CN106520700B - MDCK cell line for stably expressing bovine trypsinogen and application thereof - Google Patents

Abstract

The invention provides an MDCK engineering cell line for stably secreting and expressing bovine trypsinogen (S.pro-try), and application thereof in culturing influenza viruses and producing influenza virus vaccines, belonging to the technical field of biology. The invention also provides a method for constructing the cell line, which comprises the steps of constructing the expression gene sequences of the bovine pancreatin and the bovine pancreatin distributed by 3 kinds of subcellular distribution of secretory type, intracellular type and transmembrane type, and transiently transfecting MDCK to screen the optimal expression mode for improving the titer of the human influenza virus. The cell line provided by the invention lays a cellular foundation for the future large-scale influenza vaccine production, and solves the key problems of expansion of vaccine capacity and improvement of quality; on the other hand, the method can also be used as a research tool for effectively separating, culturing and monitoring influenza viruses and anti-influenza virus drugs in a laboratory, measuring a neutralizing antibody and the like in other aspects, and has wide application prospect.

Description

MDCK cell line for stably expressing bovine trypsinogen and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an engineering cell line clone MDCK-S.pro-try and a construction method thereof, and application of the cell line in the aspects of culturing influenza virus, preparing influenza vaccine, screening anti-influenza virus drugs, measuring neutralizing antibodies or other related researches on influenza and the like.

Background

Influenza vaccines are currently the most effective means of preventing the development and spread of influenza. Most of the human influenza vaccines in the current market are produced by taking chick embryos as a substrate, and comprise influenza A virus strains H1N1, H3N2 and influenza B virus strains. The traditional chick embryo production system has the history of about 70 years, the immune effect of the vaccine is fully proved, but the chick embryo production system has some defects, such as that the cultured influenza virus is easy to have antigen variation and cannot be completely matched with the epidemic strains of people; residual ovalbumin is easy to cause anaphylactic reaction; especially when a pandemic occurs, the supply of healthy chick embryos is limited and cannot meet the large demand for vaccines in a short time, and the like. Mammalian cell culture systems have significant advantages over chick embryos, can avoid these problems, and are the development trend for future vaccine production, and in 1995 WHO suggested the development of studies using mammalian cells as production substrates.

Mammalian cells that have been explored to be useful include African green monkey kidney cells (Vero), canine kidney epithelial cells (Madin-Darby kidney cells or Madin-darbycine kidney cells, MDCK), and human embryonic retinoblasts per.c 6. The MDCK cell is easy to culture, strong in pancreatin tolerance, susceptible to various influenza virus strains, capable of generating high virus titer and an ideal cell line for influenza vaccine production. MDCK cell-derived influenza vaccines have been approved by the European Medicines Agency (EMA). Clinical trials have shown that influenza vaccines produced using cells such as MDCK are safe and highly immunogenic, however lower virus titers are one of the bottlenecks in their large-scale production. The reasons for low titers in the complex environment of the production system are manifold, and from the standpoint of the cell matrix alone, the cells themselves do not express proteases that cleave HA, which is a limiting factor in low virus yields.

Influenza virus hemagglutinin HA is one of the major surface antigens of the virus and plays an important role in recognizing and binding to target cell receptors, mediating membrane fusion, inducing protective neutralizing antibodies. In the virus replication process, HA protein is firstly synthesized in the form of precursor HA0, and can mediate fusion of a virus envelope and a target cell membrane after being cleaved into mature HA1 and HA2 by host protease to start a virus life cycle, so that the cleavage of HA is the primary premise of virus infection cells, MDCK cells do not have HA cleavage proteases, and researches prove that a certain amount of trypsin can accelerate intercellular transmission of viruses, improve virus titer and accelerate virus multi-cycle growth, and the HA cleavage is now the conventional operation for culturing influenza virus cells.

The trypsin belongs to a serine protease family, and a serine peptidase functional domain with the C end length of 220aa can specifically hydrolyze human influenza virus or low-pathogenicity avian influenza virus HA0, so that mature HA1 and HA2 play a role in releasing virus genomes into cells to accelerate virus multi-cycle growth, but the difficulty of cell culture is increased by the added pancreatin, and the production cost of the vaccine is increased.

In human respiratory epithelial cells in a natural state, the types of proteases which really play a role of virus activation and the positions of action subcellular cells are always the research hotspots and are not determined. Various serine proteases of the same family as trypsin are known to have the function of cleaving HA, and the sites of action may be located in the processes of transport on the cell membrane, extracellular and intracellular, but further intensive studies have been lacking.

Disclosure of Invention

In a first aspect of the invention, there is provided a modified MDCK cell line stably secreting and expressing bovine trypsinogen, the cell line comprising the complete secreted bovine trypsinogen s.pro-try encoding gene integrated into its cell genome.

In one embodiment, the cell genome comprises a secreted bovine trypsinogen encoding gene, the nucleotide sequence of which is shown in Genbank LOC 780933.

In a specific embodiment, the nucleic acid coding sequence for secreted bovine trypsinogen is optimized for canine codon usage.

Preferably, the nucleic acid coding sequence of the secretory bovine trypsinogen is shown as SEQ ID No. 1:

ATGAAGACCTTCATCTTCCTGGCCCTGCTGGGCGCCGCCGTGGCCTTCCCCGTGGACGACGACGACAAGATCGTGGGCGGCTACACCTGCGGCGCCAACACCGTGCCCTACCAGGTGAGCCTGAACAGCGGCTACCACTTCTGCGGCGGCAGCCTGATCAACAGCCAGTGGGTGGTGAGCGCCGCCCACTGCTACAAGAGCGGCATCCAGGTGAGGCTGGGCGAGGACAACATCAACGTGGTGGAGGGCAACGAGCAGTTCATCAGCGCCAGCAAGAGCATCGTGCACCCCAGCTACAACAGCAACACCCTGAACAACGACATCATGCTGATCAAGCTGAAGAGCGCCGCCAGCCTGAACAGCAGGGTGGCCAGCATCAGCCTGCCCACCAGCTGCGCCAGCGCCGGCACCCAGTGCCTGATCAGCGGCTGGGGCAACACCAAGAGCAGCGGCACCAGCTACCCCGACGTGCTGAAGTGCCTGAAGGCCCCCATCCTGAGCGACAGCAGCTGCAAGAGCGCCTACCCCGGCCAGATCACCAGCAACATGTTCTGCGCCGGCTACCTGGAGGGCGGCAAGGACAGCTGCCAGGGCGACAGCGGCGGCCCCGTGGTGTGCAGCGGCAAGCTGCAGGGCATCGTGAGCTGGGGCAGCGGCTGCGCCCAGAAGAACAAGCCCGGCGTGTACACCAAGGTGTGCAACTACGTGAGCTGGATCAAGCAGACCATCGCCAGCAACTAA(SEQ IDNo.1)。

in one embodiment, the cell line is named MTY6, and is deposited at the general microbiological center of the China Committee for culture Collection of microorganisms, and is located at the institute of microbiology, Zhongkoyao institute of Siro No.1, North Cheng, south China, Beijing, and has the following culture collection numbers: CGMCC No.12681, with a preservation date of 2016, 7 and 6.

In a second aspect of the present invention, a method for constructing the modified MDCK cell line is provided, wherein the method comprises connecting an open reading frame cDNA sequence encoding s.pro-try with a eukaryotic expression vector, transfecting the eukaryotic expression vector into an MDCK cell line, and performing antibiotic pressurized screening to obtain a stably expressed cell line.

In one embodiment, the eukaryotic expression vector is pcdna3.1.

In one embodiment, the target sequences of 6 expression modes of secretory, intracellular and transmembrane zymogens or enzymes are respectively connected with eukaryotic expression vectors, transiently transfected into MDCK cell lines, inoculated with human influenza viruses to compare yield difference, and screened for an optimal expression mode for increasing virus titer.

In a preferred embodiment, the eukaryotic expression vector of the secreted bovine trypsinogen is continuously screened under pressure, and a limited dilution method is combined to obtain a stable expression monoclonal cell.

More preferably, the continuous pressure screening is screening under high concentration of G418.

Further, the concentration of G418 was 1000. mu.g/ml.

In a preferred embodiment, the nucleic acid coding sequence for secreted bovine trypsinogen is optimized for canine codon usage.

Preferably, the nucleic acid coding sequence of the secretory bovine trypsinogen is shown as SEQ ID No. 1.

More preferably, the modified MDCK cell line is MTY6, which is deposited at the general microbiology center of the china committee for culture collection management, and is located at the institute of microbiology, institute of middle school, west road 1, north chen, yang, beijing, and has the culture collection number: CGMCC No.12681, with a preservation date of 2016, 7 and 6.

In a third aspect, the invention provides an application of the modified MDCK cell line, wherein the application comprises the application of the modified MDCK cell line in the fields of culturing influenza virus, preparing influenza vaccine, screening anti-influenza drugs, measuring neutralizing antibodies or other related researches on influenza, and the like.

Preferably, the influenza is human influenza.

In a fourth aspect of the present invention, there is provided a method for culturing influenza virus, wherein the method comprises culturing influenza virus using the modified MDCK cell line described above.

In a specific embodiment, the influenza in the above method is human influenza.

In one embodiment, the method comprises inoculating human influenza virus H1, subtype H3 and type B strains into the modified MDCK cell line constructed according to the present invention.

In one embodiment, the method comprises culturing the virus on the cell at 37 ℃ in 4% CO₂Culturing in serum-free medium for 48-72 hr, and collecting virus supernatant to obtain influenza virus liquid.

Preferably, the step of propagating human influenza virus in the method does not require the addition of TPCK-pancreatin.

Preferably, TPCK-pancreatin may be further added to the step of growing human influenza virus in the method.

In a fifth aspect of the invention, a method for preparing an influenza vaccine is provided, wherein the method comprises culturing an influenza virus using the modified MDCK cell line described above.

In a specific embodiment, the influenza in the above method is human influenza.

In one embodiment, the method comprises inoculating human influenza virus H1, subtype H3 and type B strains into the modified MDCK cell line constructed according to the present invention.

In one embodiment, the method comprises culturing the virus on the cell at 37 ℃ in 4% CO₂Culturing in serum-free medium for 48-72 hr, and collecting virus supernatant to obtain influenza virus liquid.

Preferably, the step of propagating human influenza virus in the method does not require the addition of TPCK-pancreatin.

Preferably, TPCK-pancreatin may be further added to the step of growing human influenza virus in the method.

The invention adopts a genetic engineering means to simulate the distribution of three subcellular distributions of protease secretion, intracellular and transmembrane in a natural state, introduces a trypsin gene into MDCK cells, inoculates different types or subtype human influenza viruses, screens out secretory zymogen as an optimal action mode for improving the virus yield by comparing the virus yield, and screens out a cell line capable of stably and independently expressing by continuous pressurization, on one hand, the invention is used for the scale production of vaccines, simplifies the production process, more effectively improves the virus yield, lays a cellular foundation for the large-scale production of influenza vaccines in the future, and solves the key problems of the expansion of the productivity of the vaccines and the improvement of the quality; on the other hand, the method can also be used as a research tool for effectively separating, culturing and monitoring influenza viruses and anti-influenza virus drugs in a laboratory, measuring a neutralizing antibody and the like in other aspects, and has wide application prospect. The influenza virus cultured by the MDCK cell line which is obtained by screening and stably secretes and expresses the bovine trypsinogen has high production titer, and particularly the culture titer of the B/Heilong orchid/116/2010 influenza virus can reach the virus titer cultured by a common MDCK cell and TPCK-pancreatin, so that the virus culture process can be simplified by utilizing the modified MDCK cell line.

Drawings

FIG. 1 depicts the expression characterization of secreted, intracellular and transmembrane zymogens and enzyme recombinant plasmids transiently transfected with MDCK.

+: TPCK treated bovine trypsin; -: transfecting a cell sample of empty plasmids; pro-try: a secreted zymogen; and S.try: a secreted enzyme; pro-try: a transmembrane-type zymogen; try: a transmembrane enzyme; pro-try: an intracellular zymogen; try: an intracellular enzyme.

FIG. 2 shows the expression of MDCK transiently transfected by recombinant plasmid through indirect immunofluorescence assay.

FIG. 3 shows the detection of the activity of transiently transfected and expressed pancreatin.

A: a pancreatin activity standard curve; b: 6 plasmid transient transfection MDCK expresses pancreatin activity.

FIG. 4 shows the effect of transient expression of each protease on the proliferation of influenza virus.

FIG. 5 shows PCR and RT-PCR identification of target genes from monoclonal cells.

A: extracting PCR identification of genome DNA; b: RT-PCR identification of extracted cell RNA.

FIG. 6 shows the identification of the monoclonal cell line MT 21.

A: performing indirect immunofluorescence identification; b: carrying out immunoblot identification; c: measuring the activity of pancreatin;

d: PCR and RT-PCR identification of target genes of different generations of monoclonal cells;

e: and (3) measuring the pancreatic enzyme activity of the monoclonal cells of different generations.

FIG. 7 shows the growth of different influenza strains on monoclonal cells.

A: detection of NA Activity and CCID of A/Tianjinnan/15/2009 (H1N1)₅₀The titer;

b: detection of NA activity and CCID of A/Fujiangan/196/2009 (H3N2)₅₀The titer;

c: detecting NA activity and CCID of B/Heilongjiang lily/116/2010₅₀The titer;

d: detection of NA Activity and CCID of A/California/7/2009(H1N1)₅₀The titer;

FIG. 8 shows the comparison of the activity of the expression enzymes of the optimized monoclonal cell MTY6 and MT 21.

FIG. 9 shows a comparison of the growth of different viruses at MTY6 and MT 21.

A: detecting the NA activity of the attenuated live vaccine strain A1/17/California/2009/38;

b: NA activity assay of live attenuated vaccine strain A3/Switzerland/9715293/2013-CDC-LA 10A.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention. The reagents used in the examples are, unless otherwise indicated, conventional reagents available on the market.

The material sources of this example:

1. cell: MDCK is purchased from ATCC and stored in China food and drug testing institute respiratory virus vaccine room.

2. Virus: A/Tianjinnan/15/2009 (H1N1), A/Fujianggan/196/2009 (H3N2), and B/Heilongjiang Howland/116/2010, which are gifts from the professor Liao Yang, institute of medical biology, academy of Chinese medical sciences. Influenza a vaccine virus strain a/California/7/2009(H1N1) was stored in the respiratory virus vaccine room of the chinese food and drug testing institute.

Example 1 construction of eukaryotic expression vectors of recombinant bovine pancreatin genes in different expression forms and analysis of transient transfection function

(1) Vector construction: taking a bovine trypsin cDNA sequence (LOC780933) provided by GenBank as a template, designing different specific primers (tables 1 and 2), amplifying to obtain a secretory zymogen expression sequence, respectively removing an N-terminal secretory signal peptide on the basis of the secretory zymogen expression sequence, amplifying to obtain an intracellular zymogen expression sequence, and adding an HAT transmembrane functional domain to amplify to obtain a transmembrane zymogen sequence; and respectively removing the base sequence corresponding to the N-terminal APFDDDDK activated peptide on the basis of the zymogen sequence, and amplifying to obtain respective enzyme expression sequences. An upstream primer is introduced into Nhe I (shown by an italic downward sliding line), a downstream primer is introduced into EcoR I (shown by an italic downward sliding line) single enzyme cutting site, and a Kozak sequence (indicated by a square box) is added to the N end of the ATG (start codon) of the upstream primer so as to improve the expression efficiency. Mu.l of template in a 50. mu.l PCR reaction system, 32.8. mu.l of sterile double-distilled water, 5. mu.l of 10 XPCR buffer, 1. mu.l of dNTP Mix (10mmol/L), 2. mu.l of 10. mu. mol/L upstream primer, 2. mu.l of 10. mu. mol/L downstream primer, and 0.2. mu.l of Taq DNA polymerase (Invitrogen). PCR amplification conditions: pre-denaturation at 94 deg.C for 1min, denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 1min for 30 cycles, and final heat preservation at 72 deg.C for 10 min. The 6 kinds of amplified fragments are connected into pcDNA3.1 vector in double enzyme digestion mode, and after conversion and selection of single colony, double enzyme digestion and sequencing identification are carried out, the size of the result sequence is in accordance with the expectation, and the reading frame of each gene is correct.

TABLE 1 amplification primers for secreted, intracellular trypsinogen and enzyme

TABLE 2 amplification primers for transmembrane-type trypsinogen and enzyme

(2) Transient transfection: MDCK cells in logarithmic growth phase are digested and blown into single cell suspension, and the concentration is adjusted to 1 × 10⁵Cells/ml, inoculated into 6-well culture plates (2 ml per well), placed at 37 ℃ with 4% CO₂And the incubator is 24 h. Preparing liposome-DNA complexes in an optimal ratio of 1:2.5 according to the LP2000 transfection reagent protocol; adding dropwise when the cell reaches 90% confluence, at 37 deg.C and 4% CO₂Culturing for 6h, changing serum-free SFM culture solution, culturing for 48h, collecting cell supernatant for secreting expression zymogen and enzyme, adding 2ml of precooled PBS lysate containing 0.5% TritonX-100 into secretory expression cells and intracellular and transmembrane expression cells, acting for 30min at ice bath or 4 ℃, repeatedly beating for 2-3 times, centrifuging at 5000rpm and 4 ℃ for 5min, removing cell debris, and sucking supernatant and placing on ice. All collected samples were concentrated by ultrafiltration using Millipore 3K ultrafiltration tubes at 4 ℃ 5000 Xg for 20min and stored at-70 ℃.

(3) Immunoblot identification of expression: each sample is collected and loaded with 20 mu l of sample per well, SDS-PAGE electrophoresis is carried out at the stable pressure of 120V, PBS (pH7.4) is used for dissolving 5 percent skim milk for 1h at the low-speed shaking table sealing membrane at the temperature of 37 ℃, immunoblotting identification is carried out by taking rabbit anti-bovine trypsin polyclonal antibody as a primary antibody and taking goat anti-rabbit IgG antibody as a secondary antibody, and the results (figure 1) show that the proteins distributed by three cells have specific bright bands. The bands of the secretory zymogen supernatant and the cell sample are bright and are about 24-25 kD; the secretory enzyme supernatant band is more obvious than that in the cell, and the molecular size is equivalent to that of the contrast; the size of the transmembrane expression protein is close to 30kD, and the zymogen is slightly larger than the enzyme; the intracellular distribution of zymogens and enzymes is comparable to secretory expression, approximately 24-25 kD.

(4) And (3) indirect immunofluorescence expression localization identification: placing a clean glass slide in the center of each well of a 12-well cell plate, inoculating 1X 10 MDCK cells⁵Preparing cell climbing sheets; placing at 37 ℃ and 4% CO₂When the culture reaches 80% confluence, 6 plasmids are transiently transfected respectively; DMEM with 2.5% FBS was cultured for another 48 hours, and washed 3 times with preheated 1 XPBS (pH7.4) at 37 ℃; adding 2ml of 4% paraformaldehyde into each well, and fixing at room temperature for 30 min; adding 2ml of 0.1% TritonX-100 into each hole of secretory cells and intracellular cells, and allowing the mixture to permeate for 20min at room temperature; blocking 2% BSA prepared in 1ml PBS for 1h at 37 ℃; the rabbit anti-bovine trypsin polyclonal antibody is a primary antibody, 1ml of the antibody is added into each hole, and the incubation is carried out for 1h at 37 ℃; incubating FITC-labeled mouse anti-rabbit IgG monoclonal antibody at 37 ℃ for 1h in a dark place; staining the core with 0.5 mu g/ml DAPI at room temperature for 5min in dark place; and (5) observing by an inverted fluorescence microscope in a dark condition and taking a picture. Results (FIG. 2) the green fluorescent signals of the secretionally expressed zymogen and enzyme were significant, mainly concentrated around the blue fluorescent-labeled nucleus; the green fluorescence of the cells expressed in the cells is scattered around the nucleus and tends to be homogeneous, and the brightness of the zymogen is equivalent to that of the enzyme fluorescence; the peripheral brightness of the outer layer of the transmembrane cell is gathered, and the situation that the green fluorescence is gathered on the cell membrane can be basically judged according to the cell form of the same visual field under a natural light mirror, and the distribution of the subcellular of each type of expression target protein accords with the expectation.

(5) And (3) pancreatin activity detection: the commercial TPCK-trypsin with known enzyme activity is taken as a positive standard substance, and a pancreatin specific fluorescent polypeptide substrate Boc-Leu-Gly-Arg-AMC with high sensitivity is selectedAccording to EvaAdding 50mmol/L Tris-HCl buffer solution with pH value of 7.9 into each well to carry out 2-time serial dilution on TPCK-trypsin and 50 mul of each fluorescence reaction substrate with final concentration of 25 mul mol/L, uniformly mixing, placing at 35 ℃ in a dark place to react for 30min, reading the value of each well by a fluorescence microplate reader, wherein the excitation wavelength is 355nm, and the emission wavelength is 440 nm. And (3) drawing an enzyme activity standard curve by taking the enzyme activity corresponding to each dilution of the positive standard substance as an abscissa and the corresponding average fluorescence value as an ordinate, detecting 6 expressed protein samples and controls by the same method, and substituting the expressed protein samples and controls into the standard curve to obtain the expressed pancreatin activity. As a result (FIG. 3), the activity of the secreted pancreatin was as high as 10.72X 10^-2U/ml, corresponding to 1/60 with 0.5. mu.g/ml TPCK-trypsin activity added when MDCK is used in laboratory to culture influenza virus, while the secretory supernatant trypsin activity is equivalent to the zymogen and enzyme activity expressed in cell, about 6X 10^-2U/ml, minimum of proenzyme and enzyme activity expressed across the membrane of about 3X 10^-2U/ml。

(6) Effect on virus growth: the MDCK cells in the logarithmic growth phase are taken to transiently transfect 6 expression plasmids according to the method, serum-free DMEM culture medium is replaced after the MDCK cells are transfected for 6 hours, the MDCK cells are cultured for 12 hours at 37 ℃, A/California/7/2009(H1N1) influenza virus vaccine strains are inoculated according to the moi equal to 0.001, the influenza virus vaccine strains are uniformly mixed and then placed in a 37 ℃ 4% CO2 incubator to be used for virus growth. Observing the growth condition of the virus under a microscope every day, repeatedly freezing and thawing the cells for 2 times to release the virus when the growth condition is 72 hours, centrifuging for 10min at 4 ℃ and 5000rpm to remove cell fragments, carrying out virus NA activity determination, diluting the sample and the NA substrate by MES buffer solution series in each hole of a 96-hole fluorescent ELISA plate for 50 mul respectively, uniformly mixing for reacting at room temperature for 30min, stopping the reaction by 150 mul stop solution, reading the fluorescence value of each hole by a fluorescent ELISA reader, exciting the wavelength to be 355nm, emitting the wavelength to be 460nm, repeating the steps for 3 times for each sample, and carrying out data statistical analysis. The results (fig. 4) show that the viral NA activity of the experimental group for secretory expression of trypsinogen is statistically different from that of the idle control group (P <0.05), wherein the viral NA activity of the experimental group for secretory expression of trypsinogen is 1340, while that of the idle control group is only 924, and the experimental group is about 1.5 times that of the control group; the amount of the influenza A virus cultured by the other 5 expression plasmids through transient MDCK is about 949-1000 compared with that of the idle control group. We therefore selected the secreted zymogen plasmid with the highest expression activity for subsequent selection of stably transfected cells.

EXAMPLE 2 screening and identification of monoclonal cells stably expressing secretogenic trypsinogen MDCK

(1) Selection of appropriate G418 concentration in screening medium: when MDCK cells are inoculated into a 24-well plate and the confluency degree is 70%, G418 is added, the concentration is sequentially 0 mug/ml, 200 mug/ml, 400 mug/ml, 600 mug/ml, 800 mug/ml, 900 mug/ml, 1000 mug/ml and 1100 mug/ml, each concentration of the MDCK cells is repeatedly cultured in a 37 ℃ and 5% CO2 culture box, the cell death condition is observed and recorded every day, the liquid is changed every 3-4 days, a cell survival curve is drawn after two weeks of culture to determine the minimum lethal concentration of G418, and as a result, when the G418 concentration is between 800 and 900 mug/ml, the cells are all killed in 14 days, therefore, 1000 mug/ml is selected as the MDCK cell pressure in the experiment to screen the concentration of G418 in the culture medium, and the concentration of the maintenance liquid is 500 mug/ml.

(2) G418 pressure screening and limiting dilution screening of monoclonal cells: inoculation about 2X10⁵MDCK cells were plated in 6-well plates and cultured to about 90% cell density for transfection. After 24 hours, the cells are digested, the cells are subcultured in a 96-well plate at a ratio of 1:10, 5% fetal bovine serum DMEM culture solution with 200 mu 11000 mu G/ml G418 is added to each well for continuous culture, the culture solution is changed every 3-5 days, and resistant cells can grow after continuous screening for 2 weeks. Control MDCK cells were cultured in G418-containing medium only until all cells were dead without transfection with empty plasmid, resistant cell colonies with good growth status in the experimental group were selected, and after digestion, the cells were further diluted to 0.5 cells/100. mu.l with pressure-screening medium containing 1000. mu.g/ml G418 and seeded in 96-well plates at 200. mu.l per well. After continuing the G418 screening for 2 weeks, using RT-PCR (extracting genome DNA, cell RNA and RT-PCR method respectively refer to the selected kit) and pancreatin activity detection method (described above) to select the clone with highest expression level and better cell state, and then carrying out single cell clone screening by the limiting dilution method. After the screened cells are digested by pancreatin, the cells are sequentially cultured in 96-well, 24-well and 6-well cell culture plates in an expansion way and are frozen and stored according to a conventional method. The results obtained are 5 positive monoclonal antibody cell strains, and the RT-PCR identification resultSpecific bands with the expected size of about 750bp can be amplified, and the determined sequences are consistent with the target gene in comparison. Combining with the pancreatin activity measurement, 21# cell strain with highest expression enzyme activity and better cell state is selected for subsequent experiments, and is named MDCK-S.pro-try (21), MT21 for short.

(3) Identification of MT21 cell line: continuously passaging the monoclonal cell, extracting cell genome DNA of different generations for carrying out PCR identification on the secretory bovine trypsinogram gene, wherein an upstream primer is Fs: 5'-TCCGGCTAGCGCCACCATGAAGACCTTC-3' (SEQ ID No.13), the downstream primer is R: 5'-GCCGGAATTCTTAGTTGGAGGCGATGG-3' (SEQ ID No.14), using the GAPDH gene as an internal control, a specific upstream primer F was designed: 5'-GTGATGCTGGTGCTGAGTATGTT-3' (SEQ ID No.15), the reverse primer R: 5'-GGTCTTCTGGGTGGCAGTGAT-3' (SEQ ID No.16), reaction system 50. mu.l: premix Ex Taq^TM2 XPCR Solution 25. mu.l, template 5. mu.l, primers 2. mu.l each, and deionized water 15. mu.l. Amplification conditions: pre-denaturation at 94 deg.C for 1min, denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 1min for 20 cycles, and final heat preservation at 72 deg.C for 10 min. Cell RNA was extracted for RT-PCR identification with the primers as above, 10. mu.l reaction:RT Master Mix 2. mu.l, template 2. mu.l, DEPC treated water 6. mu.l. RT reaction conditions: 15min at 37 ℃; 5s at 85 ℃; the PCR reaction system and conditions were the same as above using the reverse transcription cDNA as a template. The cell expression supernatant and cell lysate of each generation were collected, subjected to immunoblotting and indirect immunofluorescence assay, and the pancreatic activity of the expression supernatant was detected (same as in example 1). Results (fig. 5, 6): the MT21 was identified to demonstrate that the secretory pancreatin gene has been successfully integrated into the MDCK genome and is correctly transcribed (see FIG. 5); no obvious morphological difference is observed between MT21 and normal MDCK cells under a common microscope, indirect immunofluorescence shows that obvious green fluorescence is visible for MT21, and cells in a control group are negative (see figure 6A); western Blot identified that MT21 had a single distinct band of about 24kD, and that the intensity of the band in the supernatant was significantly higher than that of the cell lysate without any band in the normal MDCK control (see FIG. 6B); pancreatic enzyme Activity measurement result supernatantThe activity of the pancreatic enzyme is high, about 20X 10^-2U/ml, pancreatin activity in cells about 17X 10^-2U/ml, corresponding to 1/30 plus 0.5. mu.g/ml of TPCK-trypsin activity (see FIG. 6C); the monoclonal cell strain is continuously passed to the 15 th generation, PCR and RT-PCR identification prove that the target gene between each generation is stable in heredity, and the activity of the pancreatic enzyme for detecting and expressing the supernatant has no statistical difference, about 20 multiplied by 10^-2U/ml, stable expression of the protein of interest (see FIGS. 6D and 6E).

Example 3 proliferation of influenza viruses on MDCK cells stably expressing bovine trypsinogen

3 strains of wild influenza virus A/Tianjinnan/15/2009 (H1N1), A/Fujianggan/196/2009 (H3N2), B/Heilongjiang lily/116/2010 and 1 strain of influenza A virus are subjected to massive amplification by adopting a chick embryo allantoic cavity respectively, and allantoic fluid of the obtained virus is subpackaged after centrifugation and is stored at-70 ℃ for later use. For comparison, a monoclonal cell MT21 test group (MT21), a normal MDCK control group (control), an MT21 plus 0.5. mu.g/ml TPCK-trypsin test group (MT21+), and a normal MDCK plus 0.5. mu.g/ml TPCK-trypsin control group (control +) were set up. And respectively inoculating the monoclonal cell MT21 and the normal MDCK into a 6-hole cell culture plate until the confluence is 90%, sucking out cell culture solution, washing the cells for 3 times by using sterile PBS to remove FBS, replacing 1.5mL of 1% double antibody-containing serum-free DMEM (DMEM) and culturing for 24 hours at 37 ℃, and waiting for the expression and accumulation of the monoclonal cell zymogen. Inoculating influenza virus subtype at moi of 0.001 after 24 hr, adding pancreatin group, adding TPCK-pancreatin at final concentration of 0.5 μ g/ml, mixing each group with 500 μ l per well, and placing 4% CO at 37 deg.C₂And (5) incubator culture. Freezing and thawing the cells repeatedly for 2 times at 24h, 48h and 72h respectively to release the virus, centrifuging at 4 deg.C and 5000rpm for 10min to remove cell debris, collecting supernatant virus solution, and detecting NA activity and CCID of the virus respectively₅₀The titer.

The results (FIG. 7) showed that 3 of the 4 strains tested were more sensitive to MT21 cells, and both NA activity and virus titer were higher than those measured on MDCK cells, especially the B-type strain did not require the addition of TPCK treatment trypsin for 30h to achieve the same virus yield as that of normal MDCK with 0.5. mu.g/ml TPCK-trypsin, while the MT21 expression supernatant had trypsin activity of only 0.5. mu.g/ml TPCK-trypsinMu g/ml of 1/30 of TPCK-trypsin shows that the constructed stable cell line MT21 is more sensitive to influenza virus than normal MDCK, has different effects on different types or subtypes, and particularly has obvious effect on low-growth-strength strains. In addition, the virus yield of MT21 cell culture is remarkably improved after 0.5 mu g/ml TPCK-trypsin is added, and the virus titer can reach nearly 4 times of the yield of a normal MDCK control group to the maximum, for example, after the influenza B virus strain is inoculated, the virus titer is 7.9 multiplied by 10 after MT21 cell culture is carried out for 72 hours³TCID₅₀Viral titer 2.0X 10/ml, in the same time period as ordinary MDCK cultures³TCID₅₀Pro-try cell lines we constructed were initially better suited for influenza virus propagation than MDCK cells due to the secretory expression of trypsinogen.

Example 4 identification and use of codon-optimized monoclonal cell MTY6

We optimized the gene sequence of the coding region of the bovine trypsinogen gene using the on-line Codon optimization software OPTIMIZER according to the canis Codon Usage frequency table provided by the Codon Usage Database. Optimization changes only the gene sequence and does not change the amino acid sequence. The zymogen sequence has 741 bases and 246 amino acids, and 135 bases are changed after optimization, wherein 75 bases are converted (purine-purine, pyrimidine-pyrimidine) and 60 bases are converted (purine-pyrimidine). A total of 101 amino acids of codons were changed, with a codon change rate of 40%. The specific sequence structure is shown as SEQ ID No. 1:

ATGAAGACCTTCATCTTCCTGGCCCTGCTGGGCGCCGCCGTGGCCTTCCCCGTGGACGACGACGACAAGATCGTGGGCGGCTACACCTGCGGCGCCAACACCGTGCCCTACCAGGTGAGCCTGAACAGCGGCTACCACTTCTGCGGCGGCAGCCTGATCAACAGCCAGTGGGTGGTGAGCGCCGCCCACTGCTACAAGAGCGGCATCCAGGTGAGGCTGGGCGAGGACAACATCAACGTGGTGGAGGGCAACGAGCAGTTCATCAGCGCCAGCAAGAGCATCGTGCACCCCAGCTACAACAGCAACACCCTGAACAACGACATCATGCTGATCAAGCTGAAGAGCGCCGCCAGCCTGAACAGCAGGGTGGCCAGCATCAGCCTGCCCACCAGCTGCGCCAGCGCCGGCACCCAGTGCCTGATCAGCGGCTGGGGCAACACCAAGAGCAGCGGCACCAGCTACCCCGACGTGCTGAAGTGCCTGAAGGCCCCCATCCTGAGCGACAGCAGCTGCAAGAGCGCCTACCCCGGCCAGATCACCAGCAACATGTTCTGCGCCGGCTACCTGGAGGGCGGCAAGGACAGCTGCCAGGGCGACAGCGGCGGCCCCGTGGTGTGCAGCGGCAAGCTGCAGGGCATCGTGAGCTGGGGCAGCGGCTGCGCCCAGAAGAACAAGCCCGGCGTGTACACCAAGGTGTGCAACTACGTGAGCTGGATCAAGCAGACCATCGCCAGCAACTAA(SEQ IDNo.1)

the optimized bovine trypsinogen encoding gene was successfully transiently transferred into MDCK cells in the same manner as in examples 1 and 2, and then subjected to G418 pressure screening to obtain a monoclonal cell line with stable secretory expression, which was named MTY 6. After the samples were subjected to the enterokinase activation treatment in a lump before the measurement of the expressed enzyme activity, the activity of the expression of the target protein was increased by about 3 times (about 29.12U/ml) as compared with that of MT21 monoclonal cells (about 9.37U/ml) before the optimization according to the method for detecting the pancreatic activity in example 1 (FIG. 8).

When the influenza virus is cultured under the same conditions, the yield of MTY6 influenza virus cultured by the monoclonal cell can reach about 5 times of that of the common MDCK, for example, the NA activity of the virus obtained by culturing H3N2 subtype influenza virus for 72H under the condition of not adding trypsin, the NA activity of MTY6 monoclonal cell cultured virus is 830, while the NA activity value of the virus cultured by the normal MDCK cell is only 173; compared with the MT21 monoclonal cell before optimization, MTY6 is more sensitive to influenza virus, the difference is more obvious especially when 0.5 mu g/mL of TPCK-Trypsin is added, no matter whether the TPCK-Trypsin is added or not, the MTY6 culture virus yield is about 2 times of the MT21 (see figure 9), when pancreatin is added to culture H1N1 influenza virus, the virus yield is accumulated continuously with time, the virus yield is obviously increased when 72 hours exist, the NA value of MTY6 cell is about 800, and the NA value of MT21 cell is about 220; when the H3N2 influenza virus is cultured without additional pancreatin, the virus yield is accumulated with time, and is obviously increased to 72 hours, the NA value of the virus yield of MTY6 cells is about 180, and the NA value of the virus of MT21 cells is about 80. In conclusion, the MTY6 cell line constructed by the method is more suitable for the proliferation of influenza viruses than MDCK cells due to the secretion and expression of trypsinogen, and has application value in cell influenza culture and related vaccine production. Meanwhile, the kit becomes a convenient tool for separating and culturing influenza virus, monitoring virus variation, screening antiviral drugs, determining neutralizing antibodies and the like in clinical or experimental research, and has wide application prospect.

Sequence listing

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<120> MDCK cell line for stably expressing bovine trypsinogen and application thereof

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<170> Patent In version 3.3

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atgaagacct tcatcttcct ggccctgctg ggcgccgccg tggccttccc cgtggacgac 60

gacgacaaga tcgtgggcgg ctacacctgc ggcgccaaca ccgtgcccta ccaggtgagc 120

ctgaacagcg gctaccactt ctgcggcggc agcctgatca acagccagtg ggtggtgagc 180

gccgcccact gctacaagag cggcatccag gtgaggctgg gcgaggacaa catcaacgtg 240

gtggagggca acgagcagtt catcagcgcc agcaagagca tcgtgcaccc cagctacaac 300

agcaacaccc tgaacaacga catcatgctg atcaagctga agagcgccgc cagcctgaac 360

agcagggtgg ccagcatcag cctgcccacc agctgcgcca gcgccggcac ccagtgcctg 420

atcagcggct ggggcaacac caagagcagc ggcaccagct accccgacgt gctgaagtgc 480

ctgaaggccc ccatcctgag cgacagcagc tgcaagagcg cctaccccgg ccagatcacc 540

agcaacatgt tctgcgccgg ctacctggag ggcggcaagg acagctgcca gggcgacagc 600

ggcggccccg tggtgtgcag cggcaagctg cagggcatcg tgagctgggg cagcggctgc 660

gcccagaaga acaagcccgg cgtgtacacc aaggtgtgca actacgtgag ctggatcaag 720

cagaccatcg ccagcaacta a 741

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<213> Artificial sequence

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tccggctagc gccaccatga agaccttc 28

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tccggctagc gccaccatga agaccttcat ctttctggct ctcttgggag ccgctgttgc 60

tatcgtgggc ggctacacct g 81

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tccggctagc gccaccatgt tccccgtgga cgatgatgac aag 43

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<213> Artificial sequence

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tccggctagc gccaccatga tcgtgggcgg ctacac 36

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<213> Artificial sequence

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gccggaattc ttagttggag gcgatgg 27

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<213> Artificial sequence

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tccggctagc gccaccatgt ataggccagc acgtg 35

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<213> Artificial sequence

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gtcatcatcg tccacgggga aaaaagctaa aaag 34

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<212> DNA

<213> Artificial sequence

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ctttttagct tttttccccg tggacgatga tgac 34

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<213> Artificial sequence

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gtgtagccgc ccacgataaa agctaaaaag 30

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<212> DNA

<213> Artificial sequence

<400> 11

ctttttagct tttatcgtgg gcggctacac 30

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<211> 27

<212> DNA

<213> Artificial sequence

<400> 12

gccggaattc ttagttggag gcgatgg 27

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<211> 28

<212> DNA

<213> Artificial sequence

<400> 13

tccggctagc gccaccatga agaccttc 28

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<211> 27

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<213> Artificial sequence

<400> 14

gccggaattc ttagttggag gcgatgg 27

<210> 15

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<212> DNA

<213> Artificial sequence

<400> 15

gtgatgctgg tgctgagtat gtt 23

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ggtcttctgg gtggcagtga t 21

Claims (8)

1. The modified MDCK cell line for expressing the secretory bovine trypsinogen is characterized by comprising a complete coding gene of the secretory bovine trypsinogen S.pro-try integrated into a cell genome of the cell line, wherein a Kozak sequence is added in front of the coding gene sequence of the S.pro-try, and codon optimization design suitable for dogs is carried out on a coding region of a nucleic acid sequence, the coding gene sequence of the S.pro-try is shown as SEQ ID No.1, the cell line is MTY6 and is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 12681.

2. The use of the cell line of claim 1, wherein said use comprises the use of the modified MDCK cell line in the culture of influenza virus, in the preparation of influenza vaccines, in screening of anti-influenza drugs, in neutralizing antibody assays or in other related fields of influenza research.

3. The use of claim 2, wherein the influenza virus is a human influenza virus.

4. A method of culturing influenza virus, wherein the influenza virus is inoculated into the cell line of claim 1 under suitable conditions.

5. The method of claim 4, wherein the influenza virus is a human influenza virus.

6. A method of producing an influenza vaccine, wherein influenza virus is inoculated into the cell line of claim 1 under suitable conditions.

7. The method of claim 6, wherein the influenza virus is a human influenza virus.

8. The method according to any one of claims 4 to 7, wherein no TPCK-pancreatin is added in the step of propagating influenza virus in the method, or further TPCK-pancreatin is added in the step of propagating influenza virus in the method.