CN111454910A

CN111454910A - Suspension PK15 cell line expressing porcine CD163 and CD169 molecules

Info

Publication number: CN111454910A
Application number: CN202010179414.0A
Authority: CN
Inventors: 方华明; 张冬冬; 欧仁芳; 刘小方; 潘倩
Original assignee: Ma'anshan Shiji Animal Health Management Co ltd; Shiji Biotechnology Nanjing Co ltd; Chengdu Tecbond Biological Products Co ltd
Current assignee: Chengdu Shiji Biopharmaceutical Co ltd
Priority date: 2020-03-13
Filing date: 2020-03-13
Publication date: 2020-07-28

Abstract

The invention discloses a suspended PK15 cell line for expressing porcine CD163 and CD169 molecules and a preparation method thereof, belonging to the technical field of transgenosis. The method of the invention has the following steps: 1) constructing a recombinant plasmid; 2) lipofection of PK15 cells; 3) screening puromycin; 4) and (5) carrying out suspension culture and domestication on a serum-free culture medium. The cell line prepared by the method is very sensitive to the porcine reproductive and respiratory syndrome virus, can be used for amplifying and propagating a large amount of porcine reproductive and respiratory syndrome virus, has low cost and has good application prospect in vaccine production practice.

Description

Suspension PK15 cell line expressing porcine CD163 and CD169 molecules

Technical Field

The invention belongs to the technical field of transgenosis.

Background

Porcine Reproductive and Respiratory Syndrome (PRRS) is an epidemic disease in pigs caused by the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the major symptoms of which are reproductive disorders in pregnant sows and pulmonary inflammation. The disease is introduced into China in 1996, outbreaks and epidemics occur all over the country, and due to the fact that PRRSV is fast in genetic variation and recombination, the disease becomes a worldwide significant disease nowadays, and great economic losses are caused to pig raising.

PRRSV is classified as a positive-strand RNA virus of the genus arterivirus of the family arterivirus, the family capsulaceae. The PRRSV particles are spherical, the diameter is about 50-65 nm, the nucleocapsid is icosahedral symmetrical, the diameter is about 30-35 nm, and the PRRSV mainly infects mononuclear/macrophage by strict cell tropism. Porcine Alveolar Macrophage (PAM) is the most main target cell of PRRSV, but the PAM cell is not suitable for subculturing PRRSV, and the virus used by the PRRSV vaccine at present is cultured from African green monkey kidney cell Marc145, but the PRRSV virus cultured by the Marc145 cell has low titer, and only can be cultured by adherence or microcarrier suspension, so that the requirements on PRRSV amplified production and cost control are high.

The PK15 cell is a porcine kidney cell, can be applied to the culture of certain porcine viruses (such as porcine circovirus type 2), but has low sensitivity to PRRSV, and is not suitable for PRRSV amplification culture.

Disclosure of Invention

In order to obtain a cell line which is low in cost and can produce PRRSV in a large scale, the invention provides the following technical scheme:

a method for preparing a PK15 cell line expressing porcine CD163 and CD169 and capable of suspension culture in serum-free medium, comprising the steps of:

1) construction of recombinant plasmid: connecting the CD169 gene to a PB513B vector to obtain a PB513B-CD169 plasmid, and connecting the CD163 gene to a PB513B-CD169 plasmid to obtain a PB513B-CD169-CD163 plasmid;

2) transfection of PK15 cells: the PB513B-CD163-CD169 plasmid was transfected into PK15 cells together with a transposase plasmid (a plasmid carrying a transposase gene) using a liposome 3000 transfection reagent, and cultured in a DMEM medium containing 10% (v/v) fetal bovine serum;

3) and (3) puromycin screening: screening the transgenic cells by using DMEM medium containing puromycin and 10% (v/v) fetal bovine serum;

4) culturing and domesticating: and (3) carrying out subculture on the transgenic cells, reducing the concentration of fetal calf serum in a culture medium by generations, and finally carrying out suspension culture by using a serum-free culture medium.

As the preparation method, step 3) is to screen the transgenic cells by using DMEM medium containing 2 μ g/m L puromycin and 10% (v/v) fetal bovine serum.

And/or the culture domestication method in the step 4) comprises the following steps: first, culturing for two generations in MEM medium containing 10% (v/v) fetal calf serum, then culturing for one generation in BD004 medium containing 5% fetal calf serum, then culturing for one generation in BD004 medium containing 2% fetal calf serum, then digesting PK15 cells with pancreatin to detach the culture dish, and transferring to CDPK 15259 serum-free suspension culture medium for suspension culture for more than 2 generations.

In the preparation method, the CD163 gene in the step 1) is obtained by amplifying cDNA by using a primer with a sequence shown in SEQ ID NO. 1-2 to obtain a product and then amplifying by using a primer with a sequence shown in SEQ ID NO. 7-8;

the cDNA is obtained by reverse transcription of total RNA of porcine alveolar macrophage.

In the preparation method, the CD169 gene in the step 1) is obtained by amplifying cDNA by using a primer with a sequence shown in SEQ ID NO. 3-4 to obtain a product and then amplifying by using a primer with a sequence shown in SEQ ID NO. 5-6;

The preparation method was as described above, and CD169 was ligated between the XbaI and BamHI restriction sites of the PB513B vector in step 1).

The preparation method described above, CD163 was ligated to the NotI restriction site of the PB513B vector in step 1).

The transposase plasmid described in step 2) of the preparation method as described above was PiggyBac SuperTransposase (Wuhan vast Ling Biotech Co., Ltd., Cat # P0179).

The preparation method comprises the step 2) that the mass ratio of the PB513B-CD163-CD169 plasmid to the transposase plasmid is 2: 1.

the cell line prepared by the method.

The use of the aforementioned cell line in the culture of porcine reproductive and respiratory syndrome virus.

The invention has the following beneficial effects:

1) greatly improves the susceptibility of PK15 cells to PRRSV, so that the PK15 cells can be used as an excellent host for mass propagation of the PRRSV. Experiments show that the TCID of the virus is 30 hours after inoculation₅₀Up to 8.0.

2) The PK15 cell line can be separated from microcarrier to realize suspension culture, and serum is not required to be added into a culture medium, so that the production cost is greatly reduced.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1: PB513B-CD169-CD163 transgenic vector structure diagram.

FIG. 2: the integration and expression of the CD169 gene of the transgenic PK15 cell are identified by PCR; m.1kb DNA standard molecular weight Marker; the PB513B-CD169-CD163 transgenic vector plasmid; 2. transgenic Marc145 cell genomic DNA; 3. cDNA reverse transcribed from total RNA of transgenic Marc145 cells.

FIG. 3: the CD163 gene integration and expression of the transgenic PK15 cells are identified by PCR; m.1kb DNA standard molecular weight Marker; the PB513B-CD169-CD163 transgenic vector plasmid; 2. transgenic Marc145 cell genomic DNA; 3. cDNA reverse transcribed from total RNA of transgenic Marc145 cells.

FIG. 4: transgenic PK15 cells were detected by fluorescence microscopy.

FIG. 5: PK15 cell puromycin killing curve.

Detailed Description

Example 1 construction and characterization of suspension PK15 cell line expressing porcine CD163 and CD169 molecules of the present invention

First, obtaining CD163 and CD169 genes

1. The mRNA sequences of the CD163 (SEQ ID NO: EU016226.1) and CD169 (SEQ ID NO: AF509585.1) genes were searched by the NCBI website. Primer sequences CD163F (SEQ ID NO.1) and CD163R (SEQ ID NO.2) were designed for amplification of the CD163 sequence; primer sequences CD169F (SEQ ID NO.3) and CD169R (SEQ ID NO. 4).

2. Porcine primary alveolar macrophages cultured at 2m L were extracted with total RNA using a total RNA extraction kit (DP431) from animal tissues of Tiangen Biochemical technology, Inc. (Beijing), and then reverse-transcribed into cDNA using an RT-PCR kit (Tiangen Biochemical: KR 118).

3. Using the obtained cDNA as a template, PCR-amplified CD163 gene and PCR-amplified CD169 gene molecules of CD169F (SEQ ID NO.3) and CD169R (SEQ ID NO.4) were performed using primers CD163F (SEQ ID NO.1) and CD163R (SEQ ID NO.2), respectively.

The PCR system was as follows:

the PCR procedure was as follows: 94 ℃ for 2 min; 94 ℃,15s,60 ℃,15s,72 ℃,90s,25 cycles; 72 ℃ for 5 min.

Secondly, constructing a transgenic vector expressing CD163 and CD169

1. Cloning of CD169 Gene into PB513B vector

1) Recovery of CD169 gene molecule by PCR amplification

PCR primers PBCD169F (SEQ ID NO.5) and PBCD169R (SEQ ID NO.6) were designed to amplify the CD169 gene, and the PCR system was as follows:

The obtained PCR product was subjected to 1% agarose gel electrophoresis, and then subjected to gel recovery using an ultra-thin agarose gel DNA recovery kit (DP208) from Tiangen Biochemical technology Ltd to purify the CD169DNA molecule.

2) Carrying out enzyme digestion on the PB513B vector, purifying and recovering

The PB513B vector was digested with XbaI and BamHI restriction enzymes as follows:

plasmid 40. mu. lPB513B (100 ng/. mu.l)

2μlXbaI

2μlBamHI

6 μ l of ultrapure water

The enzyme digestion is carried out for 3 hours at 37 ℃, then 6XDNA loading buffer is added for carrying out 1 percent gel electrophoresis, electrophoresis is carried out for 15 minutes at constant pressure of 100V, then DNA fragments are purified and recovered, and then the purified and digested PB513B vector fragment is recovered by using an ultrathin agarose gel DNA recovery kit (DP208) of Tiangen Biotechnology, Inc.

3) Connection of

The obtained CD169 gene fragment and the PB513B vector fragment recovered by enzyme digestion are connected by an EasyGeno rapid recombinant cloning kit, and the connection system is as follows:

incubation was carried out at 50 ℃ for 15min, then placed on ice and transformed into E.coli TOP10 competent cells. The conversion steps are as follows:

50 mu L of Escherichia coli competent cells are taken, added with a proper amount of plasmid (the volume is not more than 5 mu L) and subjected to ice bath for 30min, then heat shock is carried out at 42 ℃ for 90s, immediately the cells are put back on ice and subjected to ice bath for 2min, 400 mu L L B culture medium is added, shaking culture is carried out slowly on a shaker at 37 ℃ for 45-60min, 50-100 mu L is taken and smeared on L B solid culture medium containing ampicillin (100 mu g/m L), and inverted culture is carried out at 37 ℃ for overnight.

4) Screening, sequencing and confirmation

Picking a plurality of single colonies from a culture dish, culturing overnight in a 5m LL B culture medium, picking up plasmids by a plasmid extraction kit, carrying out enzyme digestion for identification, and finally sending the obtained plasmids to a sequencing company for sequencing to confirm that the sequence is correct, wherein the obtained plasmids are named as PB513B-CD 169.

2. Ligation of CD163 Gene to PB513B-CD169 plasmid

1) PCR amplification of CD163 Gene molecules

Designing PCR primers PBCD163F (SEQ ID NO.7) and PBCD163R (SEQ ID NO.8) to amplify the CD163 gene, wherein the PCR system is as follows:

The obtained PCR product was subjected to 1% agarose gel electrophoresis, and then gel recovery was carried out using an ultra-thin agarose gel DNA recovery kit (DP208) from Tiangen Biochemical technology Ltd to purify the CD163DNA molecule.

2) NotI digestion plasmid PB513B-CD169

Plasmid PB513B-CD169 was digested with NotI as follows:

plasmid 40. mu. lPB513B-CD169 (100 ng/. mu.l)

2μlNotI

8 μ l of ultrapure water

The enzyme digestion is carried out for 3 hours at 37 ℃, then 6XDNA loading buffer is added for carrying out 1 percent gel electrophoresis, electrophoresis is carried out for 15 minutes at constant pressure of 100V, then DNA fragments are purified and recovered, and then the purified and digested PB513B-CD169 vector fragment is recovered by using an ultrathin agarose gel DNA recovery kit (DP208) of Tiangen Biotechnology, Inc.

3) Linking the CD163 molecule to the vector PB513B-CD169

The obtained CD163 gene fragment and the PB513B-CD169 vector fragment recovered by enzyme digestion are connected by an EasyGeno rapid recombinant cloning kit, and the connection system is as follows:

3. competent transformation and validation sequences

Picking up a plurality of single colonies from a culture dish, culturing the single colonies in a 5m LL B culture medium overnight, picking up plasmids by a plasmid extraction kit, carrying out enzyme digestion for identification, obtaining the plasmids which are named as PB513B-CD169-CD163 (the structure is shown in figure 1), and sending the plasmids to a sequencing company for sequencing to confirm that the sequences are correct.

4. Extraction of endotoxin-depleted plasmids

Sequencing confirmed correct plasmid PB513B-CD169-CD163 also required endotoxin-free plasmid DNA extraction using endotoxin-free plasmid extraction kit (DP118, Tiangen Biochemical), since endotoxin affects subsequent cell transgene experiments.

Thirdly, PK15 cells are transfected

Establishment of PK15 cell puromycin killing curve

50 ten thousand PK15 cells were added to the wells of a 24-well plate, 1. mu.g/m L, 2. mu.g/m L, 3. mu.g/m L, 4. mu.g/m L, 5. mu.g/m L and 10. mu.g/m L puromycin were added, and the growth of the cells was observed every day, and the killing curve is shown in FIG. 5.

As can be seen, puromycin at 2. mu.g/m L killed all cells within 3 days, so puromycin concentration of 2. mu.g/m L was selected as the screening concentration.

Plasmid PB513B-CD163-CD169 transfection of PK15 cells

PB513B-CD163-CD169 plasmid (2. mu.g) was added to 100. mu.l buffer of liposome 3000 together with 1. mu.g transposase plasmid (PiggyBacSuperTransposase, Wuhan vast Ling Biotech Co., Ltd., Cat # P0179) in 1.5m L EP tube, gently mixed, then 8. mu.l transfection reagent for liposome 3000 was added, well mixed, left at room temperature for 15 minutes, and then mixed solution of plasmid and transfection reagent was added to PK15 cells in a six-well plate, gently mixed, and cultured in a 37 ℃ incubator (containing 5% CO)₂) After 4 hours of stationary culture, DMEM medium containing 10% fetal bovine serum was replaced.

3. Puromycin screening transfected cells;

after 48 hours of culture of the transgenic PK15 cells, culture was continued in DMEM medium containing 2. mu.g/m L puromycin (containing 10% fetal bovine serum) until all non-transgenic cells died.

4. Cloning and screening cells

The obtained transgenic Marc145 cells were trypsinized, diluted to 10 cells/m L, 100. mu.l was added to a 96-well plate and cultured, the cells in the 96-well plate were microscopically observed, culture wells having only a single cell were determined and recorded, and the medium was changed every 3 days until the 96-well plate was filled with the cells.

5. Suspension domestication

Culturing the obtained transgenic PK15 cells in MEM (with 10% of fetal calf serum) for two generations, then changing to BD004 (Gansu Jianshun Biotechnology Co., Ltd.) (containing 5% of fetal calf serum) for one generation, then changing to BD004 (containing 2% of fetal calf serum) for one generation, digesting the transgenic PK15 cells by using pancreatic enzyme, measuring the cell activity to be more than 95%, then transferring to CD PK 15259 serum-free suspension medium (Gansu Jianshun Biotechnology Co., Ltd.) for suspension culture, and performing suspension culture for two generations until the cell activity is more than 95%.

Fourth, identification of transgenic cells

1m L suspended transgenic PK15 cells were taken, genomic DNA and total RNA of the cells were extracted with a genome extraction kit (Tiangen Biochemical technology Co., Ltd.: DP304-02) and an RNA extraction kit (Tiangen Biochemical technology Co., Ltd.: DP430), respectively, wherein the total RNA was reverse-transcribed into cDNA with a reverse transcriptase, and then CD169 gene was identified by PCR with primers CD169F and CD169R, and CD163 transgene and gene expression were identified with primers CD163F and CD163RPCR (FIGS. 2 and 3), and transgenic PK15 cell transgene expression was observed with a green fluorescence microscope (FIG. 4).

Fifth, proliferation of PRRSV TB-PX strain on transgenic suspension PK15 cells

1. Cell preparation

The activity of the inoculated cells is over 95 percent generally, the cells can be passaged 1:5 before inoculation, and the cell density is 1 × 10⁶cells/mL，37℃、5％CO₂Culturing for 16-24 h in an incubator by shaking (120rpm) until the cells grow to 5 × 10⁶cells/m L are ready for use.

2. Infection inoculation

1) Taking out virus liquid (with known virus liquid titer) and putting on ice for thawing;

2) adding seed venom to be cultured according to 0.3 moi; placing at 37 ℃ and 5% CO₂Culturing for 45-72 h in an incubator by shaking (120rpm) until 70-80% of cells die.

3. Harvesting of viruses

Observing most cells to fall off under a microscope, and harvesting virus liquid (harvesting culture medium supernatant) when the cell activity is below 30%; centrifuging at 3000rpm/min for 20min, and storing the supernatant at-80 deg.C.

4. Viral fluid TCID50 assay

1) Cell digestion: selecting PK15 cells with good growth state, removing the culture medium by suction, adding 1ml of pancreatin/25T (3ml/75T), placing in an incubator at 37 ℃ for digestion, observing at any time (generally 1-2 min), removing the pancreatin, covering a cover, patting a cell bottle lightly to see that the cells fall off in a sand granule shape (if the cells do not fall off, the cell bottle can be placed back in the incubator at 37 ℃ again for continuous digestion).

2) Cell plating by suspending cells in DMEM containing 10% newborn calf serum at 3 × 10⁵The cell/m L was plated in 96 well cell culture plates at 0.1 ml/well and placed at 37 ℃ with 5% CO₂Culturing in an incubator until the cells grow to 90-95% of the bottom of the hole and the cells grow for about 16-24 h.

3) Sample dilution and inoculation: preparing sterilized 1.5ml EP tubes according to the number of samples, arranging the EP tubes in a line from left to right on a tube frame, and respectively adding 0.9ml DMEM diluted cells containing 2% fetal calf serum; taking 0.1ml of a virus sample to be detected to carry out 10-fold serial dilution to prepare 10^-1、10^-2、10^-3、10^-4、10^-5、10^-6、10^-7、10^-8、10^-9、10^-10And (4) waiting for multiple dilution samples.

4) Virus inoculation: discarding DMEM culture solution of 96-well plate, selecting proper dilution gradient as inoculation material,

6 wells were inoculated at each dilution, 0.1ml was inoculated at each well, and a negative control (DMEM with 2% fetal calf serum) and a positive control (DMEM supplemented with 2% fetal calf serum in suspension with 0.1ml of DMEM in each well before inoculation of the virus to give a final volume of 0.2ml in each well, which was then cultured in a 5% CO2 incubator at 37.0 ℃ for 96-120 h).

5) And (4) recording and calculating a result: after inoculation and culture, observing the cytopathic condition under a microscope every day; recording in detail, and calculating virus content (TCID) of the sample according to cell pathological condition by Reed-Muench method₅₀). The result shows that the virus content is highest after 30 hours of virus inoculation and can reach TCID₅₀＝8.0。

The invention constructs CD163 and CD169 into PK15 cell line, and carries out serum-free and suspension culture domestication on the PK15 cell line, thereby obtaining the PRRSV-sensitive serum-free suspension culture cell line. The cell line can overcome the defects of low sensitivity, unsuitability for passage, dependence on serum and incapability of suspension culture of PRRSV cultured cells in the prior art, can obviously improve the yield and the production efficiency of PRRSV, and has very good application prospect.

Sequences referred to in the specification:

SEQ ID NO.1：

5’-CACCATGGACAAACTCAGAATGGTGCTACATGAAAACTCT-3’

SEQ ID NO.2：

5'-TCATTGTACTTCAGAGTGGTCTCCTGAGGGATT-3'

SEQ ID NO.3：

5'-CACCATGGACTTCCTGCTCCTGCTCCTC-3'

SEQ ID NO.4：

5'-TCAGACTGTGCTTTTCACAGACTGTGTGTTTTCACAGAG-3'

SEQ ID NO.5：

5’-GTTTTGACCTCCATAGAAGATTCTAGAATGGACTTCCTGCTCCTGCTCCTC-3'

SEQ ID NO.6：

5’-GCAGATCCTTCGCGGCCGCGTCAGACTGTGCTTTTCACAGACTGT-3’

SEQ ID NO.7：

5’-AGTCTGTGAAAAGCACAGTCCGCGGGAGTCGCGGAAGCGGAGCTACTAACTTCA-3'

SEQ ID NO.8：

5’-GAGCGATCGCAGATCCTTCGCTCATTGTACTTCAGAGTGGTCTCCTGAGG-3'

SEQUENCE LISTING

<110> Chengdu Tianbang biological products GmbH

Shih Biotechnology (Nanjing) Ltd

MA'ANSHAN SHIJI ANIMAL HEALTH MANAGEMENT Co.,Ltd.

<120> suspension PK15 cell line expressing porcine CD163 and CD169 molecules

<130>GY768-2019P018652CC

<160>8

<170>PatentIn version 3.5

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<213> Artificial sequence

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tcattgtact tcagagtggt ctcctgaggg att 33

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<211>33

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<213> Artificial sequence

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tcattgtact tcagagtggt ctcctgaggg att 33

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tcagactgtg cttttcacag actgtgtgtt ttcacagag 39

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gagcgatcgc agatccttcg ctcattgtac ttcagagtgg tctcctgagg 50

Claims

1. A method for preparing a PK15 cell line expressing porcine CD163 and CD169 and capable of suspension culture in serum-free medium, comprising the steps of:

2) transfection of PK15 cells: the PB513B-CD163-CD169 plasmid was transfected into PK15 cells together with the transposase plasmid using liposome 3000 transfection reagent, cultured in DMEM medium containing 10% (v/v) fetal bovine serum;

2. The method of claim 1, wherein:

step 3) specifically, screening the transgenic cells by using a DMEM medium containing 2 mu g/m L puromycin and 10% (v/v) fetal bovine serum;

and/or the culture domestication method in the step 4) comprises the following steps: culturing for two generations in MEM culture medium containing 10% (v/v) fetal calf serum, culturing for one generation in BD004 culture medium containing 5% fetal calf serum, culturing for one generation in BD004 culture medium containing 2% fetal calf serum, digesting PK15 cells with pancreatin, separating from the culture dish, and transferring to CD PK 15259 serum-free suspension culture medium for suspension culture for more than 2 generations.

3. The method of claim 1, wherein:

the CD163 gene in the step 1) is obtained by amplifying cDNA by using a primer with a sequence shown as SEQ ID NO. 1-2 to obtain a product and then amplifying by using a primer with a sequence shown as SEQ ID NO. 7-8;

4. The method of claim 1, wherein:

the CD169 gene in the step 1) is obtained by amplifying cDNA by using primers with sequences shown as SEQ ID NO. 3-4 to obtain a product and then amplifying by using primers with sequences shown as SEQ ID NO. 5-6;

5. The method of claim 1, wherein:

in step 1) CD169 was ligated between the XbaI and BamHI restriction sites of the PB513B vector.

6. The method of claim 1, wherein:

CD163 was ligated at the NotI restriction site of the PB513B vector in step 1).

7. The method of claim 1, wherein: the transposase plasmid is PiggyBacSuperTransposase.

8. The method of claim 7, wherein:

the mass ratio of the PB513B-CD163-CD169 plasmid to the transposase plasmid in the step 2) is 2: 1.

9. a cell line produced by the method of any one of claims 1 to 8.

10. Use of the cell line of claim 9 in the culture of porcine reproductive and respiratory syndrome virus.