CN107190024A - A kind of construction method of stable expression NS1 albuminous cells strain - Google Patents
A kind of construction method of stable expression NS1 albuminous cells strain Download PDFInfo
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Abstract
The present invention relates to a kind of construction method of stable expression NS1 albuminous cells strain.NS1 genes are inserted at the EcoR I multiple cloning sites of pLenti CMV EGFP 3Flag PGK Puro carriers, with the pLenti CMV NS1 EGFP 3Flag PGK Puro recombinant plasmids built and viral packaging plasmid cotransfection 293T cells, cultivate, go out poison, collect viral supernatants, filter to obtain recombinant slow virus liquid.Packaged recombinant slow virus is infected into A549 cells, screened by puromycin, immunofluorescence and Western blot identifications must stablize expression NS1 albuminous cell strains.The present invention prepares a kind of fusion tag albumen using recombinant slow virus system and can stablize the cell line of expression NS1 albumen, and the cell line provides a good instrument to study the biological function of NS1 albumen.
Description
Technical field
The invention belongs to field of medical molecular biology.Specifically related to a kind of stable expression NS1 albumen (Non-
Structural 1Protein, NS1) lung cancer cell line construction method.
Background technology
Slow virus (Lentivirus) carrier is the gene therapy grown up based on I type human immunodeficiency virus
Carrier.Foreign gene can be effectively incorporated into host chromosome by the general retroviral vector of difference, slow virus carrier
On, so as to reach that persistence is expressed.Lentiviral contains the hereditary information required for packaging, transfection, stable integration,
Need to utilize expression vector and packaging plasmid cotransfection cells, the packaging of virus is carried out in cell.Packaged virion
It is secreted into extracellular culture medium, centrifuging and taking obtains the virion that high titre is included in supernatant, is used directly for host
The infection of cell.Target gene is entered after host cell by virus infection, genome is incorporated into, so that lasting Gao Shui
Flat expression target gene.In the related experimental implementation of cell, it is difficult to transfection according to a conventional method for some or even can not turns
The cell of dye, can greatly improve the transduction efficiency of gene, with the efficient of the gene that achieves the goal by the transfection of lentivirus mediated
Expression.
Avian influenza virus is a kind of influenza A, belongs to orthomyxoviridae family, Influenza Virus.The base of influenza A
Because group is made up of the sub-thread strand RNA of 8 sections, 11-12 kind albumen is encoded, including：HA、NA、M1、M2、NS1、NS2、NP、
PB1, PB2, PA, and NP40 and PB1-F2.Wherein NS1 albumen (non-structural protein 1) is by influenza virus
The RNA codings of 8 sections, containing 202-237 amino acid, relative molecular weight is about 28kD, variant between different virus strain.
It is relevant with its special construction that nearest structure elucidation finds that this is mainly, and NS1 albumen is made up of three parts：RNA integrated structures
Domain (RNA binding domain, RBD), effector domain (effector domain, ED) and both join domains
(linker region, LR), the join domain of flexibility and changeability can make NS1 albumen that different conformations are presented, thus complete with not
With the combination of RNA and albumen.NS1 RNA binding structural domains (RBD) are made up of 73 amino acid, can combine heterologous RNA, bag
Include virus genome RNA, virus mRNA 5 ' non-translational regions, poly (A) RNA and external source dsRNA；NS1 C-terminal effect structure
Domain (ED) is the main domains that NS1 interacts with host, with blocking host mRNA montages, Polyadenylation and consideration convey
The functions such as fortune.
The toxicity of NS1 albumen and influenza virus is closely related, plays important at the regulation and control pathogenic aspect of influenza virus
Effect.The NS1 albumen of influenza A has the effect of influence Apoptosis of Host Cells.It influences the process of Apoptosis of Host Cells
Be not set forth completely also it is clear, it is existing research find NS1 albumen simultaneously have promote apoptosis and suppress apoptosis dual work
With.Infection early stage, NS1 albumen by reducing IFN expression quantity to suppress IFN downstream reaction, so as to suppress host cell
Apoptosis；Meanwhile, NS1 can also interact with PI3K, activate PI3K/AKt signal paths, be situated between so as to suppress JNK dependences, Bax
The Apoptosis of Host Cells led.NS1 can also influence the apoptosis of cell by a lot of other approach.It is reported that in virus infection,
The PKR of state of activation plays an important role for apoptosis, and NS1 albumen can directly in conjunction with and suppress PKR activation rush apoptosis it is anti-
Should；NS1 albumen n end RNA calmodulin binding domain CaMs can be combined with intracellular dsRNA, so as to suppress the OAS/RNase of apoptosis-promoting effect
L paths；Studies have found that NS1 can suppress the activity of p53 regulatory factors, so as to suppress the apoptotic effect of p53 regulations；NS1 is straight
Connect and interacted with hGBP1 (Human guanylate-binding protein 1), cause hGBP1 GTPase activity drops
It is low, apoptotic response is inhibited indirectly.Although apoptotic process is often referred to as host cell itself in order to which limiting virus are replicated, and
A kind of antiviral response taken, but the effect of apoptotic process is also not very clear in influenza A infection cell.
The content of the invention
The purpose of the present invention is to be prepared a kind of fusion tag albumen using recombinant slow virus system and can be stablized to express NS1 eggs
White cell line, the cell line provides a good instrument to study the biological function of NS1 albumen.
The technical solution adopted by the present invention is：A kind of construction method of stable expression NS1 albuminous cells strain, including following step
Suddenly：
1) amplification of NS1 genes；
2) NS1 genes are connected with carrier pLenti-CMV-EGFP-3Flag-PGK-Puro, build recombinant slow virus table
Up to plasmid pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro；
3) by recombinant slow virus expression plasmid pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro and virus packaging matter
Grain cotransfection 293T cells, through cultivating, going out poison, collect supernatant, filter, obtain packaged recombinant slow virus CMV-GFP-
L.V.；
4) packaged recombinant slow virus CMV-GFP-L.V. is transfected into A549 cells, screens positive thin by puromycin
Born of the same parents' strain, carries out cellular immunofluorescence to positive cell strain and Western blot is identified, obtains stable expression NS1 albuminous cells
Strain.
A kind of construction method of above-mentioned stable expression NS1 albuminous cells strain, described step 1), with NS1-F and NS1-R
For specific primer, restriction enzyme site EcoR I are added, using plasmid pEGFP-N1-NS1 as template, enter performing PCR amplification；Described spy
Specific primer NS1-F and NS1-R sequence is：
NS1-F:5'CCGGAATTCATGGATTCCAACACTGTGT 3'
NS1-R:5'CCGGAATTCCGAACTTTTGACTCAATTGT 3'
A kind of above-mentioned stabilization expresses the construction method of NS1 albuminous cells strain, and PCR amplification system is：
PCR response procedures：94 DEG C of 3min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 4 DEG C of insulations, 30 circulations.
A kind of construction method of above-mentioned stable expression NS1 albuminous cells strain, step 3) in, described viral packaging plasmid
It is pLP1, pLP2 and pLP.
The beneficial effects of the invention are as follows：
1. the present invention is using recombinant slow virus as carrier, A549 human lung adenocarcinoma cells are infected, screening is obtained can stable table
Up to the cell line of NS1 albumen, NS1 protein fusion expressions EGFP, FLAG label facilitates the detection of NS1 protein expressions.
2. the present invention, which is established, can stablize the A549 cell lines for expressing NS1 albumen, the cell line is further further investigation
The biological characteristics of NS1 albumen and the mechanism of causing a disease of avian influenza virus have established solid foundation.
3. the present invention builds a kind of cell line of the overexpression NS1 albumen with label, be NS1 protein biologicals characteristic and
The research of function provides a good instrument, and also hoping turns into the ideal model that bird flu is treated.
Brief description of the drawings
Fig. 1 is pEGFP-N1-NS1 double digestion result figure.
Fig. 2 is pLenti-CMV-EGFP-3Flag-PGK-Puro carrier digestion result；
Wherein, 1：Carrier digestion post-fragment；2：1kb DNA ladder Marker：10kb、8kb、6kb、5kb、4kb、
3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp。
Fig. 3 is the structural representation of pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro expression vectors.
Fig. 4 is A549, the cell line of the empty viral vector of transfection, the identified by immunofluorescence of NS1 stably expressing cell lines；
Wherein, a：A549；b：The cell line of the empty viral vector of transfection；c：NS1 stably expressing cell lines.
Fig. 5 is the Western blot identifications of NS1 stably expressing cell lines；
Wherein, 1：Marker；2：A549Blank；3：A549H102；4：A549H4438.
Embodiment
(1) NS1 gene magnifications
1st, NS1 gene PCRs are expanded
According to NS1 gene orders (GenBank in GeneBank:AAT90838.1), software LSPrimer is utilized
(http://ccsipb.lnu.edu.cn/primer/) design NS1 genes specific primer NS1-F and NS1-R, add enzyme
Enzyme site EcoR I, specific primer NS1-F and NS1-R sequence is：
NS1-F:5'CCGGAATTCATGGATTCCAACACTGTGT 3'
NS1-R:5'CCGGAATTCCGAACTTTTGACTCAATTGT 3'
PEGFP-N1-NS1 plasmids using the preservation of this laboratory enter performing PCR amplification as template, and PCR reaction systems are：
PCR response procedures：94 DEG C of 3min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 4 DEG C of insulations, altogether 30 circulations.
2nd, DNA agarose gel electrophoresis
The agarose of configuration 1.5%, microwave stove heat is completely dissolved agarose particle；The glue plate that will be cleaned, dry
Horizontal positioned is on the table；Mix, encapsulating, insert comb, it is to avoid produce bubble；After after gel sets, comb is carefully taken out；
Glue is put into electrophoresis tank, electrophoretic buffer is added；By PCR primer loading, through 110V electrophoresis 30min.
3rd, PCR primer gel is reclaimed
DNA recovery is carried out according to the Ago-Gel DNA QIAquick Gel Extraction Kits specification of Tiangeng company.
(2) recombinant slow virus expression plasmid pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro is built
1st, pLenti-CMV-EGFP-3Flag-PGK-Puro carriers digestion
By the digestions of EcoR I of pLenti-CMV-EGFP-3Flag-PGK-Puro carriers, digestion system is in 37 DEG C of water-baths
Correct purpose band is carried out gel recovery by night, electrophoresis.Digestion system is as follows：
2nd, NS1 genes are connected with pLenti-CMV-EGFP-3Flag-PGK-Puro carriers
NS1 genes after gel in (one) is reclaimed and the carrier pLenti-CMV-EGFP-3Flag-PGK- after digestion
Puro, in 16 DEG C of reaction overnights, builds recombinant slow virus expression plasmid pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro.
T4 enzyme linked systems are as follows：
3rd, the α of plasmid transformed competence colibacillus E. coli DH 5
Take out in the α competent cells of E.coli DH 5, insertion trash ice and dissolve；The recombinant slow virus table that 10 μ l are built
50 μ l competent cells are added up to plasmid pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro, gently rotation is mixed, ice bath
30min；The heat shock Escherichia coli 45s in 42 DEG C of water-baths, is moved into rapidly in wet ice, stands 2min；Add 37 DEG C of preheating nothings
The μ l of SOC nutrient solutions 500 of antibiotic, with 180rpm rotating speeds in 37 DEG C of insulating box shaking table culture 1h.The impression for taking 120 μ l to convert
State cell is applied on the LB agar plates containing 100 μ g/ml acillins, and flat board is placed in into room temperature after after its drying, is inverted in
Incubated overnight 12-16h in 37 DEG C of incubator, checks in each culture dish whether bacterium colony occur.Select the single bacterium on agar plate
In the LB culture mediums for falling to be inoculated into 5ml ammonia benzyl resistances, 37 DEG C of shaking table 1800rpm overnight incubations, incubation time is no more than 16h.
4th, the molecular biology identification of transformant
Enter performing PCR detection by template of the genome of the conversion bacterial strain of extraction, recovery product is sequenced.Sequencing is correct
A bacterium solution part freezes guarantor bacterium, is preserved in -80 DEG C long-term.Another part bacterium solution upgrading grain.
5th, the plasmid extraction of pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro recombinant bacterial strains
Correct recombinant bacterial strain is sequenced and carries out plasmid extraction, pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro is obtained
Recombinant slow virus expression plasmid.A260 and A280 is determined with NanoDrop, it is respectively A260/ to calculate nucleic acid purity and nucleic acid concentration
280=1.92, concentration is 350ng/ μ l.
(3) recombinant slow virus CMV-GFP-L.V. acquisition
1st, the packaging of recombinant slow virus
By the recombinant slow virus expression plasmid pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro and virus that build
Packing helper plasmid pLP1, pLP2, pLP cotransfection 293T cells, (transfection pLenti-CMV-EGFP-3Flag-PGK-Puro is empty
Viral vector is used as control).24h before transfection, with the 293T cells of Trypsin Induced exponential phase, with containing 10% serum
Culture medium adjustment cell density is 5 × 105Cell, renewed vaccination 10ml is in 10cm Tissue Culture Dish, 37 DEG C, 5%CO2Incubator
Interior culture 24h, can be used to transfection when cell density reaches 90%-95%.Cell culture medium is replaced by no blood by 2h before transfection
Clear culture medium.
The prepared μ of pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro carriers 4 is added into sterilizing 2ml centrifuge tubes
G, and viral packaging plasmid each 4 μ g of pLP1, pLP2, pLP, are well mixed with 1.5ml serum-free Opti-MEM culture mediums,
Incubate 5 minutes at room temperature.Take 48 μ l FuGene HD reagents to be mixed in another Guan Zhongyu 1.5ml Opti-MEM, incubate at room temperature
5 minutes.Above two solution is mixed, incubated 20 minutes at room temperature, DNA and FuGene HD transfection composites is formed.Will
Mixed liquor is transferred in the nutrient solution of 293T cells, in 37 DEG C, 5%CO2Cultivated in cell culture incubator.Gone after 24h containing turn
Contaminate and the cell culture medium 10ml containing 10% serum is added in the culture medium of mixture, every bottle of cell, in 37 DEG C, 5%CO2Incubator
It is interior to continue to cultivate 24h.The complete medium that antibiotic-free is changed after 24h continues to cultivate.
2nd, the collection and concentration of recombinant slow virus
Transfect after 48h, collect containing virulent nutrient solution into 50ml sterile centrifugation tubes, then add the fresh complete trainings of 10ml
Nutrient solution can also be extremely produced in the cell of poison；Transfect 72h after again collect contain virulent nutrient solution, by the nutrient solution collected twice in
4 DEG C of centrifugations, 3000rpm, 15min.Vial supernatant removes cell fragment with 0.45 μm of membrane filtration, and 4 DEG C of filtrate centrifuges,
50000 × g, 90min, remove supernatant, add 100 μ l complete culture solutions, after being slowly fully resuspended with 200 μ l pipette tips, by virus
It is stored in after concentrate packing in viral pipe, it is -80 DEG C long-term to preserve, obtain recombinant slow virus CMV-GFP-L.V..
3rd, the titer determination of recombinant slow virus
First day in each hole inoculation 4 × 10 of 96 orifice plates4Individual 293T cells, do 10 times of gradients for second day dilute in EP pipes
Release, dilution process is：Every kind of virus prepares 8 1.5ml EP pipes, and often pipe adds 297 μ l complete culture solutions, into first pipe
33 μ l virus stock solution useds are added, after mixing, 30 μ l is drawn and adds second pipe mixing.The rest may be inferred, is 8 dilution factor (10-10-6).Original nutrient solution in 96 orifice plates is discarded, each dilution factor repeats 3 holes, the μ l of virus liquid 100 diluted are added per hole simultaneously
Carry out mark.Fluorescencepositive cell is counted with fluorescence microscope within 5th day.Count most latter two it is observed that fluorescence
Fluorecyte number in hole, calculates the total sum in 3 repeating holes and calculates average, it is assumed that (penultimate can be shown in for A
The fluorecyte average of fluorescence aperture) and B (last can see the fluorecyte average of fluorescence aperture).Slow virus titre meter
Calculate formula：Virus titer (TU/ml)=(A+B × 10) × 1000/2/A holes virus quantity μ l.Be computed, virus titer be 4.62 ×
107TU/ml。
(4) screening of stable transfected cells strain
1st, CMV-GFP-L.V. viruses infection A549 optimum condition optimizations
Learnt by experiment, A549 cell infection slow virus CMV-GFP-L.V., MOI 10, efficiency of infection during MOI 20
Efficiency of infection is high when 60%-70%, MOI 40, all reaches more than 80%.It is low, low to cytotoxicity with efficiency of infection height, MOI values
Premised on, optimal infectious condition：A549 cells selection MOI 20-40 carry out subsequent experimental.
2nd, stable transfected cells strain is screened
A549 cells are inoculated into 24 orifice plates by recovery culture A549 cells before transfection in good condition by 30% degree of converging,
A549 cells are made into 4 × 104Cells/ml cell suspensions, treat bed board.500 μ l, i.e., 2 × 10 are spread per hole4Cells/well, spreads 1 piece
24 orifice plates.12~20 hours postoperative infection virus, virus liquid titre 4.62 × 107TU/ml, the μ l of virus quantity 17.3.Add 10 μ per hole
L1mg/ml polybrene, finally the final concentration of 5 μ g/ml of polybrene in cell sample, training is changed after infecting 12-20 hours
Support base：Culture medium is discarded, the fresh culture mediums of 2ml are added per hole.After infection cell 72 hours, by adding and maintaining 2ug/
Ml puromycin (puromycin) kills the cell not infected effectively；A final concentration 2ug/ml was changed every 2-3 days
Puromycin fresh cultures；Observed under inverted microscope, carry out subclone screening, pick out the passage of normal growth cell mass,
Progressively amplification culture；So as to screen positive cell strain under the maintenance of puromycin medicines.
3rd, the detection of positive cell strain
Identified by immunofluorescence is carried out to positive cell with fluorescence microscope (see Fig. 4).Culture is enlarged to positive cell
After extract total protein, with FLAG monoclonal antibodies be used as primary antibody, carry out Western blot identifications (see Fig. 5).Finally obtain steady
Surely the A549 cell lines of NS1 genes are transfected.
Claims (4)
1. a kind of construction method of stable expression NS1 albuminous cells strain, it is characterised in that comprise the following steps：
1) amplification of NS1 genes；
2) NS1 genes are connected with carrier pLenti-CMV-EGFP-3Flag-PGK-Puro, build recombinant slow virus expression matter
Grain pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro；
3) recombinant slow virus expression plasmid pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro is total to viral packaging plasmid
293T cells are transfected, through cultivating, going out poison, collect supernatant, filters, obtains packaged recombinant slow virus CMV-GFP-L.V.；
4) packaged recombinant slow virus CMV-GFP-L.V. is transfected into A549 cells, positive cell is screened by puromycin
Strain, carries out cellular immunofluorescence to positive cell strain and Western blot is identified, obtains stable expression NS1 albuminous cells strain.
2. the construction method of a kind of stable expression NS1 albuminous cells strain according to claim 1, it is characterised in that described
Step 1), using NS1-F and NS1-R as specific primer, add restriction enzyme site EcoR I, using plasmid pEGFP-N1-NS1 as mould
Plate, enters performing PCR amplification；Described specific primer NS1-F and NS1-R sequence is：
NS1-F:5'CCGGAATTCATGGATTCCAACACTGTGT 3'
NS1-R:5'CCGGAATTCCGAACTTTTGACTCAATTGT 3'。
3. the construction method of a kind of stable expression NS1 albuminous cells strain according to claim 2, it is characterised in that PCR expands
Increasing system is：
PCR response procedures：94 DEG C of 3min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 4 DEG C of insulations, 30 circulations.
4. a kind of construction method of stable expression NS1 albuminous cells strain according to claim 1, it is characterised in that step
3) in, described viral packaging plasmid is pLP1, pLP2 and pLP.
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