Extracellular system for translating viral mRNA
Extracellular system for translating viral mRNA
Translation of poliovirus and hepatitis C virus mRNA with mammalian cell S10 extracts.
Operation method
Extracellular system for translating viral mRNA
Materials and Instruments
ATP GTP Plasmid DNA Creatine Phosphokinase 10% SDS Gum Creatine Phosphokinase Nuclease tRNA HeLaS3 Cells TgSVA Cells Lx Cells Derived from Ltk Cells Expressing Human Poliovirus Receptor NS20Y Cells Human Liver Cancer Derived HepG2 Cells Move -Materials and equipment Caveat 1) S10 and S100 fractions are supernatants obtained by centrifuging the cytoplasm at 18000 g and 100000 g for 20 minn.2) RNase-free and high-grade reagents should be used, and glassware should be washed 3 times with 2 mpL/L NaOH or immersed in 0.05 moL/L NaOH overnight and then rinsed completely with fresh deionized water.3) High iron hemoglobin is used to prevent the phosphorylation of elF2α and the pH should be carefully adjusted and should be free of RNaSe.4) S10 extracts are prepared from TgSVA cells and homogenized more than 80 times to lyse the cells. Cell homogenate preparation varies with cell type.5) Nuclease treatment decreases the activity of S10 extracts. eGTA and Ca2+The concentration of EGTA and Ca2+ , incubation time (5~l5 min) and temperature (15~20°C) are very important.2Inappropriate concentrations of EGTA and CaCl2 are often the main cause of failure, as free calcium will allow the nuclease to retain its activity.6) Well-grown cells are essential for the efficient preparation of S10 extract, for monolayer cells, trypsin digestion can be used, and then transfer to flasks for further incubation for 5-8 hours.7) The success rate of preparing active S10 extracts is 70%, the success rate of preparing S10 extracts from virus-infected HeLa cells is relatively high, while the success rate of preparing translationally inactive extracts from non-infected monolayers is only 30%~50%.8) In the cell-free translation system, K+Mg2+, Mgions vary with the type of extract and mRNA. For example, in HeLaS10, for poliovirus mRNA, 100 mmol/L K+ and 0,8 mmol/LMand 0,8 mmol/LMg2+ for poliovirus mRNA, and 0,8 mmol/LMgand 0,8 mmol/LMg 2+ for poliovirus mRNA, while for HCV virus mRNA 176 mmol/LK+ and 1.6 mmol/LMg 2+ for HCV virus mRNA.and 1.6 mmol/LMg2+ for HCV virus mRNA.The appropriate amount of poliovirus mRNA for this case is 0.5ug For more product details, please visit Aladdin Scientific website.
HEPES-KOH Tris-HCl Isotonic solution Hypotonic solution 10X Salt solution Cleavage solution Creatine phosphate Ferritin Spermidine EGTA DNaseI LiC1 Precipitation solution TE S10 Flipze mixture [32S]Met Electrophoresis buffer L 2X Sampling buffer Gel fixation solution
High Speed Centrifuge Dounce Homogenizer SDS Gel Electrophoresis Unit
(i) Cells
1) HeLaS3 cells were cultured in RPMI1640 medium containing 5% Neonatal Cow Serum (NCS) and 0.15% NaHCO3 at 37℃ in rotary flasks, and cell passaging was performed daily to maintain a cell density of 2~5X105 cells per milliliter.
2) TgSVA cells were derived from the kidneys of transgenic mice expressing the poliovirus receptor and maintained in monolayer culture in DMEM medium containing 5% fetal bovine serum and 0.15% NaHCO3.
3) Lx cells were derived from Ltk cells expressing the human poliovirus receptor and maintained in monolayer culture with DMEM medium containing 5% fetal bovine serum, 104 mol/L hypoxanthine, 4×10-3 mol/L aminopurine, 8×10-4 mol/L thymine deoxyribonucleoside and 0.15% NaHCO3.
4) NS20Y cells (murine adult neuronal cell tumor) were maintained in high-sugar DMKM medium containing 10% fetal bovine serum and 0.15% NaHCO3.
5) Human hepatocellular carcinoma-derived HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum and 15% NaHCO3.
(ii) Preparation of S10 extracts
The reagents and water used were RNase-free, and the consumables were disposable.
1) lmol/LHEPES-KOH (PH7.5), autoclaved, stored at room temperature
2) lmol/LTris-HCl (pH 7.5), autoclaved and stored at room temperature.
3) Isotonic solution: 35 mmol/L HEFESKOH (pH 7.5), 146 mmol/L NaCl, 11 mmol/L glucose. Autoclave and store at 4°C.
4) hypotonic solution: l0 mmol/LHEPES-KOH (pH7.5), 15 mmol/LKC1, 1.5 mmol/LMgAC2, 6 mmol/L mercaptoethanol, -20℃.
5)10X salt solution: 200 mmol/LHEPESKOH(pH7.5), 1.2 mol/LKC1, 50 mmol/LMgAc2, 6 mmol/L mercaptoethanol. -20℃ Storage
6) Cleavage solution: l0 mmol/LHEPES-KOH(pH7.5), 120 mmol/LKC1, 1.5 mmol/LMgAc2, lmmol/L dithiothreitol-
7) 0.2mol/L phosphocreatine (CP): Phosphocreatine was dissolved in RNase-free water, dispensed in 0.2~0.3 ml portions and stored at 20°C.
8)10 mg/ml Creatine Phosphokinase (CPK): CPK is dissolved in 20 mmol/L HEPES, pH7.5 and 50% glycerol solution, dispensed in 0.2rnl portions and stored at 20℃.
9) 15OOOU/ml Nuclease: 15000U (=0.667 mg) of Nuclease S1 dissolved in lml of RNase-free water, stored at -20℃.
10)15 mg/mltRNA: Dissolve ISmgtRNA in lml water, phenol extract several times, ethanol precipitate. Then dissolve the precipitate in RNase-free water at 15 mg/ml and store at -20°C.
11)lmmol/L ferritin: 6.5 mg of ferritin was dissolved in 0.5 ml of 1mol/LKOH, added 0.5 mLTris-HCl (pH7.5), adjusted to ph7.8 with lmol/LHCl, added 8.5 ml of hexanediol. 18000 g centrifugation for 5 min, the supernatant was collected, and stored in 20℃.
12) l00 mmol/L spermidine: dissolve in RNase-free water and store at 20℃.
13)0.2mol/LEGTA: dissolve in RNase-free water, adjust to PH7.5 with 5mol/LKOH, store at 4℃.
14)20 mmol/LATP: Dissolve 122 mg of ATP in 10 ml of RNasc-free water, adjust to pH7.0 with lmol/LNaOH, and store in 0.2~0.5 ml portions at -20℃.
15)20 mmol/LGTP: Dissolve 96 mgGTP in 10 ml of RNase-free water, adjust to pH7.0 with lmol/LNaOH, divide into 0.2~0.5 ml portions and store at -20℃.
16) High speed centrifuge.
17) Dounce homogenizer.
(ii) In vitro transcription
1) Plasmid DNA: Plasmid DNA containing T7 or T3 promoter and IRES derived from poliovirus or HCV.
2) DNaseI: RNase-free DNaseI (2U/ul).
3)LiC1 precipitation solution: 7.5 mol/L, LiC1, 50 mmol/LEDTA.
4)TE: 10 mmol/L Tris-HCl(pH7.6),lmmol/LEDTA
(iv) In vitro translation
1)S10 Flipper mixture; 4ul0.5mol/LHEPESKOH(pH7.5),26UL15mol/LKAc,0.8ulMgAc2,50ul20 mmol/LATP,2.7UL20mmol/LGTP,45ul0.2mol/LCP.2ul1mol/LDTT, 2ul0.1mol/L spermidine, 11ullmmol/L amino acid mixture (no methionine). Add RNase-free water to I8Oul,30~50ul in portions and store at 80℃.
2) [32S]Met: 10mCi/ml of [32S]Met (120Ci/mmol) (translation grade), stored at 80℃.
3) Creatine phosphokinase (CPK): 0.5ug/ul of CPK, freshly prepared with 10 mg/ml CPK and RNase-free water.
4) SDS gel electrophoresis device
5)10% SDS gel: 9.8% acrylamide, 0.2% methylenebisacrylamide, 1% SDS, 0.375 mol/L Tris-HCl (pH 8.8)
6) Electrophoresis buffer: 3 g Tris base, 14.4 g glycine, lg SDS, add water to 1L.
7) 2X Sampling buffer: 100 mmol/:LTris-HCKpH6.8),200 mmol/LDTT, 4% SDS, 0.2% bromophenol blue, 20% glycerol.
8) Gel fixative:50% methanol, 10% HAc dissolved in water.
II. Methods of operation
(-) Preparation of in vitro translation extracts
1. Uninfected HeLa cells
Unless otherwise specified, all steps must be performed at 0~4℃.
1) Centrifuge HeLa cells at 300 g for 7 min in a low-temperature centrifuge, and collect HeLa cells in a suspension larger than 2L.
2) Wash with isotonic solution 3 times. Infected and uninfected HeLa cells are treated in the following steps.
3) Resuspend the cells in a small volume of isotonic solution and transfer to a graduated conical centrifuge tube.
4) After centrifugation at 300 g and adjusting the cell density to 2X109/7~8 ml, resuspend the cells in 1.5 times the cell volume (10 ml) of the hypotonic solution, and allow to stand for 10 min at TC.
5) Homogenize the homogenate 50 times with an ice-cold Dounce homogenizer and microscopically examine the homogenate for complete cell lysis.
6) Add 5/18 volume of 10X salt solution, centrifuge at 18,000g and discard the precipitate (including nucleus, mitochondria, membrane structure and other cellular fractions), and the supernatant is the crude S10 extract.
7) Add ATP to a final concentration of lmmol/L, GTP to a final concentration of 0.2nnnol/L, CP to a final concentration of 8 mmol/L, and CPK to a final concentration of 0.2 mg/ml, and incubate at 37°C for 30 min to remove endogenous mRNA bound to ribosomes.
8) 100-fold volume of lysis solution was incubated at 4°C for lh, or the extract was passed through a G-25 column instead of lysis.
9) If the S10 extract becomes cloudy, centrifuge at 6600 g for 5 min and collect the milky supernatant for the following steps.
10) Incubate 0.2 mllmmol/L methaemoglobin, 50ul10 mg/ml CPK, 0.lml0.1mol/LCaCl2, 0.lml15000U/ml nuclease S7 at 15-20°C for 5-15 min to destroy endogenous mRNA.
11) Add 0.lml0.2mol/LEGTA,40uL15 mg/mltRNA to stop the nuclease reaction. If necessary, centrifuge at 6600 g for 5 min and freeze at -80°C in 0.2~0.3 ml portions.
2. spinal cord poliovirus infected HeLa cells
1) Collect HeLa cells (5L, 2X109 cells) and suspend them in 1/20 volume (250 ml) of serum-free RPM11640.
2) Infected with poliovirus at a complex MOI of 20 ( 4Xl010pfu ) (pfu empty spot forming units)
3) Absorb for 20 min at room temperature, then incubate for 40 min at 37°C.
4) Add 3 times the volume (750 ml) of RPMI1640 containing 7.5% NCS and incubate the infected cells in a transfer flask at 37°C for 4 h. Prepare S10 in the same way as for Hela cells not infected with the virus.
3. Infected and non-infected monolayers
1) The monolayers of cells (TgSVA, Lx, NS20Y and HepG2) used to prepare S10 extracts should be in good growth condition. After digestion with 0.02mol/LEDTA and 0.1% trypsin, 2X109 cells were collected from 60-100 dishes (150 mm in diameter), suspended in serum-containing DMEM culture medium and continued to be cultured in a rotary flask for 5-8 hours.
2) If the cells are to be infected with poliovirus, add MOI of 20 and incubate in suspension for 5 h. Absorption method is the same as for HeLa cells.
3) After infection, continue incubation in suspension for 4 h. Prepare S10 teak extract as for HeLa cells.
(ii) In vitro RNA synthesis
1) Linearize the gene with a suitable endonuclease and purify with phenol: chloroform. DNA fragments need not be isolated, and transcripts should be prepared by standard in vitro transfection methods or by commercial kits.
2) After the RNA is transcribed, digest the template DNA, add 1U/sample of RNase-free DNaseI and incubate at 37℃ for 15 min.
3) Add 1/2 volume of 7.5mol/L LiCl and incubate at 20℃ for 30 min.
4) Centrifuge at 10,000 g for 10 min, wash the RNA precipitate with 80% ethanol, air-dry the precipitate and dissolve it in 0.5 ml of RNase-free TE (final concentration 1ug/ul). Usually 1ug of template DNA is used to transcribe 5o~100ug of RNA, which is dispensed in 10~20ul batches and stored at -80℃.
5) RNA product size is analyzed by agarose gel electrophoresis and yield is analyzed spectrophotometrically.
(iii) Cell-free translation
S10 extracts stored at 8o C can only be freeze-thawed once, and the appropriate K+, Mg2+ ion concentration is different for each extract and each mRNA.
1) Add all components to the tube, the final reaction consists of 20ul (or l0 ul) of SlO flushing mixture and RNA template, add RNase-free water to a final volume of 25ul (or 12.5ul).
For example, the reaction mixture for poliovirus mRNA in HeLaS10 plants: 13.5ulS10,4.5ul Translation Mixture, 1ul0.5ug/uLCPK,0.1ulRNasin,0.5uCi[32S]Met, Template RNA (0.5ug), add RNasin-free water to 25ul, and the final concentration of each reagent is 1.0 mmol/LATP,57umol/LGTP,9 mmol/L creatine (CP),0.02ug/ul creatine phosphokinase (CPK),2 mmol/LDTT,0.2 mmol/L spermine,20 mmol/LHEPES,120 mmol/L (150 mmol/L for HCVmRNA) KAc,0,8 mmol/L (1.5 mmol/L for HCVmRNA) MgAc2, 19 amino acids each 60umol/L, 0.5pCi[32S]Met and 0.5ugRNA
2) incubate at 30~32°C (usually 60 min), add sample buffer to the translation mixture, boil for 2 min, and analyze by 10% SDS-PAGE.
3) If necessary, an autoradiographyenhancer can be used during exposure.