CN115433719A - Sheep origin immortalized cell line for preparing various viruses - Google Patents
Sheep origin immortalized cell line for preparing various viruses Download PDFInfo
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Abstract
The invention provides a sheep derived immortalized cell line for culturing and preparing viruses. The hTERT gene is introduced into sheep embryonic turbinate cells (OFTu) to construct an immortalized OFTu cell line. The cell line can be used for amplifying and preparing at least one of sheep infective pustule virus (ORFV), sheep Pox Virus (SPV), goat Pox Virus (GPV) and pimple skin disease virus (LSDV). The sheep-derived immortalized cell line provided by the invention has better application and transformation prospects: the cell line can be continuously passaged, and the proliferation performance is stable; (2) The cell line is sensitive to the contagious ecthyma virus of sheep, the titer of the virus harvested after infection is higher, and the virus form is stable. And meanwhile, the virus has better sensitivity to other viruses. The cell line is used for preparing the virus, so that the production cost can be reduced, the batch difference can be reduced, and the guarantee is provided for obtaining high-quality vaccine.
Description
Technical Field
The invention relates to establishment and application of a sheep embryonic turbinate immortalized cell line, in particular to application of sheep infective pustule virus, capripoxvirus and pimple dermatosis virus in culture and preparation of the cell line.
Background
There are various construction schemes for immortalized cell lines. More common schemes include overexpression of human telomerase reverse transcriptase gene (hTERT), SV40 large T antigen fragment, HPV early gene fragment, etc. The construction of the immortalized cell line can lead the cells to be in a continuous division state, simultaneously maintain the genetic and phenotypic characteristics of the cells and be beneficial to the basic research of cell molecular biology. Furthermore, immortalized cell lines can also be used for viral vaccine production. Under the premise of maintaining the stability of the genetic characteristics of the virus, the virus is efficiently amplified and prepared, and is used for the large-scale production of vaccines.
The application relates to the field of amplification preparation of poxviruses such as contagious pustular dermatitis virus, capripoxvirus, pimple skin disease virus and the like. Early studies showed that the viruses can be cultured in primary ovine embryonic turbinate cells. However, the primary cells have limited passability, and according to the experience of different batches, the cells can pass through 20-30 generations and then undergo differentiation, thereby remarkably prolonging the cell division time and reducing the virus yield. The repeated material drawing of the primary cells not only has larger batch difference, but also has high cost and long time consumption. Has great application limitation.
Furthermore, it has been reported that primary cells of sheep and cattle testis can also amplify the above viruses. However, the above-mentioned tissue acquisition process involves the risk of infecting brucella, and requires strict detection for each sampling, which is costly and time-consuming in terms of biological safety. It also has certain limitations.
Disclosure of Invention
The invention provides an immortalized sheep embryo turbinate cell line for culturing and preparing poxviruses, wherein the poxviruses comprise sheep infective pustule virus, sheep poxvirus, goat poxvirus and pimple dermatosis virus. Also includes attenuated strain based on the artificial passage domestication of the virus and attenuated strain with virulence gene deletion.
The invention provides a sheep derived immortalized cell line, which is preserved and numbered as follows: CCTCC NO: c2022191, the deposit name is: the immortalized cell line OFTu of sheep turbinate bone, which is preserved in China center for type culture Collection with the address as follows: china, wuhan university, phone (027) 68752319, the culture was received from the collection at 23/6/2022 and registered in the book. Viability of the cultures the depository was tested at 2022, 6 months and 27 days and the results were survival. Namely an ovine immortalized cell line, also called as OFTu immortalized cell line or OFTu cell line. The cell line is a sheep origin immortalized cell line used for virus culture and preparation, and the cell line is an immortalized sheep embryo turbinate cell line. Preferably, the cell line is used for culturing and preparing poxvirus, wherein the poxvirus comprises contagious pustular virus, ovine poxvirus, caprine poxvirus and pimple dermatosis virus, and the poxvirus comprises attenuated strains based on the viruses through artificial subculture and attenuated strains with virulence gene deletion.
In the present invention, the sheep derived immortalized cell line is also called OFTu immortalized cell line or OFTu cell line.
Preferably, the sheep-derived immortalized cell line is an immortalized sheep embryo turbinate cell line prepared by transfecting human source telomerase reverse transcriptase gene. The hTERT gene of the sheep immortalized cell line can be effectively expressed after multiple passages. And (3) carrying out expression detection on the OFTu cell line at the transcription level until twenty generations of hTERT gene expression maintains high expression.
Any one of the above is preferably that the virus is at least one of contagious pustular virus (ORFV), sheep Pox Virus (SPV), goatpox virus (GPV), and pimple skin disease virus (LSDV), and the immortalized cell line of ovine origin is used for the culture or production of the virus. The capacity of the sheep-derived immortalized cell line in culturing and preparing the virus is partially superior to that of common cell lines such as VERO, MDBK and the like.
Preferably, any one of the above, said immortalized cell line of ovine origin is used for the preparation of an attenuated strain of said virus, said attenuated strain of said virus comprising a deletion of at least one virulence gene of said virus. The good virus preparation capacity of the sheep-derived immortalized cell line is particularly the high-efficiency preparation capacity of attenuated strains with corresponding virus virulence gene deletion (single or multiple). The sheep immortalized cell line has no significant difference with the primary OFTu cell in the aspect of virus amplification capacity, and is significantly stronger than the current commercialized cell line, such as VERO cell line. And the sheep immortalized cell line can also effectively amplify gene deletion virus.
Preferably, in any of the above, the immortalized cell line of ovine origin may be passaged for at least 80 passages. The cell proliferation speed of the sheep immortalized cell line is obviously higher than that of the primary OFTu cell after multiple passages, at least eighty passages of the sheep immortalized cell line still keeps a good proliferation state, and the chromosome morphology is normal.
Preferably, in any one of the above cases, the generation ratio of the sheep-derived immortalized cell line is 1:1-10, and the optimal passage ratio is 1:5. it may be preferable that the ratio of 1: 1. 1: 2. 1: 3. 1: 4. 1: 6. 1: 7. 1: 8. 1: 9. 1:10.
any one of the above is preferred, the sheep derived immortalized cell line has the characteristics of faster division, less differentiation tendency, low spontaneous apoptosis rate and high tolerance to digestive enzymolysis process compared to primary cells.
Preferably, any one of the above, the immortalized cell line of ovine origin is capable of cell transfection. Basic research and drug development can continue with the method of cell transfection. Cells may be transfected using a conventional transfection reagent such as liposome, PEI (polyethyleneimine) or calcium phosphate, or may be transfected using an electrotransfection method. The activity of the cells after transfection of the transfection reagent is more than 80 percent, and the activity of the cells after electrotransfection is more than 60 percent. After transfection, the cell line can continue to survive and proliferate.
Preferably, in any of the above, the immortalized cell line of sheep origin is further genetically modified. And carrying out genetic modification again on the basis of the sheep derived immortalized cell line so as to further improve the culture capacity of the immortalized cell line subjected to genetic optimization to the viruses: or is suitable for more poxviruses to be cultured and prepared efficiently.
The invention provides application of any one of the sheep-derived immortalized cell lines in virus culture and preparation.
Preferably, the use further comprises the preparation and culture of an attenuated strain of the virus comprising a deletion of at least one virulence gene of the virus.
Preferably, in any of the above cases, the virus is at least one of impetigo ovis, sheep pox virus, goat pox virus, and pimple skin disease virus.
The invention provides application of any one of the sheep-derived immortalized cell lines in a cell transfection experiment.
The sheep-derived immortalized cell line is subjected to cell transfection. The transfection may be performed by using a common transfection reagent such as liposome, PEI (polyethyleneimine) or calcium phosphate, or by using an electrotransfection method. The activity of the cells after transfection of the transfection reagent is more than 80 percent, and the activity of the cells after electrotransfection is more than 60 percent. After transfection, the cell line can continue to survive and proliferate.
In a preferred embodiment of the present invention, the sheep-derived immortalized cell line is prepared by the following steps:
first, infectious diseases such as Peste des petits ruminants, tuberculosis, foot and mouth disease, sheep pox, brucellosis and the like are detected in healthy pregnant sheep with 3-5 months of gestation, and the detection result is negative. The pregnant sheep were then anesthetized, removed aseptically, and the nasal concha tissue of the foetal sheep was isolated and washed 3-5 times with D-Hank's solution containing the diabody (100U/mL penicillin and 100. Mu.g/mL streptomycin). After the tissue is sterilized in the laboratory, it is introduced into the laboratoryPerforming aseptic cutting to obtain fine tissue blocks, performing enzymolysis with 0.25% trypsin at 37 deg.C for 3 times, each time for 15-20min, and shaking every 5min, and stopping when the liquid becomes turbid and viscous. Transfer the liquid to a new 50mL centrifuge tube and add serum to stop the digestion. The 70 μm mesh was placed in a new 50mL centrifuge tube for filtration and the tissue mass was removed. After the filtered cell suspension is centrifuged at 1000rpm for 10min, D-Hank's containing double antibody is added for blowing and resuspension, and the washing is repeated for 3 times. Finally, the cell pellet was blown out with a DMEM medium containing 20% FBS and 1% double antibody, and transferred to a cell culture flask. At 37 ℃ C, 5% CO 2 Culturing in an incubator, observing the growth condition of the cells every day, and performing liquid change or passage operation according to the confluence degree of the cells.
When the fetal sheep turbinate tissue cells overgrow the culture bottle, the tissue blocks and the culture solution are discarded. Simultaneously, primary cell digestion is carried out by trypsin, and the digested primary cells 1: and 2, subculturing. And (3) performing partial cryopreservation on the primary cells after the cells are overgrown, wherein the cryopreservation condition is a culture solution containing 5% of fetal calf serum and 10% of DMSO. After the frozen cells are recovered, the cell state and growth vigor are good. Primary ovine embryonic turbinate cells (OFTu cells) were thus obtained.
Primary cell transfection conditions were then screened. Firstly, laying primary cells on a 96-well plate, and transfecting when the cells reach 50-60%, 60-70%, 70-80%, 80-90% and 90-100% respectively. The transfection conditions were: liposome: the ratio of DNA (pEGFP-C3, genBank: U57607.1) was 1:1-10:1. it was found that liposomes (volume. Mu.L) at cell densities of 70-80%: the ratio of DNA (mass. Mu.g) was 2: 1. the state was best for this primary cell, and the highest transfection efficiency was obtained.
The transfection assay of the hTERT expression vector was performed under the transfection conditions described above. (1) The cells to be transfected are subcultured to a 100mm large dish, and the old culture solution is discarded when the confluency of the cells is 70-80%, and is replaced by 10mL of fresh culture solution; (2) Adding 50 mu L of Lipo3000 transfection reagent into 1mL of serum-free and double-antibody-free culture solution, gently pumping and uniformly mixing, and standing for 5min at room temperature; (3) Adding 25 mu g of plasmid DNA into 1mL of serum-free and double-antibody-free culture solution, blowing, beating and uniformly mixing; (4) Adding the plasmid DNA mixed solution into the Lipo3000 mixed solution, gently blowing and beating for 4-5 times, and incubating for 20min at room temperature; (5) Adding the Lipo-DNA compound into a 100mm dish along the side wall of the culture plate, and slightly shaking the culture dish according to the cross to uniformly mix the liquid; (6) After culturing for 6h in the incubator, the culture solution containing the Lipo-DNA complex is discarded, and the culture is continued by replacing with a complete culture solution containing serum. 48 hours after transfection, the cells were subjected to G418 drug screening at a screening concentration of 300. Mu.g/mL. After 3 days of cell selection, cells in the control group that were not transfected with plasmid all died, and the G418 selection concentration was reduced to 150. Mu.g/mL for the following 1 week. After the screening, single cell clone is picked and enlarged for culture.
After the cell line obtained by the scheme is subjected to amplification culture, total RNA is extracted and is subjected to reverse transcription to form cDNA, and the transcription level of the hTERT gene is detected by a PCR method. The hTERT transcription level detection is respectively carried out on the cells of the first generation, the fifth generation, the tenth generation, the fifteenth generation, the twentieth generation and the twenty-fifth generation, and the transcription of the hTERT gene is detected in the cell lines, which indicates that the gene is effectively expressed in the constructed cell lines.
And analyzing the cell viability and the cell growth speed of the fifteenth generation cell line, the twentieth generation cell line and the fifteenth generation primary cell by a CCK8 cell viability detection test and a cell counting method respectively. The research finds that the cells of each generation have no significant difference in cell viability, cell growth and the like. The successful construction of the immortalized cell line is proved.
And (3) transmitting the constructed OFTu immortalized cell line to a six-pore plate, and detecting the cell seed separation rate. After seeding the cells in the plate, the growth vigor of the cells was examined and the seeding rate of the cells was recorded at 1:3 to 1: growth after cell passage at 10 o. When the cell seeding rate is 1:3 hours, the time required for the cell line to grow full of the monolayer is between 48 and 50 hours; when the cell's seed separation rate is 1:5 hours, the time required for the cell line to overgrow the monolayer is between 56 and 58 hours; when the cell's seed separation rate is 1:6 hours, the time required for the cell line to grow full of the monolayer is between 60 and 62 hours; when the cell's seed separation rate is 1: at 10 hours, the time required for the cell line to grow full of the monolayer was between 72-74 hours.
Subsequent detection of cytopathy following infection of OFTu immortalized cell lines with Epstein-Barr Virus (ORFV)Time required for Change (CPE). The results of the experiments showed that the time required for CPE production was 3-4 days for 80% of the cells after inoculation of the ORFV immortalized cell line with OFTu. Simultaneous detection of viral titre (logTCID) 50 ) Above 6.5, indicating that the OFTu immortalized cell line can efficiently expand ORFV. After infecting the OFTu immortalized cell line with capripoxvirus (SPV), the time required for 80% of the cells to produce CPE is 5-6 days; after infecting the OFTu immortalized cell line with Lumpy Skin Disease Virus (LSDV), 80% of the cells required 4-5 days to produce CPE, and the virus titer (logTCID) was measured 50 ) Above 6.5, the OFTu immortalized cell line can effectively amplify SPV and LSDV.
Selecting primary cells showing logarithmic growth and a sixty-five generation OFTu immortalized cell line, adding colchicine into a cell culture solution, incubating for 5 hours, digesting the cells by pancreatin, and centrifuging to obtain cell precipitates. The cell pellet was resuspended in 50mM KCl solution and treated for 40 minutes. The cells were fixed by adding methanol/glacial acetic acid (3). And adding methanol/glacial acetic acid fixing solution again, fully resuspending the cells, and then dripping the cell suspension on a 4-DEG C precooled slide for drying. Giemsa staining was then performed and the chromosomes were observed under a microscope. The result shows that the chromosome morphology of the primary cell and the OFTu immortalized cell line is normal, 2 times of the chromosome morphology and has 54 chromosomes.
Selecting primary cells and sixty-five generations of OFTu immortalized cell lines, selecting Hela cells as positive control, selecting PBS solution as negative control, and performing nude mouse subcutaneous inoculation test, wherein the number of inoculated cells is 10 7 Cell/cell. The formation of tumor masses could be detected in situ on the seventh day after the inoculation of HeLa cells into nude mice, and the average diameter of the tumor masses was 1cm 20 days after the inoculation of Hela cells. In contrast, no lumps were formed 30 days after injecting primary, sixty-five passages OFTu immortalized cell lines subcutaneously into nude mice. After the breeding is continued for 3 months, the lumps still do not appear. The experiment proves that the sheep derived immortalized cell line (namely, the OFTu immortalized cell line) constructed by the invention has no tumorigenicity.
The sheep-derived immortalized cell line has good application and transformation prospects: (1) The sheep-derived immortalized cell line can be continuously passaged, and the proliferation performance is stable; (2) The sheep-derived immortalized cell line is sensitive to the contagious ecthyma virus of sheep, the titer of the virus harvested after infection is higher, and the virus form is stable; (3) The sheep immortalized cell line has no tumorigenicity, and is safe and reliable. The sheep-derived immortalized cell line is used for preparing the virus, so that the production cost can be reduced, the batch difference can be reduced, and the guarantee is provided for obtaining high-quality vaccine.
Drawings
FIG. 1 is a plasmid map of pCI-neo-hTERT in example 1 of the present invention.
FIG. 2 shows the PCR identification of hTERT gene transcript in different generations of OFTu cell line in example 1 of the present invention.
FIG. 3 is a comparison of growth status of different generation primary OFTu cells and OFTu cell lines observed under microscope in example 2 of the present invention.
FIG. 4 is a comparison of the viability of primary OFTu cells of different generations and cells of the OFTu cell line tested by the CCK8 method in example 2 of the present invention.
FIG. 5 shows the cytopathic state of ORFV infected primary OFTu cell and OFTu immortalized cell line in example 3 of the present invention.
FIG. 6 is an electron microscope observation of virus particles formed after ORFV inoculation of primary OFTu cells and OFTu immortalized cell lines in example 3 of the present invention.
FIG. 7 is a comparison of the virus titers of ORFV inoculated with primary OFTu cells, OFTu cell lines and VERO cell lines in example 3 of the present invention.
FIG. 8 shows the cytopathic state of SPV infected primary OFTu cells and OFTu cell lines under microscope observation in example 4 of the present invention.
FIG. 9 shows the cytopathic state of primary OFTu cells and OFTu cell lines infected with LSDV under microscope in example 5 of the present invention.
FIG. 10 is a comparison of virus titers after inoculation of LSDV in primary OFTu cells and OFTu cell lines in example 5 of the present invention.
FIG. 11 shows the transfection efficiency of primary OFTu cells and OFTu cell lines observed by fluorescence microscopy in example 6 of the present invention.
FIG. 12 shows fluorescence microscopy of primary OFTu cells and preparation and amplification of ORFV-. DELTA.120-GFP attenuated strains of OFTu cell lines in example 6 of the present invention.
FIG. 13 is a diagram showing the cytopathic state of primary OFTu cells and OFTu cell lines infected with ORFV-. DELTA.120-GFP attenuated strain under a microscope in example 6 of the present invention.
FIG. 14 is a microscopic observation of primary OFTu cells and OFTu cell line chromosomes in example 7 of the present invention.
FIG. 15 shows the tumorigenesis of the control cells of PBS, primary OFTu cells, OFTu cell lines and control Hela cells inoculated into nude mice in example 8.
Detailed Description
The present invention will be more clearly and completely described in the following embodiments, but the described embodiments are only a part of the embodiments of the present invention, and not all of them. The examples are provided to aid understanding of the present invention and should not be construed to limit the scope of the present invention.
Example 1
Identification of expression level of hTERT gene in OFTu cell line:
as described above, a well-conditioned primary OFTu cell was first obtained by a primary cell culture technique in which the culture conditions were DMEM medium containing 20% FBS, 1% diabody (with penicillin at 100U/mL and streptomycin at 100. Mu.g/mL), and after the cell state was stabilized, FBS was maintained at 10%. Subsequently subjecting the primary cells to 37 ℃, 5% CO 2 Culturing in an incubator, and observing the growth and proliferation conditions of the cells every day. When the primary cells were passaged, the cells from which the culture medium was discarded were washed 2 times with PBS and digested with an appropriate amount of 0.25% trypsin. Subjecting the trypsinized primary cells to a reaction of 1: and 2, subculturing. When the state and the growth vigor of the primary cells are good, a cell transfection test is carried out.
In terms of cell density of 70-80%, liposomes (volume. Mu.L): the ratio of plasmid DNA (mass. Mu.g) was 2:1, and plasmid DNA is a recombinant plasmid pCI-neo-hTERT (FIG. 1) capable of overexpressing hTERT gene (GenBank: AB 085628.1). If the cell transfection efficiency is low, secondary transfection can be carried out after the first transfection of the cells, the old culture solution in a 10cm dish is discarded before transfection,the medium was replaced with 10mL of fresh medium. And adding the recombinant plasmid DNA mixed solution for over-expressing the hTERT gene into the Lipo3000 mixed solution, gently blowing and beating for several times, incubating at room temperature for 20min, then dropwise adding the Lipo-DNA compound into a 100mm cell culture dish, and gently shaking the culture dish to uniformly mix the transfection compound. 6h after addition of the transfection complex, replacing the fresh complete medium, 5% CO at 37 ℃% 2 Culturing in an incubator for 48h, and observing the growth and proliferation conditions of the cells every day. Meanwhile, empty plasmids were transfected into primary cells as a control group. And then, carrying out G418 drug screening on the transfected primary cells and untransfected primary cells, wherein the screening concentration is 300 mu G/mL. After 3-4 days of cell selection, the G418 selection concentration was reduced to 150. Mu.g/mL after all untransfected cells had died. Then picking single cell clone and carrying out cell amplification culture.
The single cell clone obtained by the scheme is subjected to amplification culture, cell samples are obtained at different generations to extract total RNA, and then the total RNA is reversely transcribed into cDNA. The transcription level of the plasmid-transfected hTERT gene was detected by PCR. The results of the experiments suggest that transcription of hTERT gene was detected in all of the above cell lines (269 bp band), indicating that telomerase gene was efficiently expressed in the cell lines of the above generations (fig. 2, m.
Example 2
Identification of growth and proliferation states of OFTu cell lines:
the primary OFTu cells and the OFTu immortalized cell lines obtained in example 1 were compared in terms of cell growth status, and fig. 3 shows the comparison in terms of the growth status of different generations of primary OFTu cells and OFTu immortalized cell lines observed under a microscope, where a: twentieth generation B of primary OFTu cells: thirty th generation C of primary OFTu cells: the twentieth generation D of the OFTu cell line: the eighty-th passage of the OFTu cell line. First comparing primary OFTu cells that also underwent 20 passages (fig. 3A) and an OFTu immortalized cell line (fig. 3C), it was found that the proliferation rate of hTERT transfected OFTu cells was significantly faster than primary OFTu cells. The growth rate of primary OFTu cells after 30 passages (fig. 3B) was significantly slowed down and even stopped proliferating, while the immortalized cell line of OFTu (fig. 3D) remained well proliferating after 80 passages.
The viability of the cells of different generations was examined by the CCK8 method. First comparing primary OFTu cells and OFTu immortalized cell lines, which were also subjected to 15 passages, it was found that the proliferation rate of the OFTu immortalized cell lines was significantly faster than primary OFTu cells at different time points (48 h, 72h, 96 h) (fig. 4). Also, when comparing primary OFTu cells subjected to 20 passages with OFTu immortalized cell lines, it was found that the proliferation rate of the OFTu immortalized cell lines was significantly faster than primary OFTu cells (fig. 4). Meanwhile, the activity states of the OFTu immortalized cell lines of 15 and 20 generations are not obviously different; whereas the activity status of the primary OFTu cells of passage 15 was superior to that of the primary OFTu cells of passage 20 (fig. 4).
The CCK8 assay used in the present invention is a method disclosed in the prior art.
Example 3
And (3) identifying the level of the amplified contagious ecthyma virus of the OFTu cell line:
and (3) inoculating the OFTu cell line (35 generations) and the OFTu primary cell (10 generations) obtained by the scheme with the contagious ecthyma virus (ORFV), and detecting the sensitivity of the cells or the cell line to the virus. Cytopathic effect (CPE) was detected 72h after infection of cells with wild-type ORFV. The experimental results showed that both primary OFTu cells and OFTu cell lines produced typical CPE within 72h after inoculation with ORFV, indicating that the OFTu cell line was sensitive to ORFV (fig. 5, where 5A was primary OFTu cells and 5B was OFTu cell line).
Further, it was examined whether ORFV produced cytopathic effects in the ovine immortalized cell line and also produced morphologically intact viral particles. After inoculation for 96h, the diseased cell line and the primary cells (as a control) are collected, viruses are concentrated by an ultracentrifuge, and then a high-purity virus solution is obtained by a density gradient centrifugation method. The cell line infected by the virus was found to produce virus particles with the same morphology as the primary cells by transmission electron microscopy (fig. 6, wherein 6A is the primary OFTu cell and 6B is the OFTu cell line), confirming that the virus particles obtained by culturing after immortalizing the primary OFTu cell into the OFTu cell line were not changed in morphology and potential performance.
The ability of primary OFTu cells, OFTu cell lines and VERO cell lines to produce ORFV virions was also compared. Lesion OFTu cell lines, primary OFTu cells (as control) and VERO cell lines (as control) were collected 96h after the inoculation. By TCID 50 The method detects the virus titer. The results show that the OFTu cell lines did not differ significantly from the primary OFTu cells in their ability to amplify viruses and were all significantly stronger than the VERO cell lines (fig. 7). The sheep-derived immortalized cell line can be used for amplifying ORFV in a large amount. The sources of the wild strains of ORFV are as follows. ( 1.Zhong J #, guan J #, zhou Y, cui S, wang Z, zhou S, xu M, wei X, gao Y, ZHai S, song D, he W, gao F, ZHao K, genomic characterization of two or more microorganisms from J protein protocol in China Genes,2019,55: 490-501.2.ZHao K, song D, he W, lu H, zhang B, li C, chen K, gao F.identification of a polymeric analyte from an outer sample in a sheath sample in the sheath protocol of the microorganism, and 19. Main 19;142 (3-4):408-15. )
Example 4
Identification of the level of amplified sheep pox virus of OFTu cell line:
the OFTu cell line (35 generations) and the OFTu primary cell (10 generations) obtained by the above-mentioned protocol are inoculated with Sheep Pox Virus (SPV), and the sensitivity of the above-mentioned cell or cell line to the virus is detected. After 96h of SPV infection of cells, cytopathic effect (CPE) was detected. The experimental results showed that both primary OFTu cells and OFTu cell lines produced typical CPE within 96h after inoculation of SPV, indicating that OFTu cell lines were sensitive to SPV (fig. 8, where 8A was primary OFTu cells and 8B was OFTu cell lines).
Example 5
And (3) identifying the level of the OFTu cell line amplified pimple skin disease virus:
the OFTu cell line (35 generations) and the OFTu primary cell (10 generations) obtained by the scheme are inoculated with the pimple skin disease virus (LSDV), and the sensitivity of the cell or the cell line to the virus is detected. Cytopathic effect (CPE) was detected 72h after infection of the cells with LSDV. The experimental results showed that both primary OFTu cells and OFTu cell lines produced typical CPE within 72h after inoculation with LSDV, indicating that OFTu cell lines were sensitive to LSDV (fig. 9, where 9A was primary OFTu cells and 9B was OFTu cell lines).
The ability of primary OFTu cells and OFTu cell lines to produce LSDV virions was also compared. Lesion OFTu cell lines and primary OFTu cells (as controls) were collected 120h after the inoculation. By TCID 50 The method detects the virus titer. The results show that the OFTu cell line does not differ significantly from primary OFTu cells in its ability to amplify viruses (fig. 10).
Example 6
Detecting and preparing the transfection efficiency of the OFTu cell line, and identifying the capability of amplifying ORFV virulence gene deletion strains:
the cell line OFTu cell line obtained in the above protocol (35 passages) and OFTu primary cells (10 passages) were passaged into 100mm dishes, liposomes (μ L): plasmid (pEGFP-C3) (μ g) was 2:1 conditions for transfection. The results of the experiments showed that the transfection efficiency of the cell lines was higher than that of the primary OFTu cells (FIG. 11, in which 11A is the primary OFTu cell and 11B is the OFTu cell line)
And detecting the preparation and amplification capability of the cell line on ORFV virulence gene deletion strains. The experimental results showed that both primary OFTu cells and OFTu cell lines were able to produce ORFV120 gene-deleted strain carrying green fluorescence (ORFV- Δ 120-GFP attenuated strain) (fig. 12, where 12A is ORFV- Δ 120-GFP produced by primary OFTu cells and 12B is ORFV- Δ 120-GFP produced by OFTu cell line). The obtained ORFV-delta 120-GFP attenuated strain is used for infecting primary OFTu cells and OFTu cell lines at the same time, and the test result shows that the ORFV-delta 120-GFP attenuated strain can be effectively amplified in the cell lines (FIG. 12, wherein 12C is ORFV-delta 120-GFP infected primary OFTu cells 48h, and 12D is ORFV-delta 120-GFP infected OFTu cell line 48 h). Meanwhile, after ORFV-delta 120-GFP infects cells for 72h, cytopathic effect (CPE) was detected. The experimental results showed that both primary OFTu cells and OFTu cell lines produced typical CPE (fig. 13, where 13A was primary OFTu cells and 13B was OFTu cell line). The obtained OFTu immortalized cell line is proved to be capable of effectively amplifying gene deletion viruses, thereby increasing the application range of the cell line. The ORFV-delta 120-GFP attenuated strain is constructed by the following method. (Zhou, yanlong et al, ORF Virus ORF120 Protein Positively Regulation the NF-. Kappa.B path by interaction with G3BP1.Journal of virology vol.95,19 (2021))
Example 7
Chromosome observation of OFTu cell line:
in order to examine the chromosome stability of the OFTu cell line obtained by the above protocol, a chromosome of the cell was prepared. Selecting primary cells presenting logarithmic growth and a sixty-fifth generation OFTu cell line, adding colchicine into a cell culture solution, incubating for 5 hours, digesting the cells by pancreatin, and centrifuging to obtain cell precipitates. The cell pellet was resuspended in 50mM KCl solution and treated for 40 minutes. The cells were fixed by adding methanol/glacial acetic acid (3). And adding methanol/glacial acetic acid fixing solution again, fully resuspending the cells, and then dripping the cell suspension on a 4-DEG C precooled slide for drying. Then giemsa staining was performed, and the chromosomes were observed under a microscope (100-fold oil-scope). The results showed that the primary cell, the OFTu immortalized cell line, was morphologically normal, 2-fold, with 54 chromosomes (fig. 14, where 14A was the primary OFTu cell and 14B was the OFTu cell line).
Example 8
Identification of potential tumorigenicity of OFTu cell line:
in order to test the safety of the OFTu cell line obtained by the scheme, the potential tumorigenicity of the cell line is tested. PBS (negative control), primary OFTu cells, sixty-five generation OFTu cell line and Hela cells (positive control) were inoculated subcutaneously into nude mice, and the number of inoculated cells was 10 7 Cell/cell. Tumor growth was measured day by day after one week of cell injection. After 7 days of cell injection, the nude mice inoculated with Hela cells form obvious tumor masses, and the tumor masses grow rapidly within 20 days of Hela cell inoculation, and the average diameter of the tumor can reach 1cm. In contrast, the co-injection of primary OFTu cells, a sixty-five generation OFTu cell lineNo significant lumps were observed in situ in nude mice 30 days after cell injection (fig. 15, where 15A is PBS control, 15B is primary OFTu cells, 15C is OFTu cell line, 15D is Hela cells). No lump was formed after 3 months. The experiment proves that the constructed OFTu immortalized cell line has no tumorigenicity and is safe and reliable.
The sheep origin immortalized cell line of the invention has the preservation number of: CCTCC NO: C2022191. obtained by the method described in example 1 and tested via examples 2-8. The hTERT gene of the sheep-derived immortalized cell line can be effectively expressed after being passed for multiple times. And (3) carrying out expression detection on the OFTu cell line at the transcription level until twenty generations of hTERT gene expression maintains high expression. The virus amplification capacity of the cell line is not significantly different from that of the primary OFTu cell, and the cell line is significantly stronger than that of the current commercialized cell line, such as VERO cell line. And the sheep immortalized cell line can also effectively amplify gene deletion virus. After multiple passages, the cell proliferation speed of the sheep immortalized cell line is obviously higher than that of the primary OFTu cell, at least eighty generations of the sheep immortalized cell line still keep a good proliferation state, and the chromosome morphology is normal. No tumorigenicity and safe and reliable cell line.
Claims (10)
1. An immortalized cell line of ovine origin, preserved under the accession number: CCTCC NO: C2022191.
2. the cell line of claim 1, wherein the sheep derived immortalized cell line is an immortalized sheep embryonic turbinate cell line prepared by transfection of a human source telomerase reverse transcriptase gene.
3. The cell line of claim 1 or 2, wherein the sheep derived immortalized cell line is passaged at a ratio of 1:1 to 10.
4. The cell line of claim 1 or 2, wherein the immortalized cell line of ovine origin can be passaged for at least 80 passages.
5. The cell line according to claim 1 or 2, wherein the sheep derived immortalized cell line divides more rapidly, is less prone to differentiation, has a low spontaneous apoptosis rate and is more resistant to digestion processes than primary sheep embryonic turbinate cells.
6. The cell line of claim 1 or 2, wherein the sheep derived immortalized cell line is further genetically engineered.
7. Use of the sheep-derived immortalized cell line according to any one of claims 1 to 6 for the culture and production of viruses.
8. The use of claim 7, further comprising preparing and culturing an attenuated strain of said virus comprising a deletion of at least one virulence gene of said virus.
9. The use of claim 7 or 8, wherein the virus is at least one of impetigo ovis, poxvirus capripoxvirus, dermatoma lumpy virus.
10. Use of the ovine immortalized cell line according to any one of claims 1 to 6 in cell transfection experiments.
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