CN117625516A - Cell strain highly sensitive to C1-233 strain replication, screening method and application - Google Patents

Abstract

The invention discloses a cell strain which is highly sensitive to C1-233 replication, a screening method and application thereof, and belongs to the technical field of biological products for animals. The invention discloses a high-sensitivity monoclonal cell strain PK-15B6E7 for copying chimeric porcine circovirus live vaccine strain, the preservation number is CGMCC N _O .45729. The PK-15B6E7 strain and the parent PK-1After the 5B6 cell strain is inoculated with the C1-233 strain respectively, the proliferation titer of the C1-233 strain in PK-15B6E7 cells is about 10 times higher than that of parent cells, which indicates that the PK-15B6E7 cells are more sensitive to the replication of the C1-233 strain and the replication efficiency of viruses is higher. The PK-15B6E7 monoclonal cell strain disclosed by the invention is suitable for the industrial production of chimeric porcine circovirus live vaccines, improves the production efficiency and further reduces the vaccine production cost.

Description

Cell strain highly sensitive to C1-233 strain replication, screening method and application

Technical Field

The invention belongs to the technical field of biological products for livestock, and particularly relates to a cell strain which is highly sensitive to C1-233 replication, a screening method and application.

Background

Porcine circovirus disease (porcine circovirus-associated disease, PCVAD) is a disease which seriously harms the pig industry worldwide and brings great economic loss to pig industry in various countries. Porcine circovirus (porcine circovirus, PCV) belongs to the family of circoviridae, members of the genus circoviridae, genotypes are largely divided into four types PCV1 (porcine circovirus type, PCV 1), PCV2 (porcine circovirus type 2, PCV 2), PCV3 (porcine circovirus type 3, PCV 3) and PCV4 (porcine circovirus type 3, PCV 3), wherein PCV2 is the major etiology of weaned pig multisystemic wasting syndrome (postweaning multisystemic wasting syndrome, PMWS), pigskin inflammation and nephrotic syndrome (porcine dermatitis and nephropathy syndrome, PDNS), porcine respiratory disease complex (porcine respiratory disease complex, PRDC).

To date, no specific drug has been used to treat this disease, and vaccine immunization is considered to be the most effective prevention and control method. With the use of PCV2 vaccine, the prevention and control effect on PCVAD is obvious clinically. However, commercial porcine circovirus vaccines are mainly whole virus inactivated vaccines and subunit vaccines. The commercial whole virus inactivated vaccine has poor immune protection effect, large immune dosage and short protection period, and has the potential risk of incomplete inactivation; subunit vaccines also have the problems of slow production of protective antibodies, high production cost, easy adverse reaction caused by added adjuvants and the like, and both mainly induce humoral immunity of pig bodies. The chimeric porcine circovirus live vaccine (C1-233 strain) developed in the early stage of the applicant can induce cellular immunity and humoral immunity of organisms at the same time, and has excellent virus removal function and can obviously reduce the horizontal transmission risk of PCV2 compared with commercial vaccines after the immune pigs are detoxified. However, in the production of chimeric porcine circovirus live vaccines (strain C1-233), the viral replication titer was low, which is also a problem faced in the development of PCV2 whole virus inactivated vaccines. Therefore, there is an urgent need to obtain a method and cell strain having high sensitivity to replication of chimeric porcine circovirus live vaccine C1-233 strain.

Disclosure of Invention

The invention aims to provide a cell strain which is highly sensitive to the replication of a C1-233 strain, a screening method and application thereof, wherein the C1-233 strain has high proliferation titer in PK-15B6E7 cells, the PK-15B6E7 cells are more sensitive to the replication of the C1-233 strain, and the replication efficiency of viruses is higher.

The invention provides a method for replicating a highly sensitive cell strain PK-15B6E7 of a chimeric porcine circovirus live vaccine strain C1-233, wherein the monoclonal cell strain is preserved with the preservation number of CGMCC N _O .45729。

The invention also provides a screening method of the cell strain PK-15B6E7, which comprises the following steps: (1) Recovering the parent PK-15B6 cells without PCV1 pollution, and then subcloning to obtain monoclonal cell strains;

(2) And screening out a monoclonal cell strain which is highly sensitive to the replication of the chimeric porcine circovirus live vaccine strain C1-233 from the monoclonal cell strain, so as to obtain the cell strain PK-15B6E7.

Preferably, the method of subcloning the cells in step (1) comprises limiting dilution.

Preferably, the limiting dilution method comprises: 1) Recovering the parent PK-15B6 cells without PCV1 pollution, culturing until the cell confluence reaches 70% -80%, and digesting the cells into single cells by pancreatin; mixing a proper amount of cells with a DMEM culture medium containing 20% of fetal calf serum, and placing the mixture in a cell culture plate, wherein each hole of the cell culture plate contains 0.5-0.8 cell/100 mu L;

2) Marking the hole containing the monoclonal cells according to the growth condition of the cells in each hole, and performing expansion culture when the cells in the hole grow to 1/4-1/3 of the bottom of the hole.

Preferably, the method of screening in step (2) comprises direct immunofluorescence.

Preferably, the direct immunofluorescence method comprises: a) Inoculating PK-15B6 cells and monoclonal cell strains subjected to subcloning and expansion culture to a cell culture plate for culture, and discarding a cell culture medium when the cells grow to 60-70% confluence;

b) Inoculating C1-233 strain virus liquid into each hole of the cell culture plate in the step a), adsorbing the virus, discarding the liquid and culturing;

c) After methanol fixation, incubating with fluorescent labeled monoclonal antibodies against PCV2 Cap protein, judging positive cells according to fluorescence, and screening monoclonal cell strains susceptible to replication against C1-233 strains.

Preferably, the mixed inoculation in step a) is performed in an inoculum size of 2X 10 ⁴ cells/wells.

Preferably, the C1-233 strain broth in step b) is diluted with serum-free DMEM medium and inoculated at moi=0.01;

preferably, the liquid is discarded and the culture medium is supplemented with DMEM medium containing 2% fetal calf serum.

Preferably, the mab in step c) comprises mab 2A5 against PCV2 Cap protein;

preferably, the mab is labeled with FITC.

The invention also provides application of the cell strain PK-15B6E7 in passaging, expanding propagation and/or proliferation of chimeric porcine circovirus live vaccine strain C1-233.

The beneficial effects are that: the invention provides a method for copying a highly sensitive cell strain PK-15B6E7 to a chimeric porcine circovirus live vaccine strain C1-233, and when the PK-15B6E7 is screened, the PK-15B6 parent cell preserved in a laboratory is firstly confirmed to contain no PCV1 virus, so that the pollution of PCV1 is avoided. Obtaining a plurality of monoclonal cell strains; after the screened cell lines are subjected to expansion culture, the sensitivity of the cell lines to replication of the C1-233 strain is detected by direct immunofluorescence. Finally obtaining a cell strain PK-15B6E7, PK-15B6E7 cells with uniform morphology and good growth performance aiming at the C1-233 strain replication sensitivity, and carrying out passage to F ₃₀ The characteristic of high sensitivity to C1-233 strain replication is still maintained, and the obtained virus titer is about 10 times higher than that of the parent cell. The cell strain is regulated by the pharmacopoeia of animal of the people's republic of China (2020 edition)The purity test shows that the cell strain has no bacterial, fungal, mycoplasma and exogenous virus pollution. The PK-15B6E7 has important significance for the industrialized production of chimeric porcine circovirus live vaccines, cost reduction and synergy, and simultaneously provides new power for the prevention and control of PCVAD. The PK-15B6E7 strain is suitable for the industrial production of chimeric porcine circovirus live vaccines, improves the production efficiency, and is expected to further reduce the vaccine production cost.

Biological preservation information

The monoclonal cell strain PK-15B6E7 is preserved in 2023 and 10 and 11 days to China general microbiological culture Collection center (CGMCC), and the specific preservation address is the North Chenxi Lu No. 1 and No. 3 in the Chaoyang area of Beijing city, and the preservation number is CGMCC No.45729.

Drawings

FIG. 1 is a graph showing the results of direct immunofluorescence detection of PK-15B6 and PK-15B6E7 monoclonal cell lines infected with C1-233 strain, and is annotated: PK-15B6 cells; PK-15B6E7 cells;

FIG. 2 is a graph showing TCID determination after infection of PK-15B6 and PK-15B6E7 cells by C1-233 strain ₅₀ Partial pictures in time;

FIG. 3 is a graph showing the results of direct immunofluorescence assay of PK-15B6 and PK-15B6E7 after culturing C1-233 strains in different cell lines and collecting 10-fold dilution of virus liquid, wherein PK-15B6 cells are shown in the graph A; PK-15B6E 7F 10 cells; PK-15B6E 7F 15 cells; PK-15B6E 7F 20 cells; PK-15B6E 7F 25 cells; f, PK-15B6E 7F 30 cells;

FIG. 4 shows the growth curves of PK-15B6 and PK-15B6E7 cells.

Detailed Description

The invention provides a method for replicating a highly sensitive cell strain PK-15B6E7 of a chimeric porcine circovirus live vaccine strain C1-233, wherein the monoclonal cell strain is preserved with the preservation number of CGMCC N _O .45729。

After the PK-15B6E7 strain and the parent PK-15B6 cell strain are inoculated with the C1-233 strain in various modes, the proliferation titer (half tissue infection amount, TCID) of the C1-233 strain in the PK-15B6E7 cells ₅₀ ) Are about 10 times higher than the parent cells, indicating PK-15B6E7Cells are more sensitive to replication of the C1-233 strain, and the replication efficiency of viruses is higher.

The invention also provides a screening method of the cell strain PK-15B6E7, which comprises the following steps: (1) Recovering the parent PK-15B6 cells without PCV1 pollution, and then subcloning to obtain monoclonal cell strains;

(2) And screening out a monoclonal cell strain which is highly sensitive to the replication of the chimeric porcine circovirus live vaccine strain C1-233 from the monoclonal cell strain, so as to obtain the cell strain PK-15B6E7.

The present invention preferably performs cell subcloning by limiting dilution methods, preferably comprising: 1) Recovering the parent PK-15B6 cells without PCV1 pollution, culturing until the cell confluence reaches 70% -80%, and digesting the cells into single cells by pancreatin; mixing a proper amount of cells with a DMEM culture medium containing 20% of fetal calf serum, and placing the mixture in a cell culture plate, wherein each hole of the cell culture plate contains 0.5-0.8 cell/100 mu L;

2) And marking the hole containing the monoclonal cells according to the growth condition of the cells in each hole, and performing expansion culture when the cells in the hole grow to 1/4-1/3 of the bottom of the hole, so as to be used for detecting the replication sensitivity of the C1-233 strain by a subsequent direct immunofluorescence method.

The method of screening in step (2) of the present invention preferably comprises a direct immunofluorescence method, preferably comprising: a) Inoculating PK-15B6 cells and monoclonal cell strains subjected to subcloning and expansion culture to a 96-well plate for culture, and discarding a cell culture medium when the cells grow to 60-70% confluence;

b) Inoculating C1-233 strain virus liquid into each hole of the cell culture plate in the step a), adsorbing the virus, discarding the liquid and culturing;

c) After methanol fixation, incubating with fluorescent labeled monoclonal antibodies against PCV2 Cap protein, judging positive cells according to fluorescence, and screening monoclonal cell strains susceptible to replication against C1-233 strains.

The invention preferably uses 2X 10 after expanding the PK-15B6 cells and subcloned monoclonal cell lines ⁴ cells/well were seeded in 96 well cell plates and placed in 5% CO ₂ In an incubator at 37 DEG CCulturing for 24-36 h. After the cells grew to about 70% confluence, the cell culture medium was discarded and washed 3 times with PBS, C1-233 strain virus liquid diluted with DMEM medium without serum was inoculated into the wells at moi=0.01, placed in an incubator to adsorb the virus for 1.5 hours, and then the liquid in the wells was discarded, and DMEM medium with 2% fetal bovine serum was supplemented for maintenance culture for 72 hours. After the culture was completed, the maintenance solution was discarded, washed 3 times with PBS, and then the mixture was dried by shaking, 150. Mu.L/well of precooled methanol was added, and the mixture was fixed in a refrigerator at 4℃for 15 minutes. After fixation, the plate was discarded, washed 3 times with PBS, and then the plate was dried by pipetting, and FITC-labeled monoclonal antibody (hereinafter abbreviated as MAB) 2A5 against PCV2 capsid protein (Cap protein) was added in a 1:400 dilution, 50. Mu.L/well, and incubated in an incubator at 37℃for 1 hour in the absence of light. After incubation, the plate was discarded, washed 3 times with PBS, and then the plate was dried, and the 96-well plate was observed under a fluorescence microscope, photographed, and recorded.

The monoclonal antibody 2A5 is developed and stored by the laboratory, and specific information is disclosed in Chinese patent CN 115725510A.

The invention also provides application of the cell strain PK-15B6E7 in passaging, expanding propagation and/or proliferation of chimeric porcine circovirus live vaccine strain C1-233.

In the embodiment of the invention, after the PK-15B6E7 strain and the parent PK-15B6 cell strain are inoculated with the C1-233 strain in various modes, the proliferation titer (half tissue infection amount, TCID) of the C1-233 strain in the PK-15B6E7 cells ₅₀ ) All are about 10 times higher than the parent cells, which indicates that PK-15B6E7 cells are more sensitive to the replication of C1-233 strain and the replication efficiency of viruses is higher. The PK-15B6E7 monoclonal cell strain disclosed by the invention is suitable for the industrial production of chimeric porcine circovirus live vaccines, improves the production efficiency, and is expected to further reduce the vaccine production cost.

In order to further illustrate the present invention, a replication-competent cell strain of C1-233, and a screening method and application thereof, provided by the present invention, will be described in detail with reference to examples, which should not be construed as limiting the scope of the present invention.

Example 1

1. Materials and methods

1.1 materials

1.1.1 cells and strains

PK-15B6 cells were kept in this laboratory. Chimeric porcine circovirus strain C1-233 has been disclosed in Chinese patent CN106834242A.

1.1.2 major reagents

Fetal bovine serum, available from Lonsera company (cat# S711-001S); dulbecco's Modified Eagle's Medium-high glucose (DMEM) available from Sigma company (cat# D5648); FITC fluorescein labeled monoclonal antibody 2A5 against PCV2 Cap protein was studied and prepared by the present laboratory (working concentration 1:400); CCK-8 kit was purchased from Biyun Tian Biotechnology Co., ltd (cat# C0041); 2 XTaqMastermix (cat# P112) and Mycoplasma contamination rapid detection reagent (cat# D101-01) were purchased from Northenan Biotechnology Co., ltd.

2. Method of

2.1 Method for obtaining PK-15B6 monoclonal cells (limiting dilution method)

(1) The PK-15B6 cells which are free of PCV1 pollution and good in state are inoculated into a T25 cell bottle, after the cell confluency reaches 70% -80%, PBS is used for 3 times, 1mL of 0.25% pancreatin is added, standing and digestion are carried out for about 5min, pancreatin is removed, 5mL of DMEM (DMEM medium containing 4% fetal bovine serum) is added for stopping digestion, then cells are repeatedly blown and dispersed into single cells, a cell counter is used for counting, a proper amount of cells are taken and added into the DMEM medium containing 20% fetal bovine serum, and the cells are limited and diluted into a 96-well plate, so that each well contains 0.5-0.8 cells, namely 0.5-0.8 cells/100 mu L.

(2) And observing the growth condition of the cells under a microscope about day 5 after subcloning, namely, whether monoclonal cell strains exist or not, marking a hole containing the monoclonal cells, and performing expansion culture when the cells grow to 1/4-1/3 of the bottom of the hole, so as to be used for detecting the replication sensitivity of the C1-233 strain by a subsequent direct immunofluorescence method.

2.2 screening for C1-233 replication-insensitive monoclonal cell lines

(1) Respectively digesting PK-15B6 cells with cell confluence of 70% -80% and subcloned monoclonal cell strain with 0.25% pancreatin, blowing and mixing with DMEM culture medium containing 4% fetal bovine serum uniformly, and mixing with 2×10 ⁴ cell/well inoculationIn 96-well cell plates, 5% CO ₂ Culturing in an incubator at 37 ℃ for 24-36 hours.

(2) After the cells grew to about 70% confluence, the cell culture medium was discarded and washed 3 times with PBS, the C1-233 strain virus liquid diluted with serum-free DMEM medium was inoculated into the plates at moi=0.01, placed in an incubator to adsorb the virus for 1.5 hours, then the liquid in the wells was discarded, and the DMEM medium containing 2% fetal bovine serum was supplemented for maintenance culture for 72 hours. The number of positive cells is observed by a direct immunofluorescence method, and monoclonal cell strains which are susceptible to replication aiming at the C1-233 strain are screened.

2.3 direct immunofluorescence assay

(1) The maintenance solution is discarded from the 96-well plate, washed 3 times with PBS, then the mixture is patted dry, 150 mu L/well of precooled methanol is added, and the mixture is fixed for 15min in a refrigerator at 4 ℃.

(2) After fixation, the plate was discarded, washed 3 times with PBS and then patted dry, FITC-labeled PCV2 Cap protein mab 2A5 was added at 1:400 dilution, 50. Mu.L/well, and incubated in an incubator at 37℃for 1h in the absence of light.

(3) After the incubation, the liquid in the plate is discarded, the plate is washed for 3 times by PBS and then is patted dry, and the 96-well plate is placed under a fluorescence microscope for observation, photographing and recording.

According to the invention, monoclonal cells obtained by a limiting dilution method are subjected to direct immunofluorescence detection, screening is performed in a manner of observing the number of positive cells, and a PK-15B6E7 cell with high replication sensitivity aiming at a C1-233 strain is screened as shown in a figure 1.

2.4 Proliferation effect detection of PK-15B6E7 cell strain on C1-233 strain

2.4.1 Detection of replication effect of PK-15B6E7 cell strain on C1-233 strain

(1) PK-15B6 and PK-15B6E7 cells were isolated at 4X 10 ⁴ cells/well were seeded in 96 well cell plates and placed in 5% CO ₂ Culturing in an incubator at 37 ℃ for about 12 hours.

(2) 6 1.5mL EP tubes were placed on a tube rack, 900. Mu.L of serum-free DMEM medium was added to each tube, and 100. Mu. L C1 strain 1-233 virus liquid (TCID) ₅₀ ＝1×10 ^-4.6 Per mL) was added to the 1 st EP tube, thoroughly mixed by shaking, and then 10 was aspiratedmu.L was added to the 2 nd EP tube and so on, diluted to the 6 th tube.

(3) After cells in 96-well plates grew to about 70% confluency, cell culture media were discarded and washed 3 times with PBS, the viral fluid diluted in (2) was inoculated sequentially into the plates, 8 duplicate wells were set for each dilution, 100 μl was added per well, and positive and negative controls were set.

(4) The 96-well plate was placed at 37℃in 5% CO ₂ Incubator incubate for 1.5h, then discard the liquid in the wells and supplement DMEM with 2% fetal bovine serum for 72h.

(5) After the culture is finished, detecting by a direct immunofluorescence method and calculating TCID of the virus by using a Reed-Muench method ₅₀ 。

TCID of C1-233 strain in PK-15B6 cells was calculated ₅₀ ＝1×10 ^-4.5 TCID in PK-15B6E7 cells/mL ₅₀ ＝1×10 ^-5.6 As shown in FIG. 2, the viral titer of the C1-233 strain replicated in PK-15B6E7 cells was about 10-fold higher than that of the parent cells.

2.4.2 Detection of proliferation effect of PK-15B6E7 cell strain on passage expansion of C1-233 strain

(1) And respectively inoculating PK-15B6 and PK-15B6E7 cells into T25 cell bottles, when the cells grow to 65-75% confluence, rinsing with PBS for 3 times, and discarding the PBS for later use.

(2) Inoculating the virus liquid of the C1-233 strain into a T25 cell bottle according to MOI=0.02 virus amount, placing the T25 cell bottle in an incubator to adsorb for 1.5h, directly supplementing DMEM (medium-electron microscope) with 2% fetal bovine serum to 5mL after the adsorption is finished, and placing the T25 cell bottle in the incubator to culture for 72h for passage.

(3) After culturing for 72h, the supernatant is harvested and kept below-70 ℃ for later use. After washing 3 times with PBS, the cells are digested with 0.25% pancreatin, the digestion is stopped by adding DMEM culture solution containing 4% fetal calf serum, the cells are gently blown to disperse, the cells are passaged according to the proportion of 1:3, after culturing for 72 hours, the cell bottles are repeatedly frozen and thawed 3 times, and then centrifuged, and the supernatant is collected.

(4) PK-15B6 was inoculated into 96-well cell plates and the TCID of the supernatant collected was assayed by direct immunofluorescence as described above ₅₀ 。

PK-15B6 and PK-15Inoculating B6E7 cells into T25 cell bottle, subjecting C1-233 strain to passage and virus expansion, and performing TCID ₅₀ And (5) detecting. The results are shown in Table 1, where the viral titer in PK-15B6E7 cells was about 10-fold higher than in the parent cells, regardless of the first-generation cell supernatant or the second-generation freeze-thaw cell supernatant.

TABLE 1 proliferation effect of PK-15B6 and PK-15B6E7 cells on C1-233 strain passaging and expanding

2.5 Proliferation stability detection of PK-15B6E7 cell strain on C1-233 strain

The 10 th, 15 th, 20 th, 25 th and 30 th generation cells of PK-15B6E7 cell strain are respectively inoculated with the C1-233 strain, and after the cells are cultured for 72 hours, the cells are repeatedly frozen and thawed for 3 times to collect virus liquid, and the stability of the proliferation of the C1-233 strain of the cell strain in different generations is detected by a direct immunofluorescence method.

The PK-15B6E7 cells of different generations are respectively inoculated with the C1-233 strain, and the collected virus liquid is detected by a direct immunofluorescence test, so that the result is shown in figure 3, and the proliferation effect of the PK-15B6E7 cells of different generations on the C1-233 strain is not obviously different.

2.6 PK-15B6E7 cell strain growth rate detection

PK-15B6 and PK-15B6E7 cells were cultured in 1X 10 cells ⁴ The cells/wells were inoculated into 96-well plates and incubated with DMEM containing 4% fetal calf serum at 37℃with 5% CO ₂ Culturing in an incubator for 72 hours. At intervals of 12h, 10. Mu.L of CCK-8 was added to each well, incubated for 1h, and OD was measured with an ELISA reader ₄₅₀ And 6 wells were set for each group and the detection was continued for 72h. The absorbance values are plotted on the vertical axis and the time on the horizontal axis.

The growth rates of PK-15B6 and PK-15B6E7 cells were examined by CCK-8 kit, and as shown in FIG. 4, the growth rates of PK-15B6E7 cells were significantly slower than those of PK-15B6 cells (P < 0.01).

2.7 Purity detection of PK-15B6E7 cell strain

PK-15B6E7 cells are taken, DNA extraction is carried out according to a method of virus DNA extraction, then PCR detection is carried out on viruses such as PCV1, PCV2, PCV3, PPV, PRV, CSFV and the like, and positive and negative controls are set. Cell supernatants were added to sterile assay medium for detection. And meanwhile, taking a proper amount of cell supernatant according to the operation instruction of the mycoplasma pollution rapid detection kit, and determining whether the cell has mycoplasma pollution.

TABLE 2 PCR primer sequences

Wherein, the PCR reaction system is a 25 mu L system: 2 XTaqMastermix 12.5. Mu.L, forward primer (10. Mu.M) 1. Mu.L, reverse primer (10. Mu.M) 1. Mu. L, cDNA 1. Mu.L and ddH ₂ O 9.5μL；

Reaction procedure for DNA viruses (except ASFV): pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 1min,30 cycles; extending at 72 ℃ for 7min;

reaction procedure for ASFV: pre-denaturation at 95℃for 10min; denaturation at 95℃for 15s, annealing at 62℃for 30s, extension at 72℃for 30s,40 cycles; and then the extension is carried out for 7min at 72 ℃.

The PCR results show that the detection of exogenous viruses of PK-15B6E7 cells is negative. The sterility test result shows that the bacteria and mould pollution is avoided, and the mycoplasma detection is negative.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (10)

1. A strain PK-15B6E7 which replicates a chimeric porcine circovirus live vaccine strain C1-233 and is highly sensitive to cells is characterized by having a preservation number of CGMCC N _O .45729。

2. The method for screening cell strain PK-15B6E7 according to claim 1, comprising the steps of: (1) Recovering the parent PK-15B6 cells without PCV1 pollution, and subcloning to obtain monoclonal cell strains;

(2) And screening out a monoclonal cell strain which is highly sensitive to the replication of the chimeric porcine circovirus live vaccine strain C1-233 from the monoclonal cell strain, so as to obtain the cell strain PK-15B6E7.

3. The method of claim 2, wherein the method of subcloning cells of step (1) comprises limiting dilution.

4. A screening method according to claim 3, wherein the limiting dilution method comprises: 1) Recovering the parent PK-15B6 cells without PCV1 pollution, culturing until the cell confluence reaches 70% -80%, and digesting the cells into single cells by pancreatin; mixing a proper amount of cells with a DMEM culture medium containing 20% of fetal calf serum, and placing the mixture in a cell culture plate, wherein each hole of the cell culture plate contains 0.5-0.8 cell/100 mu L;

2) Marking the hole containing the monoclonal cells according to the growth condition of the cells in each hole, and performing expansion culture when the cells in the hole grow to 1/4-1/3 of the bottom of the hole.

5. The method of screening according to claim 2, wherein the method of screening in step (2) comprises direct immunofluorescence.

6. The screening method of claim 4 or 5, wherein the direct immunofluorescence method comprises: a) Inoculating PK-15B6 cells and monoclonal cell strains subjected to subcloning and expansion culture to a cell culture plate for culture, and discarding a cell culture medium when the cells grow to 60-70% confluence;

b) Inoculating C1-233 strain virus liquid into each hole of the cell culture plate in the step a), adsorbing the virus, discarding the liquid and culturing;

c) After methanol fixation, incubating with fluorescent labeled monoclonal antibodies against PCV2 Cap protein, judging positive cells according to fluorescence, and screening monoclonal cell strains susceptible to replication against C1-233 strains.

7. The method according to claim 6, wherein the mixed inoculation in step a) is performed in an amount of 2X 10 ⁴ cells/wells.

8. The screening method according to claim 6, wherein the C1-233 strain of virus solution in step b) is diluted with serum-free DMEM medium and inoculated at moi=0.01;

preferably, the liquid is discarded and the culture medium is supplemented with DMEM medium containing 2% fetal calf serum.

9. The screening method of claim 6, wherein the mab in step c) comprises mab 2A5 against PCV2 Cap protein;

preferably, the mab is labeled with FITC.

10. Use of the cell strain PK-15B6E7 according to claim 1 for passaging, propagating and/or proliferating chimeric porcine circovirus live vaccine strain C1-233.