AU2020102162A4 - Pig tonsil cell line susceptible to jev and construction method thereof - Google Patents

Abstract

The present invention discloses a pig tonsil cell line that is susceptible to Japanese encephalitis JEV. The cell line is obtained by infecting a primary pig tonsil cell line with lentiviral particles, screening cells in a DMEM/F12 culture solution containing serum and 5 added with puromycin and then performing amplification culture on positive cells using a culture solution containing no puromycin; the lentiviral particles are obtained by using pHY sv4-Puro as a lentiviral vector, using psPAX2 and pCMV-VSV-G as an auxiliary package vector and co-transfecting into 293T cells. The cell line of the present invention has the function of infinite passage, is sensitive to JEV, and can be used for correlated studies. 10 (FIG. 2) 2/6 100X-DAPI 10~X-CI9 FIG.2 A B 3 inS2f D 1540 01 24187HZ4M4U72It24H~UR72K 12H38H6GH 12H36H63H 12H39HSCH Tim i FIG.3

Description

2/6

100X-DAPI 10~X-CI9

FIG.2

A B 3

inS2f D 1540

01

24187HZ4M4U72It24H~UR72K 12H38H6GH 12H36H63H 12H39HSCH Tim i

FIG.3

PIG TONSIL CELL LINE SUSCEPTIBLE TO JEV AND CONSTRUCTION METHOD THEREOF

Field of the Invention

The present invention relates to a pig tonsil cell line, in particular to a pig tonsil cell line

susceptible to JEV and a construction method thereof.

Background of the Invention

Pig Japanese B encephalitis (referred to as encephalitis) is a common viral encephalitis

caused by Japanese Encephalitis viruses (JEV), is a zoonotic disease, and can infect multiple livestock species, bird species and humans. For the disease, viruses are mainly propagated by

the bite of blood sucking insects such as mosquitoes. Any variety and gender of pigs are

susceptible to the disease. The main clinical symptoms are abortion or stillbirth of pregnant sows, encephalitis, ataxia, fever and death of newborn piglets, obvious enlargement of testicles

of boars, neurological symptoms of some sick pigs, and infection abortion of some pregnant animals.

PK-15 cells (pig renal epithelial cells), ST cells (pig testicular cells, PIEC cells (pig hip arterial endothelial cells) and the like are common cell lines for researching infection of pig JEV, but the organ origins of these cells themselves do not infect or seldom infect pig JEV. The process of researching the injection of pig JEV using cells such as PK-15, ST and PIEC has situations, for example, infection proliferation is different from that of target organs, innate immune response is different that of target organs, acquired immune response is different from target organs, interaction between viruses and cells is different from that of target organs. Change and interaction of these viruses and genes are not completely consistent with actual situations of target organs, and even completely different. Such the result may cause misleading to lead to different experiment results and clinical situations, thereby affecting the development of clinical vaccines and drugs.

Pig tonsil is a target organ for multiple diseases of pig, and is also a lesion of in-vivo infection. However, there are no available pig tonsil cell lines for researching the effect of the pig tonsil on infection and propagation of pig diseases.

Summary of the Invention

The object of the present invention is to provide a pig tonsil cell line susceptible to JEV. This method solves the problem that there is no pig tonsil cell line infected with porcine viruses at present. The pig tonsil cell line susceptible to JEV has the function of infinite passage, can maintain the features of pig tonsil cells, can keep karyotype to be normal in the process of passage, has no change of chromosome structure and number, is sensitive to JEV, and can be used for passage of JEV and related researches.

In order to achieve the above object, the present invention provides a pig tonsil cell line susceptible to JEV, wherein the cell line is obtained by infecting a primary pig tonsil cell line with lentiviral particles, screening cells in a DMEM/F12 culture solution containing serum and added with puromycin and then performing amplification culture on positive cells using a culture solution containing no puromycin;

wherein, the lentiviral particles are obtained by using pHY-sv40-Puro as a lentiviral vector,

using psPAX2 and pCMV-VSV-G as an auxiliary package vector and co-transfecting into 293T cells.

Preferably, the primary pig tonsil cell line is cultured in a 5%FBS DMEM/F12 culture

medium, and 100 U/mL penicillin and 100 gg/mL streptomycin are added in the culture medium.

Preferably, the lentiviral particles are cultured using 2%FBS DMEM.

Preferably, the medium for amplification culture is a 5%FBS DMEM/F12 medium

containing no puromycin.

Preferably, when the infection is performed, 5gg/mL polybrene is added.

The present invention provides a construction method of a pig tonsil cell line susceptible

to JEV, the method including: obtaining primary pig tonsil cells, and culturing in a 5%FBS

DMEMF12 medium in which 100 U/mL penicillin and 100 gg/mL streptomycin are added; obtaining lentiviral particles containing a pHY-sv40-Puro lentiviral vector by using pHY-sv40

Puro as a lentiviral vector, using psPAX2 and pCMV-VSV-G as an auxiliary package vector,

co-transfecting into 293T cells, and culturing using a 2%FBS DMEM; infecting the cultured the primary pig tonsil cells with the lentiviral particles; after the infection, carrying out cell screening on the pig tonsil cells in a DMEM/F12 culture solution containing serum and added with puromycin to obtain positive cells, wherein the concentration of the puromycin is the minimum lethal concentration of normal pig tonsil cells; carrying out amplification culture on the positive cells with a 5%FBS DMEM/F12 medium containing no puromycin, and constructing to obtain the pig tonsil cell line susceptible to JEV.

Preferably, the preparation of the primary pig tonsil cells includes: removing connective

tissues of the needed pig tonsil part in a sterile plate, washing with Hank's liquid, cutting into pieces, adding Hank's liquid, putting the obtained mixture into a sterile conical flask so as to

disperse cells, standing, and collecting cell suspension on the upper layer.

Preferably, the preparation of lentiviral particles includes: culturing the transfected 293T

cells for 48h, then collecting supernatant for the first time, then culturing for 24h, and harvesting

supernatant and cells to be mixed with the previously collected supernatant; carrying out

multigelation so that cells are broken, filtering, collecting supernatant, mixing the supernatant with PEG8000, obtaining precipitate, and dissolving the obtained precipitate with DMEM

culture solution to obtain the lentiviral particles.

Preferably, when the infection is performed, polybrene is added.

Preferably, the concentration of polybrene is 5 g/mL.

Preferably, the amplification culture is carried out by more than 50 generations.

The pig tonsil cell line susceptible to JEV of the present invention solves the problem that there is no porcine tonsil cell line infected with porcine viruses at present, and has the following advantages.

The cell line of the present invention takes the pig tonsil cell as a host cell for the first time and transfects lentivirus expression plasmids with SV40 antigen genes, the pig tonsil cell line susceptible to JEV is obtained through Puromycin screening. The cell line has the function of infinite passage, can maintain the features of pig tonsil cells, can keep karyotype normal in the process of passage, and has no change of chromosome structure and number; furthermore, the pig tonsil cell line is sensitive to JEV, JEV can be replicated in the cell to be used for passage of JEV and related researches; a new cell line is provided for culture of JEV, the range of host cells of JEV research is expanded, which is of important significance to study the research of JEV on pathogenesis of an immune system.

Brief Description of Drawings Fig. 1 is a culture growth situation graph of a pig tonsil cell susceptible to JEV according to the present invention. Fig. 2 is an imunofluorescence identification graph of a pig tonsil cell susceptible to JEV according to the present invention. Fig. 3 shows optimization of growth conditions of a pig tonsil cell susceptible to JEV according to the present invention.

Fig. 4 is a cell proliferation graph of a pig tonsil cell susceptible to JEV according to the present invention.

Fig. 5 is a technology route map of a pig tonsil cell line constructed according to the present invention.

Fig. 6 shows a pig tonsil cell susceptible to JEV and pig nephrocyte infected JEV.

Fig. 7 is an infection effect graph of a pig tonsil cell susceptible to JEV.

Fig. 8 shows that a green fluorescent protein is transfected and expressed by a pig tonsil cell susceptible to JEV.

Detailed Description of the Invention

The technical solution in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention but not all the embodiments. Based on the embodiments of the present invention, all other embodiments made by those skilled in the art without creative efforts belongs to the protective scope of the present invention.

Provided is a pig tonsil cell line susceptible to JEV. The cell line is obtained by infecting a primary pig tonsil cell line with lentiviral particles, screening cells in a DMEM/F12 culture solution containing serum and added with puromycin and then performing amplification culture on positive cells using a culture solution containing no puromycin.

Wherein, the lentiviral particles are obtained by using pHY-sv4O-Puro as a lentiviral vector,

using psPAX2 and pCMV-VSV-G as an auxiliary package vector and co-transfecting into 293T cells.

The primary pig tonsil cell line is cultured in a 5%FBS DMEMF12 culture medium, and 100 U/mL penicillin and 100 Rg/mL streptomycin are added in the culture medium.

The lentiviral particles are cultured using 2%FBS DMEM.

The medium for amplification culture is a 5%FBS DMEM/F12 medium containing no

puromycin.

When the infection is performed, 5Rg/mL polybrene is added.

Example 1 Construction of cell line with pig tonsil epithelial cells

As shown in Fig.5, Fig. 5 is a technology route map for constructing a pig tonsil cell line according to the present invention. The construction process is as follows:

1. Culture of pig tonsil cells

The fresh pig tonsil was put into a sterile plate on a super-clean bench, connective tissues such as coating were removed, the rest pig tonsil was washed with Hank's solution for 3-5 times until the Hank solution was transparent and clear, then the tonsil tissue was cut into pieces to be pasty, the diameter of the tissue fragment is about 1-2 mm, a little of Hank's solution was added, the resulting tissue fragments were put into a sterile conical flask with glass beads, the cells are dispersed by shaking and then stood for a moment so that tissue fragments descended, the cell suspension on the upper layer was collected to be inoculated to a cell culture dish, 100 U/mL penicillin and 100 jig/mL streptomuycin (which prevented contamination of bacteria) were added, and solution was changed every one day.

2. Measurement of minimum lethal concentration of puromycin in pig tonsil epithelial cells

Puromycin was used as a screening drug. Since Puromycin resistance gene Puro was present in expression vector Phy-SV40-Puro, the expressed pig tonsil epithelial cells obtained by exogenous genes were capable of surviving in the culture solution DMEM/F12 (purchased from gibco) containing a certain concentration ofPuromycin, and the untransfected normal cells were died at this concentration. Therefore, it was needed to determine the minimum lethal concentration of the Puromycin in the normal cells to serve as a screening concentration.

Measurement of minimum lethal concentration of Puromycin was performed as follows:

Puromycin having concentration gradient was added in the culture solution ofthe pig tonsil epithelial cells to be screened for one week, and the minimum lethal concentration of Puromycin in the normal cells was measured. When the concentration of Puromycin in the culture solution reached 2 g/mL, it was seen under the microscope that all the cells were died, so the minimum lethal concentration of Puromycin was 2 g/mL.

3. Acquisition of lentiviral particles containing lentiviral vector pHY-SV40-Puro

psPAX2 (6 g, purchased from addgene, #12260), pCMV-VSV-G (6 g, purchased from addgene, #8454), and pHY-SV40 Puro (7.5 gg) (purchased from addgene, Hh-Lv-005) were co-transfected into 293T cells. Where, pHY-SV40-Puro was a lentiviral expression plasmid (namely as a lentiviral vector), psPAX2 (a plasmid expressing lentiviral coat) and pCMV-VSV G were used as auxiliary packaging vectors. The Puromycin resistance gene was contained on the expression vector pHY-SV40-Puro.

Cells were added into the mixture to continue culture for 48 h, and the cell supernatant sample was collected; a new culture medium was changed, and the cell supernatant sample was collected when 72 h elapsed.

The supernatant was centrifuged for 10 min at 3500 rpm to remove cell fragments. After being filtered with a 0.45 M filter, the cells were sub-packed (ImL/tube) and stored at - 80 C.

After transfection for 48 h, the fluorescence generated by expression of mark gene EGFP (enhanced green fluorescent protein, carried by pHY-SV40-Puro) was observed under a fluorescence microscope, and meanwhile the supernatant was harvested. After transfection for 72 h, the supernatant and cells were harvested and mixed with the above-mentioned supernatant.

The obtained mixture was subjected to multigelation for three times so that cells were broken and then filtered using 0.45 m disposable filter, the supernatant was collected and mixed with 5 x PEG 8000, settled overnight at 4 °C and centrifuged at 4000 g for 10 m so as to remove the supernatant. The precipitate was dissolved with DMEM culture solution and stored at - 80 °C for later use.

4. Infection of pig tonsil epithelial cells with lentiviral particles

When growing to the seventh day, the primary pig tonsil epithelial cells were subjected to lentivirus infection, and polybrene (51tg/mL, polybrene) was added to improve the ability of virus infection.

After infection for 24 h, fresh culture solution was changed. After infection for 72 h, Puromycin was added until the final concentration was 2gg/mL.

5. Screening of positive expression of pHY-SV40-Puro vector

After pHY-SV40-Puro lentiviral particles infected pig tonsil epithelial cells, Puromycin having a final concentration of2 gg/mL was added in the DMEM 12 culture solution containing serum for cell screening.

After lentivirus infected cells for 72 h, the expression vector is expressed and green fluorescence was seen under the fluorescence microscope. When the pig tonsil epithelial cells infected with lentivirus emitted green fluorescence, pLVX-EGFP-T2A-Puro-SV40T had been entered into cells and positively expressed. At this moment, the concentration of Puromycin was reduced to a half, screening was continued until cells fully grew at the bottom of the dish, so that it can be seen that positive cells are island-shaped cell populations and still green under the fluorescence microscope. Then, cells were digested, and subjected to amplification culture with the DMEM/F12 culture solution containing Puromycin.

The screened positive cells are cells stably transfected with pHY-SV40-Puro. Target genes SV40 were integrated to genome of the cells, rather than outside the genome. In the stable transfection process, genes carried on the plasmid pHY-SV40-Puro were integrated to the genome of the host, screening was carried out for 14 days through Puromycin and then continued at half dose to realize the purpose of stable transfection, exhibiting that report genes were continuously expressed in the transfected positive cells. Therefore, the screened positive cells continuously emitted green fluorescence and presents Puromycin resistance.

When the screened positive cells were subjected to amplification culture to 50 generations using the culture solution containing no Puromycin, the pig tonsil epithelium cell line was constructed.

The promoter of the green fluorescent protein (EGFP) label introduced in the constructed pig tonsil epithelium cell line had been methylated. There was no fluorescence under the fluorescence microscope, which facilitates the subsequent fluorescence experiment of the cell line not to be affected.

Example 2: Characteristic verification of primary cells of pig tonsil epithelium cells and constructed cell line

Characteristic verification was carried out on primary cells of pig tonsil epithelium cells and the constructed cell line using immunofluorescence, including purity of primary cells, growth and culture characteristics of immortalized cell lines, and some biological functions. The susceptibility of PT cells to JEV was also found, and the specific steps were as follows:

(1) Cells climb slides

Glass sheets were put in a 24-well plate, 1 mL of DMEM/F12 culture medium was added in each well, with 0.02 million cells/well. The cells were placed in an incubator for 2 h or overnight.

(2) The cells were inoculated with 5MOI JEV viruses, and 2% FBS DMEM culture medium was changed after 2 h, and cell samples were harvested at different time points.

(3) Immobilization

After the cells climb the slices, the culture medium was sucked, the cells were washed with PBS (phosphate buffer solution), added with 4% PFA (paraformaldehyde) and immobilized at 4 °C for 30 min. PBS for 3 x 5min/time was used for washing.

(4) Rupture membrane and seal

Water was removed from glass slices, the glass slices without water were placed on the support ofthe culture dish. The glass slide sealing solution was prepared as follows: 0.5% Triton X-100 was mixed with PBS (the final concentration of Triton X-100 was 0.25%) in a ratio of 1:1, and then 10% serum (when the membrane was ruptured and sealed, the source of serum should be the same as that of the second antibody, if the second antibody was derived from sheep, sheep blood was used) was added, 50 L of membrane rupturing and sealing solution was dropped onto a waterproof membrane, and one side of the glass slide where cells were present was covered for 2 h.

(5) Primary antibody incubation

Preparation of primary antibody: the antibody was diluted with PBS 1:100 (200). After the membrane was ruptured, 50 pL of primary antibody (2H4) was put on the waterproof membrane (wet box), and one side of the glass slide where cells were present was covered and placed at 4 °C (for a week at most).

(6) Second antibody incubation

Washing was carried out with PBS for 3 x 5min / time, and a second antibody mixed solution was prepared as follows: second antibody (Alexa Flour 488 goat anti-mouse IgG): PBS = 1:500, DAPI(4', 6-diaminol-2-phenylindole): PBS= 1:1000. 50 L of second antibody mixed solution was dropped on the waterproof membrane, the glass slide was covered and placed for 2 h at room temperature under the dark condition.

(7) Embedment

The glass slide was washed with PBS for 3 x 5min / time. One drop of Fluoromount-G was dropped on the glass side, and one side of the glass side where cells were present was covered.

As shown in Fig. 1, Fig. 1 is a culture growth situation graph of a pig tonsil cell susceptible to JEV according to the present invention. In the figure, A is growth situation of PT cells for 24 h, B is growth situation of PT cells for 48 h, C is a morphology of PT cells for 12 h, D is a morphology of PT cells for 24 h, E is a morphology of PT cells for 48 h, and F is a morphology of PT cells for 72 h. It can be seen from the figure that the cell was oval and spindle in morphology, with a diameter of 15-20 pm. The morphologies of the cells were similar to the morphologies of the most epithelial cells.

As shown in Fig. 2, Fig. 2 is an imunofluorescence identification graph of a pig tonsil cell susceptible to JEV according to the present invention (A: DAPI stained nucleus, B: CK-19 stained keratin). It can be seen from the figure that all the cells are epithelial cells, and there are no other cell varieties.

As shown in Fig. 3, Fig. 3 shows optimization of growth conditions of a pig tonsil cell susceptible to JEV according to the present invention. In the figure, A is growth of PT cells in different concentrations of fetal bovine serum (FBS), and B is growth of PT cells in different media (MEM, minimum Eagle's medium; DMEM, Dulbecco's Modified Eagle's medium). It can be seen from the figure that the optimum growth condition of cells is an MEM culture medium with 5% fetal bovine serum concentration.

As shown in Fig. 4, Fig. 4 is a cell proliferation graph of a pig tonsil cell susceptible to JEV according to the present invention. It can be seen from the figure that the cells enter a logarithmic growth phase between 12 and 24 hours, and then enter a stable growth phase after 24 hours.

As shown in Fig. 6, Fig. 6 shows a pig tonsil cell (PT cell) susceptible to JEV and pig nephrocyte (PK cell) infected JEV (MOCK is a blank control group and JEV is an experimental group for virus inoculation); A: a growth curve of viruses on PT cells and PK cells (pig kidney epithelial cells) when inoculated with 5MOI JEV; B: a growth curve of viruses on PT cells and PK cells when being inoculated with 0.5MOI JEV; C: replication of viruses on PT cells when being inoculated with 5MOI JEV; D: the replication of virus in PK cells when inoculated with 5MOI JEV. It can be seen from the figure that JEV viruses can be normally amplified in PT cells.

As shown in Fig. 7, Fig. 7 is an infection effect graph of a pig tonsil cell susceptible to JEV. (A: expression of JEV E protein after virus inoculation for 24h; B: nucleus; C: co localization of E protein and nucleus; D: expression of JEV E protein after virus inoculation for 48h; E: nucleus; F: co-localization of E protein and nucleus). It can be seen from the figure that all the PT cells have been infected with JEV at 48h.

As shown in Fig. 8, Fig. 8 is a fluorography of a green fluorescent protein transfected and expressed by a pig tonsil cell susceptible to JEV (A: expression of GFP after PT cells are transfected with GFP for 24 h; B: nucleus; C: co-localization of GFP and nucleus; D: expression of GFP after PT cells are transfected with GFP for 48 h; E: nucleus; F: co-localization of GFP and nucleus). It can be seen from the figure that the effectiveness of transfecting PT cells with GFP plasmids is relatively high.

Although the contents of the present invention have been described in detail through the above preferred embodiments, it should be understood that the above description should not be deemed as limiting the present invention. After those skilled in the art read the above contents, multiple modifications and substitutions of the present invention will become apparent. Therefore, the protective scope of the present invention should be limited by the appended

Claims (12)

claims. CLAIMS What is claimed is:

1. A pig tonsil cell line susceptible to JEV, wherein the cell line is obtained by infecting a

primary pig tonsil cell line with lentiviral particles, screening cells in a DMEM/F12 culture

solution containing serum and added with puromycin and then performing amplification culture

on positive cells using a culture solution containing no puromycin;

wherein, the lentiviral particles are obtained by using pHY-sv40-Puro as a lentiviral vector,

using psPAX2 and pCMV-VSV-G as an auxiliary package vector and co-transfecting into 293T cells.

2. The pig tonsil cell line susceptible to JEV according to claim 1, wherein the primary pig

tonsil cell line is cultured in a 5%FBS DMEM/F12 culture medium, and 100 U/mL penicillin

and 100 gg/mL streptomycin are added in the culture medium.

3. The pig tonsil cell line susceptible to JEV according to claim 1, wherein the lentiviral particles are cultured using 2%FBS DMEM.

4. The pig tonsil cell line susceptible to JEV according to claim 1, wherein the medium for

amplification culture is a 5%FBS DMEM/F12 medium containing no puromycin.

5. The pig tonsil cell line susceptible to JEV according to claim 1, wherein 5Rg/mL

polybrene is added when the infection is performed.

6. A construction method of a pig tonsil cell line susceptible to JEV, the method comprising:

obtaining primary pig tonsil cells, and culturing in a 5%FBS DMEM/F12 medium in which 100 U/mL penicillin and 100 g/mL streptomycin are added; obtaining lentiviral particles containing a pHY-sv40-Puro lentiviral vector by using pHY sv40-Puro as a lentiviral vector, using psPAX2 and pCMV-VSV-G as an auxiliary package vector, co-transfecting into 293T cells, and culturing using a 2%FBS DMEM; infecting the cultured the primary pig tonsil cells with the lentiviral particles; after the infection, carrying out cell screening on the pig tonsil cells in a DMEM/F12 culture solution containing serum and added with puromycin to obtain positive cells, wherein the concentration of the puromycin is the minimum lethal concentration of normal pig tonsil cells; carrying out amplification culture on the positive cells with a 5%FBS DMEM/F12 medium containing no puromycin, and constructing to obtain the pig tonsil cell line susceptible to JEV.

7. The construction method of the pig tonsil cell line susceptible to JEV according to claim

6, wherein the preparation of the primary pig tonsil cells comprises: removing connective

tissues of the needed pig tonsil part in a sterile plate, washing with Hank's liquid, cutting into

pieces, adding Hank's liquid, putting the obtained mixture into a sterile conical flask so as to disperse cells, standing, and collecting cell suspension on the upper layer.

8. The construction method of the pig tonsil cell line susceptible to JEV according to claim

6, wherein the preparation of lentiviral particles comprises:

culturing the transfected 293T cells for 48h, then collecting supernatant for the first time,

then culturing for 24h, and harvesting supernatant and cells to be mixed with the previously collected supernatant; and

carrying out multigelation so that cells are broken, filtering, collecting supernatant, mixing the supernatant with PEG8000, obtaining precipitate, and dissolving the obtained precipitate with DMEM culture solution to obtain the lentiviral particles.

9. The construction method of the pig tonsil cell line susceptible to JEV according to any

one of claims 6-8, wherein when the infection is performed, polybrene is added.

10. The construction method of the pig tonsil cell line susceptible to JEV according to claim 9, wherein the concentration of polybrene is 5 g/mL.

11. The construction method of the pig tonsil cell line susceptible to JEV according to any

one of claims 6-8, wherein the amplification culture is carried out by more than 50 generations.

12.The construction method of the pig tonsil cell line susceptible to JEV according to any one of claims 6-8, wherein after the infection, the pig tonsil cells are in a 5% Bioind dialysis

type fetal calf serum DMEM/F12 culture medium.