CN109486769A - A kind of couple of PRRSV susceptible pig endometrial epithelial cell system and its construction method - Google Patents
A kind of couple of PRRSV susceptible pig endometrial epithelial cell system and its construction method Download PDFInfo
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Abstract
The invention discloses a kind of couple of PRRSV susceptible pig endometrial epithelial cell system and its construction methods, it is using pig endometrial epithelial cell as host cell, transfection has the slow virus expression plasmid of SV40T antigen gene, obtains after Puromycin drug screening, cell expansion culture.The pig endometrial epithelial cell system that the present invention constructs has the function of infinitely passing on, and can maintain the characteristic of pig endometrial epithelial cell, and it is normal to be able to maintain caryogram in succeeding generations, and there is no chromosome structure sum number purposes to change；Moreover, the pig endometrial epithelial cell system is sensitive to PRRSV virus, it can be used for the passage and relevant research of PRRSV.
Description
Technical field
The present invention relates to field of transgenic technology, and in particular to a kind of couple of PRRSV susceptible pig endometrial epithelial cell
System and its construction method.
Background technique
Pig breeding and disordered breathing syndrome, are called pig blue-ear disease, bring about great losses in the production process of pig.The disease
Caused by pig breeding and disordered breathing syndrome viral (PRRSV).The disease only infects pig, other animals will not infect this virus,
And the pig of each age and kind can infect this virus, wherein the most susceptible with the piglet in farrowing sow and January.
The clinically property symptom characterized by pregnant sow miscarriage and the premature labor of fetus.Early-stage study shows to have infected the tire of PRRSV virus
Youngster can cause to miscarry because of the apoptosis of placenta cells.And endometrium and fetal placenta play the foundation and maintenance of gestation
Important function.It researchs and proposes, the cell membrane of fetus, the apoptosis of the endometrial epithelial cell of parent and the termination and mistake of gestation
Losing has important relationship.But be limited to continuous variation of the pig endometrial epithelial cell in the oestrous cycle each time, so far for
Only, the endometrial epithelial cell system of pig is not still successfully established.
In the method for building up of progenitor cells system, the method for Telomerase and SV40 large T antigen is two kinds of most common methods.
The method of Telomerase is the extension based on telomere to achieve the purpose that cell line constructs, and the building mode of Telomerase is more advantageous to
Maintain the biological characteristics of the cell line built.But it is limited to the passage number of endometrial epithelial cell, currently with end
The technology of granzyme is still immature to construct.And the construction method of SV40 large T antigen cell line is then to make cell to anti-tumor transformation
To reach the building condition of cell line, but the cell line of SV40 large T antigen method building is during Long Term Passages, possible
It can be with the change of caryogram.
Due to so far still without treatment pig blue-ear disease specific drug, so vaccine become control PRRSV outburst and propagate
Effective ways and main means.PRRSV has very strong host cell tropism, currently used for separating and cultivating the cell of PRRSV
Mainly porcine alveolar macrophage (PAM) and African green monkey kidney cell (Marc-145), but PAM is not and uterus and placenta phase
The cell of pass；Marc-145 derives from monkey, rather than pig derived cell, will lead to PRRSV in vitro study be limited to cannot to react from
The infection conditions of right host cell.Therefore, need to construct the endometrial epithelial cell system in the susceptible pig source new PRRSV,
To simulate the process of PRRSV infection natural reservoir (of bird flu viruses) cell, expand research range.
Summary of the invention
For the above-mentioned prior art, the object of the present invention is to provide the susceptible pig endometrial epithelium of a kind of couple of PRRSV is thin
Born of the same parents system and its construction method.The pig endometrial epithelial cell system that the present invention constructs has the function of infinitely passing on, and can maintain
The characteristic of pig endometrial epithelial cell, it is normal to be able to maintain caryogram in succeeding generations, and chromosome structure and number is not present
Variation；Moreover, the pig endometrial epithelial cell system is sensitive to PRRSV virus, it can be used for the passage and correlation of PRRSV
Research.
To achieve the above object, the present invention adopts the following technical scheme:
The first aspect of the present invention provides the susceptible pig endometrial epithelial cell system of a kind of couple of PRRSV, is with pig uterus
Intimal epithelium cell is as host cell, and transfection has the slow virus expression plasmid of SV40T antigen gene, through Puromycin medicine
It is obtained after object screening, cell expansion culture.
Preferably, the slow virus expression plasmid includes: SV40 large T antigen gene, antibiotic-screening gene Puro gene
And EGFP gene.
It is furthermore preferred that the slow virus expression plasmid is pLVX-EGFP-T2A-Puro-SV40T.
The second aspect of the present invention provides the construction method of above-mentioned pig endometrial epithelial cell system, comprising the following steps:
(1) primary pig endometrial epithelial cell is obtained, and is cultivated；
(2) using pLVX-EGFP-T2A-Puro-SV40T as slow virus carrier, using psPAX2 and pCMV-VSV-G as
Package carrier is assisted, lentiviral particle is obtained；
(3) using the primary pig endometrial epithelial cell after lentiviral particle transfection culture；
(4) it after lentiviral particle transfection pig endometrial epithelial cell, is added in the DMEM/F12 culture solution containing serum
The Puromycin of final concentration of 2 μ g/ml carries out cell screening, obtains positive cell；
(5) culture solution without Puromycin is used to expand culture the positive cell of screening, i.e. building is obtained to PRRSV
Susceptible pig endometrial epithelial cell system.
Preferably, in step (1), the method that obtains primary pig endometrial epithelial cell and culture are as follows: acquisition pig uterus
Pig endometrium is shredded into small fragment of tissue, is directly seeded to Tissue Culture Dish by inner membrance, and culture solution is not added and is put into incubator
Middle 10-20min；A small amount of culture solution is added after taking-up, continues to cultivate in incubator, fluid infusion adds simultaneously to tissue block is not crossed after 4h
Add penicillin and streptomysin, changes the liquid once every other day.
It is furthermore preferred that the additional amount of the penicillin is 100U/ml, the additional amount of streptomysin is 100 μ g/ml.
Preferably, in step (2), pLVX-EGFP-T2A-Puro-SV40T, psPAX2 and pCMV-VSV-G are turned jointly
Dye enters 293T cell, harvests supernatant after 48h；Supernatant cell is harvested after 72h, and is mixed with the supernatant harvested before, repeatedly
Freeze thawing makes cell rupture, and filtering collects supernatant, supernatant is mixed with 5 × PEG8000, and overnight, 4000g is centrifuged for 4 DEG C of sedimentations
1h removes supernatant, and precipitating DMDM culture solution is dissolved, i.e. acquisition lentiviral particle.
Since the angling of epithelial cell, especially pig endometrial epithelial cell is serious, therefore transfect extremely difficult.
In order to transfect to pig endometrial epithelial cell, the present invention has carried out preferably the lentiviral particle for transfection, knot
Fruit discovery, using pLVX-EGFP-T2A-Puro-SV40T as slow virus carrier, using psPAX2 and pCMV-VSV-G as auxiliary
Package carrier, the lentiviral particle constructed in this way are best to the transfection of pig endometrial epithelial cell.
Preferably, it in step (3), when primary pig endometrial cell culture was to the 7th day, carries out lentiviral particle and turns
Dye.The research of the invention finds that cell viability is best, is conducive to slow disease when primary pig endometrial cell culture was to the 7th day
The transfection of malicious particle.
Preferably, in step (3), when carrying out slow-virus transfection, while the polybrene of 5 μ g/ml is added；To improve
The transfection abilities of virus.
Preferably, in step (5), the culture solution without Puromycin is used to expand 50 generations of culture the positive cell of screening
More than.When the positive cell of screening is when expanding after 50 generations of culture, the green fluorescent protein tag being transferred in cell line is opened
Mover has been methylated, therefore cell line unstressed configuration under fluorescence microscope, is conducive in this way using cell line of the invention
It carries out unaffected when subsequent fluorescence experiments.
The third aspect of the present invention provides above-mentioned pig endometrial epithelial cell and ties up in separation and/or culture PRRSV
Using.
Beneficial effects of the present invention:
(1) building obtains the pig endometrial epithelial cell system susceptible to PRRSV to the present invention for the first time.Pig uterus of the invention
Intimal epithelium cell line has puromycin selection markers；It can infinitely pass on；It is able to maintain that pig endometrial epithelial cell
Characteristic；It is able to maintain that caryogram is normal in succeeding generations, there is no chromosome structure sum number purposes to change；It is transferred in cell line
The promoter of green fluorescent protein tag has been methylated, the unstressed configuration under fluorescence microscope, after being conducive to cell line progress
Continuous fluorescence experiments are interference-free；More it is essential that the pig endometrial epithelial cell system is sensitive to PRRSV virus, PRRSV
It can result in its apoptosis, while PRRSV can also be replicated into the cell herein, can be used for the passage of PRRSV and relevant grind
Study carefully, provide a new cell line for the culture of PRRSV, expands the range of the host cell of PRRSV research, for
PRRSV has important meaning to the pathogenesis of reproductive system.
(2) present invention contains target gene SV40T exogenous expression's carrier by transfection, makes pig endometrial epithelial cell
Skipping the G1 phase enters the S phase, to extend the cell cycle.
(3) present invention will be transfected into pig endometrial epithelial cell, structure containing target gene SV40T exogenous expression's carrier
Build the pig endometrial epithelial cell system of transgenosis；The expression of fluorescence microscope marker gene EGFP, and pass through
Puromycin screening obtains positive cell, and carries out PCR identification confirmation target gene SV40T and be integrated into pig endometrial epithelium
The genome of cell.
Detailed description of the invention
Fig. 1-A is the figure after tissue block method's cell culture seven days；
Fig. 1-B is primary cell identified by immunofluorescence figure；
Fig. 1-C is the growth curve chart of primary cell；
Fig. 2 is the Technology Roadmap of pig endometrial epithelial cell system building of the invention；
Fig. 3-A is to transfect the monoclonal cell figure obtained after pLVX-EGFP-T2A-Puro-SV40T plasmid；
Fig. 3-B is that the cell line obtained passes on the figure in the 5th, 10,20,30,40,50 generation；
Fig. 4-A is the figure that the growth curve of the cell line of primary cell and acquisition compares；
Fig. 4-B is the cell cycle figure of the cell line of primary cell and acquisition；
Fig. 4-C is the result figure of the detection of cell line immunoblotting analysis the ER α and PR of primary cell and acquisition；
Fig. 4-D is PCR detection Sn, CD163, SV40, the result figure of GAPDH；
Fig. 4-E is the cell line karyotyping figure obtained；
Fig. 4-F is the cell line identified by immunofluorescence figure obtained；
Fig. 5-A~B is apoptosis rate variation diagram and statistical analysis column after primary cell and the cell line infection PRRSV of acquisition
Figure.
Fig. 5-C is the measurement result of PRRSV infection cell line restrovirus titre.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
As background technology part is introduced, due to pig endometrial epithelial cell in the oestrous cycle each time not
Disconnected variation, and the passage number of pig endometrial epithelial cell is limited, and therefore, it is difficult to construct pig using telomere zymotechnic
Endometrium epithelial cell line.And use SV40 large T antigen method building cell line may companion during Long Term Passages
There is the change of caryogram；Pig endometrial epithelial cell angling is serious in addition, therefore transfect extremely difficult.Above-mentioned factor is led
Cause the building of pig endometrial epithelial cell system very difficult.
In addition, PRRSV has very strong host cell tropism, the cell currently used for separating and cultivating PRRSV is mainly
Porcine alveolar macrophage (PAM) and African green monkey kidney cell (Marc-145), if the pig intrauterine susceptible to PRRSV can be constructed
Film epithelial cell line then has great importance for the research and development of the culture of PRRSV and PRRSV vaccine.
Based on this, the object of the present invention is to provide the pig endometrial epithelial cell system that a kind of couple of PRRSV is susceptible, the present invention
By exogenous expression's carrier pLVX-EGFP-T2A-Puro-SV40T (exogenous expression by the way of slow virus packaging infection
Carrier is provided by Southwest University for Nationalities；Those skilled in the art can also slow virus carrier construction method routinely construct
To) import in pig endometrial epithelial cell, the expression of fluorescence microscope marker gene EGFP, and pass through
Puromycin screening obtains positive cell, and identifying through PCR confirms that target gene SV40T is integrated into pig endometrial epithelium really
In the genome of cell；The significant PROTEIN C K18 of epithelial cell is detected by immunofluorescence technique and endometrial epithelium is thin
The expression of born of the same parents specific receptor ER α and PR, identification confirm that the cell obtained is strictly endometrial epithelial cell；Pass through Diagnosis of Sghistosomiasis
Note technology (WesternBlotting, WB) confirms that endometrial epithelial cell specific receptor ER α and PR are implicitly present in；Pass through
Karyotyping counts the chromosome quantitative in endometrial epithelial cell system；Through PCR identification PRRSV receptor Sn and CD163 in structure
The expression in cell line built；By flow cytomery, its apoptosis after infecting PRRSV changes；It is replicated compared with PAMs
The ability of PRRSV.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.
Test material used in the embodiment of the present invention is the test material of this field routine, can pass through commercial channel
It is commercially available.The experimental method that detailed conditions are not specified is said according to conventional methods or according to operation proposed by supplier
What bright book carried out.
The source of portion of reagent used in the embodiment of the present invention and material is as follows:
DMEM/F12(gibco)；Trypsin (gibco) containing EDTA；Fetal calf serum (Chinese holly, Zhejiang day Hangzhoupro biology section
Skill limited liability company)；Tissue culture plate and culture dish (Corning)；(the raw work bioengineering in Shanghai is public for plasmid extraction kit
Department)；Puromycin；PsPAX2 (addgene, #12260)；PCMV-VSV-G (addgene, #8454)；pLVX-EGFP-T2A-
Puro-SV40T (Southwest University for Nationalities offer)；0.45 μm of disposable filter (-HV)；Polybrene, PEG8000；
4% paraformaldehyde, PBS；TBST；0.5%Triron X-100；Rabbit-anti mouse CK18, ER α, PR polyclonal antibody (abcam)；
The donkey anti-rabbit IgG (Beyotime) that Alexa Fluor 555 is marked；Hoechst 33342(Beyotime)；TRIzol
Reagent (Invitrogen), PrimeScriptTMRT reagent Kit with gDNA Eraser(Perfect Real
Time)(TaKaRa)；2 × EasyTaq PCR SuperMix (Transgen Biotech), Biorad PCR；RIPA lysate
With PMSF (Beyotime)；BCA Protein Assay Kit(Beyotime)；Goat anti-rabbit igg (H&L)；75% and 95%
Ethyl alcohol；PI(sigma)；BD flow cytometer；0.075mol/L KCl；Colchicine (0.1 μ g/ml)；Methanol；Glacial acetic acid；BD
FACSCalibur Flow cytometer using BD PharmingenTM FITC Annexin V Apoptosis
Dection Kit。
Embodiment 1: the building of pig endometrial epithelial cell building cell line
1. the culture of pig endometrial epithelial cell:
Fresh pig uterine tissue is taken, splits Cavity surface after rinsing well.The dead cell that PBS washes off mucus and falls off.It will
Endometrium is cut, and is put into PBS and is flushed three times.The endometrium cut is shredded into small fragment of tissue (1-2mm3Greatly
It is small), it is directly seeded to 6cm Tissue Culture Dish, culture solution is not added and is put into 15min in incubator.Add a small amount of culture solution after taking-up, after
It is cultivated in continuous incubator.Four hours, rear fluid infusion to no mistake tissue block, while adding mycillin (penicillin 100U/ml, strepto-
100 μ g/ml of element).Changed the liquid once after pig endometrial epithelial cell converges around tissue block every other day, continue culture four days to
With.
The present invention is using Puromycin as screening drug, due on expression vector pLVX-EGFP-T2A-Puro-SV40T
There is Puromycin resistant gene Puro, the pig endometrial epithelial cell that foreign gene is expressed can be containing certain dense
It spends in the culture solution DMEM/F12 of Puromycin and survives, and the normal cell of untransfected can be dead under the concentration, it is therefore desirable to
Determine the minimum lethal concentration (MLC) of normal cell Puromycin as screening concentration.
The minimum lethal concentration (MLC) of Puromycin measures: concentration gradient is added in the culture of pig endometrial epithelial cell
Puromycin screen one week, measurement normal cell Puromycin minimum lethal concentration (MLC).As Puromycin in culture solution
When concentration reaches 2 μ g/ml, visible cell is all dead under microscope, so the minimum lethal concentration (MLC) of Puromycin is 2 μ g/
ml。
2. obtaining the lentiviral particle containing slow virus carrier pLVX-EGFP-T2A-Puro-SV40T:
By psPAX2 (6 μ g), pCMV-VSV-G (6 μ g), pLVX-EGFP-T2A-Puro-SV40T (7.5 μ g) are transfected jointly
Into in 293T cell.After 48 hours, the fluorescence microscopy fluorescence that expression EGFP is generated under the microscope, while harvesting supernatant.72 is small
When after harvest supernatant cell, mixed with supernatant before.Multigelation makes cell rupture three times, then with 0.45 μm of filter mistake
Supernatant is collected in filter.Supernatant is mixed with 5 × PEG8000, overnight, 4000g is centrifuged one hour for 4 DEG C of sedimentations.Supernatant is removed,
Precipitating DMDM culture solution is dissolved, -80 DEG C of storages are stand-by.
3. lentiviral particle infects pig endometrial epithelial cell:
When primary pig endometrial epithelial cell grows into the 7th day, slow-virus infection is carried out, is added simultaneously
Polybrene (5 μ g/ml) improves virus infection ability；After infection 24 hours, fresh medium is replaced；After infection 72 hours, add
Enter Puromycin to 2 μ g/ml of final concentration.
The screening of 4.pLVX-EGFP-T2A-Puro-SV40T carrier positive expression:
After pLVX-EGFP-T2A-Puro-SV40T lentiviral particle infects pig endometrial epithelial cell, containing serum
The Puromycin that final concentration of 2 μ g/ml is added in DMEM/F12 culture solution carries out cell screening.
After slow virus infected cell 72 hours, expression vector is expressed and under fluorescence microscope it can be seen that green is glimmering
Light.When infection slow virus pig endometrial epithelial cell can issue green fluorescence, illustrate pLVX-EGFP-T2A-Puro-
SV40T comes into cell and positive expression.Puromycin concentration is halved into lasting screening until cell covers with ware at this time
Bottom, it is seen that positive cell is in island shape cell mass, still in green under fluorescence microscope；Then vitellophag, with containing
The DMEM/F12 culture solution of Puromycin expands culture.
The positive cell screened is all the cell of stable transfection pLVX-EGFP-T2A-Puro-SV40T, target gene
SV40T is integrated on the genome of cell, rather than is free on except genome；During stable transfection, plasmid pLVX-
The gene carried on EGFP-T2A-Puro-SV40T can be integrated on the genome of host；14 days are screened again by Puromycin
Half-value dose, which persistently screens, shows as reporter gene lasting table in the positive cell being transfected to achieve the purpose that stable transfection
It reaches, therefore the positive cell screened continuously sends out green fluorescence and Puromycin resistance is presented.
The culture solution without Puromycin is used at least to expand 50 generations of culture the positive cell of screening, i.e. building obtains pig
Endometrial epithelial cell system.
The promoter for the green fluorescent protein tag being transferred in the pig endometrial epithelial cell system that the present invention constructs
It has been be methylated that, the unstressed configuration under fluorescence microscope, it is interference-free to be conducive to the subsequent fluorescence experiments of cell line progress.
Embodiment 2: the cell line signature verification of pig endometrial epithelial cell primary cell and building
1. test method:
It is verified through immunofluorescence technique in 1.1 pig endometrial epithelial cell sources
Detect Keratin 18 in the cell line (building of embodiment 1) of pig endometrial epithelial cell primary cell and building
(CK18), estrogen receptor ɑ (ER ɑ), the expression of PgR (PR).Pig endometrial epithelial cell primary cell and building
Cell line fixes one hour through immunostaining fixer room temperature.Permeabilization is carried out to cell using Triron-X100.PBS cleaning three
It is secondary, 5 minutes every time.Then 10% fetal calf serum is covered to be closed.Serum is sopped up, primary antibody, concentration 1:100,4 DEG C of mistakes are covered
Night is incubated for.Primary antibody is cleaned three times with PBS, 5 minutes every time.The fluorescence secondary antibody that Alexa Fluor 555 is marked in covering, concentration
For 1:100, room temperature is protected from light incubation one hour.It is cleaned three times with PBS, 5 minutes every time.It is dyed with PI dye liquor, 10 minutes, so
It is washed off afterwards with PBS, every time 5 minutes three times.Fluorescence microscopy microscopic observation cell issues red fluorescence, and it is glimmering that nucleus issues blue
Light.
1.2 WesternBlotting detect ER ɑ and PR
Pig endometrial epithelial cell primary cell and the cell line of building (building of embodiment 1) carry out total protein extraction.
Then protein electrophoresis, transferring film are carried out.The film to take a turn for the better carries out closing 100 minutes with 5% skimmed milk power, is washed after closing with TBST
Three times, every time 5 minutes.Then film is immersed in primary antibody, 4 degree of overnight incubations.Primary antibody is recycled, then film is washed three times with TBST,
7 minutes every time.Then it is incubated for HRP secondary antibody room temperature 50 minutes.Film is washed three times with TBST, 7 minutes every time.Finally shone with ECL
Liquid carries out chemiluminescence detection.
1.3 RT-PCR detect PRRSV receptor
Extract the RNA of the cell line (building of embodiment 1) of pig endometrial epithelial cell primary cell and building.With reversion
Record the synthesis that kit carries out cDNA.Then PCR detection is carried out.
The reaction system of 25 μ l are as follows: 12.5 μ l PCR SurperMix；1 μ l upstream primer (10 μm of ol/)；Draw in 1 downstream μ l
Object (10 μm of ol/)；cDNA 1μl；Add ultrapure water to 25 μ l.
PCR program is: 94 DEG C initial denaturation 5 minutes；94 DEG C are denaturalized 30 seconds, and 60 DEG C are annealed 30 seconds, and 72 DEG C extend 30 seconds, 34
Circulation；72 DEG C 5 minutes fully extended.Wherein SV40T annealing temperature is 63 DEG C.Design of primers is as follows:
SV40T- upstream primer: AGTGGCTGGGCTGTTCTTTT；(SEQ ID NO.1)
SV40T- downstream primer: ATGGGAGCAGTGGTGGAATG；(SEQ ID NO.2)
Sn- upstream primer: GGATTCGGGCTTCTACTTCTG；(SEQ ID NO.3)
Sn- downstream primer: TACCAGGAAAAACGGGTGTC；(SEQ ID NO.4)
CD163- upstream primer: TGTGGAAGTGCTGTCAGTTTCT；(SEQ ID NO.5)
CD163- downstream primer: AAATGTGTCCAGTTCCCTCACT；(SEQ ID NO.6)
GAPDH- upstream primer: TGGTGAAGGTCGGAGTGAAC；(SEQ ID NO.7)
GAPDH- downstream primer: GGAAGATGGTGATGGGATTTC；(SEQ ID NO.8)
1.4 cell cycles and growth curve statistics
Cell is harvested in centrifuge tube, the PBS cleaning cell of pre-cooling is added.1000rpm is centrifuged 10 minutes, abandons supernatant.By
It is added dropwise to 75% ethyl alcohol of 5ml pre-cooling, mixes cell, 4 DEG C are protected from light overnight.800g centrifuge cell 10 minutes, abandon supernatant.Later
Cleaning cell 2 times to remove all ethyl alcohol.Then cell dyeing is carried out, cell is resuspended in 0.5mLPI/RNase dyeing liquor,
Room temperature is protected from light incubation 15 minutes.Sample is kept in dark place in first 4 DEG C of analysis.Flow cytometer detects within an hour.
Pig endometrial epithelial cell primary cell and the cell line of building (building of embodiment 1) are passed on to 24 orifice plates
In, 1000, every hole cell.It counts daily, continuous 7 days.Cell growth curve figure is drawn according to result.
The pig endometrial epithelial cell system karyotyping of 1.5 buildings
Cell terminates first four hours addition colchicines (0.1ug/ml) of culture.Cell dissociation is collected, supernatant is abandoned in centrifugation.
Hypotonic solution (0.075mol/L KCl) 5ml of 37 DEG C of preheatings is added, piping and druming is uniform, 37 DEG C of water-bath 20min.Supernatant is abandoned in centrifugation.
1ml fixer (methanol: glacial acetic acid=3:1, ready-to-use) is added along tube wall, shake when being added dropwise uniformly, stands 5min and is centrifuged
Abandon supernatant.3ml fixer is added, stands 30min, supernatant is abandoned in centrifugation.3ml fixer is added again, stands 30min, centrifugation is abandoned
Supernatant.1ml fixer (additional addition PI dyeing liquor) is added to be resuspended.Dropper draws cell suspension, and high-altitude drop is in frost slide
On (95% ethyl alcohol impregnates for 24 hours, 4 DEG C of preservations), over-fires under alcolhol burner flame several times immediately, set and bake piece 2h in baking oven.
The pig endometrial epithelial cell system of 1.6 PRRSV infections building causes Apoptosis
The pig endometrial epithelial cell system of PRRSV infection building is after 48 hours, using apoptosis detection kit to cell
It is handled.Flow cytometer detects within an hour.
The ability of the pig endometrial epithelial cell system duplication PRRSV of 1.7 buildings
The pig endometrial epithelial cell system of building and PAMs infect PRRSV simultaneously.It is collected after 48 hours and contains viral supernatants.
Respectively by two kinds of supernatant gradient dilutions (10 containing virus2、103、104、105), then the normal PAMs of subinfection, it observes within continuous seven days
Respective virus titer TICD is calculated after cell50。
2. the cell line signature verification result of pig endometrial epithelial cell primary cell and building
2.1 immunofluorescence results
After tissue block method's cell culture seven days, it is seen that epithelial cell is around this tissue block compact growth, in paving stone shape (figure
1-A).The fibroblast being wherein mingled with can remove through differential digestion three times.
Pig endometrial epithelial cell primary cell and the cell line of the building visible CK18 after Immunofluorescence test, ER ɑ,
PR has expression (Fig. 1-B) in cell.
The pig endometrial epithelial cell system cell performance test results of 2.2 buildings
It is detected through immunoblotting analysis, it was demonstrated that functional protein ER ɑ and PR on endometrial epithelial cell can be with normal expressions；
It can be that the SV40T of cell immortality has presence in PCR result；As the important virus receptor of PRRSV, Sn and CD163
It can be detected through PCR；Karyotyping is the results show that the pig endometrial epithelial cell system chromosome quantitative form of building is normal；
G0 the and S phase for the pig endometrial epithelial cell system that flow cytometer constructs as the result is shown is obviously prolonged compared to primary cell.
The pig endometrial epithelial cell system of 2.3 buildings is to PRRSV neurological susceptibility
After the pig endometrial epithelial cell of building ties up to infection PRRSV 48 hours, the apoptosis through flow cytomery
Rate obviously rises；Supernatant Test Virus titre results after virus infection show that the pig endometrial epithelial cell system of building obtains
Obtaining titre is 3.23logTICD50 containing viral supernatants, and the 3.45logTICD50's lower than PAMs contains viral supernatants.Illustrate structure
The pig endometrial epithelial cell system built can be used for apoptosis correlation caused by PRRSV infection pig endometrial epithelial cell and grind
Study carefully, and can be used for the preservation and amplification of virus.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair
Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
SEQUENCE LISTING
<110>Shandong Agricultural University；Member Bioisystech Co., Ltd, Ningbo German side
<120>a kind of couple of PRRSV susceptible pig endometrial epithelial cell system and its construction method
<130> 2018
<160> 8
<170> PatentIn version 3.5
<210> 1
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<212> DNA
<213>artificial sequence
<400> 1
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<210> 2
<211> 20
<212> DNA
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<400> 2
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<212> DNA
<213>artificial sequence
<400> 3
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<213>artificial sequence
<400> 4
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<212> DNA
<213>artificial sequence
<400> 5
tgtggaagtg ctgtcagttt ct 22
<210> 6
<211> 22
<212> DNA
<213>artificial sequence
<400> 6
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<212> DNA
<213>artificial sequence
<400> 8
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Claims (10)
1. a kind of couple of PRRSV susceptible pig endometrial epithelial cell system, which is characterized in that be with pig endometrial epithelial cell
As host cell, transfection has the slow virus expression plasmid of SV40T antigen gene, expands through Puromycin drug screening, cell
It is obtained after big culture.
2. pig endometrial epithelial cell according to claim 1 system, which is characterized in that the slow virus expression plasmid packet
Contain: SV40 large T antigen gene, antibiotic-screening gene Puro gene and EGFP gene.
3. pig endometrial epithelial cell according to claim 2 system, which is characterized in that the slow virus expression plasmid
pLVX-EGFP-T2A-Puro-SV40T。
4. the construction method of claim 1-3 described in any item pig endometrial epithelial cells system, which is characterized in that including with
Lower step:
(1) primary pig endometrial epithelial cell is obtained, and is cultivated；
(2) using pLVX-EGFP-T2A-Puro-SV40T as slow virus carrier, using psPAX2 and pCMV-VSV-G as auxiliary
Package carrier obtains lentiviral particle；
(3) using the primary pig endometrial epithelial cell after lentiviral particle transfection culture；
(4) it after lentiviral particle transfection pig endometrial epithelial cell, is added in the DMEM/F12 culture solution containing serum dense eventually
The Puromycin that degree is 2 μ g/ml carries out cell screening, obtains positive cell；
(5) culture solution without Puromycin is used to expand culture the positive cell of screening, i.e. building obtains susceptible to PRRSV
Pig endometrial epithelial cell system.
5. construction method according to claim 4, which is characterized in that in step (1), obtain primary pig endometrial epithelium
Cell and the method for culture are as follows: pig endometrium is shredded into small fragment of tissue by acquisition pig endometrium, is directly seeded to thin
Born of the same parents' culture dish is not added culture solution and is put into 10-20min in incubator；A small amount of culture solution is added after taking-up, continues to train in incubator
It supports, fluid infusion while being added penicillin and streptomysin, changed the liquid once every other day to tissue block is not crossed after 4h；
Preferably, the additional amount of the penicillin is 100U/ml, and the additional amount of streptomysin is 100 μ g/ml.
6. construction method according to claim 4, which is characterized in that in step (2), by pLVX-EGFP-T2A-Puro-
SV40T, psPAX2 and pCMV-VSV-G are transfected jointly enters 293T cell, harvests supernatant after 48h；It is thin that supernatant is harvested after 72h
Born of the same parents, and mixing with the supernatant harvested before, multigelation makes cell rupture, and supernatant is collected in filtering, by supernatant with
PEG8000 mixing, overnight, centrifugation removes supernatant for sedimentation, precipitating DMDM culture solution is dissolved, i.e. acquisition lentiviral particle.
7. construction method according to claim 4, which is characterized in that in step (3), trained to primary pig endometrial cell
When supporting to the 7th day, lentiviral particle transfection is carried out.
8. construction method according to claim 4, which is characterized in that in step (3), when carrying out slow-virus transfection, together
When the polybrene of 5 μ g/ml is added.
9. construction method according to claim 4, which is characterized in that in step (5), by the positive cell of screening with being free of
It is more than generation to expand culture 50 for the culture solution of Puromycin.
10. the described in any item pig endometrial epithelial cells of claim 1-3 tie up to separation and/or cultivate answering in PRRSV
With.
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