in T75 culture flasks twice with PBS, add 5 mL of diluted H3N2 virus solution, and incubate at 37°C and 5% CO2 for 1 h; after incubation, add 5 mL of serum-free DMEM medium containing 2 μg / mL TPCK-trypsin, and continue culturing for 48-72 h (the peak period of viral replication determined in preliminary experiments, when cells have not shed in large quantities and the viral concentration is the highest);

[0093] 4. Hemagglutination titer method for detecting viral adaptability

[0094] Virus supernatant collection: Use a pipette to aspirate the cell supernatant from the T75 culture flask, centrifuge at 4°C and 12,000 rpm for 5 min to remove cell debris, and collect the supernatant;

[0095] Preparation of red blood cell suspension: Fresh chicken blood (SPF grade Leghorn chicken, stored at 4°C for no more than 24 hours after collection) and guinea pig blood (SPF grade Hartley guinea pig, stored at 4°C for no more than 12 hours after collection) were used respectively. The blood cells were washed three times with physiological saline (0.9% NaCl) (centrifuged at 2000 rpm for 5 min each time) to remove plasma components, and finally prepared into a 5% (v / v) red blood cell suspension (volume ratio: 5 mL red blood cells + 95 mL physiological saline).

[0096] Hemagglutination titer determination (96-well V-plate method): ① Add 50 μL of physiological saline to each well of a 96-well V-plate; ② Add 50 μL of virus supernatant to well 1, mix by pipetting 5 times, then transfer 50 μL to well 2, and perform serial dilutions of 2-fold (dilution gradient: 1:2, 1:4, 1:8, ..., 1:256), with well 12 serving as a blank control (50 μL of physiological saline only); ③ Add 50 μL of 5% erythrocyte suspension (chicken blood or guinea pig blood, to be tested separately) to each well, and gently shake the plate to mix the liquid; ④ Observe the results after incubation at 37℃ for 30 min: the highest dilution that shows "complete erythrocyte agglutination without precipitation" is the hemagglutination titer;

[0097] Repeatability control: Three replicate wells are set for each sample, and the average of the three test results is taken as the final titer. If the titer difference between replicate wells exceeds two gradients (e.g., 1:64, 1:128, 1:256), the test must be repeated.

[0098] 5. Screening of highly adaptable MDCK monoclonal cell lines

[0099] Screening threshold setting: First, detect the H3N2 virus hemagglutination titer of parental MDCK cells (ATCC NBL-2 strain) under the same infection conditions (3 replicates, the average value is usually 1:32), and set the screening threshold as "the hemagglutination titer of the monoclonal cell line is ≥1:64 (i.e., more than twice that of the parental cells)".

[0100] Strain screening and validation: For all monoclonal cell lines that have completed the test, their hemagglutination titers are compared to screen out the qualified strains; the qualified strains are validated once (re-amplified to T75 bottles, and the virus infection and hemagglutination titer test are repeated). If the difference in titers between the two tests is ≤2 gradients (e.g., 1:64 for the first test and 1:128 for the second test), they are judged as "high virus adaptability MDCK monoclonal cell lines".

[0101] Example of effect

[0102] 1. The detection cycle for the same candidate strain in a 24-well plate was 12 days, while that for the control group was 22 days, a reduction of approximately 50%. Furthermore, the correlation R between the viral concentration detected in the 24-well plate and the T75 vial for the HA gene was [not specified]. 2 =0.9952 (see) Figure 3 This demonstrates that early detection results are reliable and can predict performance after expanded culture.

[0103] 2. ① Specificity: The melting curve shows a single peak (see...) Figure 1 The absence of heterogeneous bands indicates that only the HA gene was amplified.

[0104] ② Sensitivity: Minimum detection limit 10 1copies / μL (lowest detection limit for comparative example 10 copies / μL) 4 (copies / μL)

[0105] ③Stability: Intra-batch CV ≤ 10%, inter-batch CV ≤ 10% (comparative example CV ≥ 20%).

[0106] ④ No blood-borne interference: The concentration of H3N2 IVR-227 strain detected by q-PCR was 7.8 × 10⁻⁶. 8 The ratio of copies / μL was unique, while the comparative ratio of chicken blood yielded no results, and guinea pig blood yielded a 1:32 ratio (contradictory results).

[0107] 3. For example Figure 4 As shown, using non-specific forward primer F1: 5'-CTATTGGACAATAGTAAAACCGGGRGA-3' / reverse primer R1: 5'-GTCATTGGGRATGCTTCCATTTGG-3', the primer melting curve of the fluorescent dye q-PCR method (horizontal axis: temperature (°C), vertical axis: fluorescence signal value) shows a double peak (peak temperature below 80°C), indicating the presence of non-specific amplification products or primer dimers.

[0108] 4. For example Figure 5 As shown, the PCR detection results of the HA gene are compared with the traditional hemagglutination titer method, and the linear correlation between the two is also shown.

[0109] 5. For example Figure 6 The following diagram shows the MDCK cell monoclonal culture screening flowchart (steps: parental cells → 96-well plate monoclonal culture → 24-well plate culture → q-PCR preliminary screening → T75 bottle verification → stable lineage), which visually demonstrates the screening process.

[0110] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for culturing MDCK monoclonal cell lines, characterized in that, Includes the following steps: S1: Culture the pretreated cells to the logarithmic growth phase; S2: Dilute the logarithmic phase cells described in S1 to a concentration of 1 cell / 100 μL and then seed them into the first culture medium for culture. S3: After digesting the single cell colonies in the first culture vector in S2, they are inoculated into the second culture vector until the cell confluence reaches 70-80%, thus obtaining the MDCK monoclonal cell line.

2. The cultivation method as described in claim 1, characterized in that, The culture time described in S2 is 7-10 days.

3. The cultivation method as described in claim 1 or 2, characterized in that, The incubation period described in S3 is 3 to 4 days.

4. The MDCK monoclonal cell line obtained by the culture method according to any one of claims 1 to 3.

5. A method for screening highly adaptable MDCK monoclonal cell lines, characterized in that, Includes the following steps: S1: Infect the MDCK monoclonal cell line as described in claim 4 with a virus, culture it, and obtain virus-infected cells; S2: Extract RNA from the virus-infected cells, perform reverse transcription, and q-PCR to obtain the viral RNA copy number; S3: Compare the viral RNA copy number obtained in S2 with the threshold. Cells with a copy number higher than the threshold are considered highly adaptable MDCK monoclonal cell lines.

6. The screening method as described in claim 5, characterized in that, The viral MOI at the time of infection was 0.01; the transfection time was 48~72h.

7. The screening method as described in claim 6, characterized in that, When the transfection time is 48 hours, the threshold is ≥5.1×10⁻⁶. 9 copies / μL; When the transfection time is 60 hours, the threshold is ≥6×10⁻⁶. 9 copies / μL; When the transfection time is 72 hours, the threshold is ≥5.4 × 10⁻⁶. 9 copies / μL.

8. The screening method according to any one of claims 5 to 7, characterized in that, The primer set for the q-PCR has: (1) A nucleotide sequence as shown in any of SEQ ID NO:1 to SEQ ID NO:4; or (2) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (1), and which has the same or similar function as the nucleotide sequence shown in (1); or (3) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (1) or (2).

9. A primer set for screening highly adaptable MDCK monoclonal cell lines, characterized in that, The primer set has: (1) A nucleotide sequence as shown in any of SEQ ID NO:1 to SEQ ID NO:4; or (2) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (1), and which has the same or similar function as the nucleotide sequence shown in (1); or (3) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (1) or (2).

10. The product being tested, characterized in that, include: The primer set as described in claim 9 and acceptable auxiliaries.

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