पुनः संयोजक इन्फ्लुएंजा वायरस के डीएनए प्लाज्मिड से पीढ़ी
Summary
प्लास्मिड डीएनए से इन्फ्लूएंजा ए वायरस के बचाव एक बुनियादी और आवश्यक प्रयोगात्मक तकनीक है कि इन्फ्लूएंजा शोधकर्ताओं उत्पन्न करने के लिए पुनः संयोजक वायरस इन्फ्लूएंजा वायरस के जीव विज्ञान में कई पहलुओं का अध्ययन करने के लिए, और संभावित वैक्टर या टीके के रूप में इस्तेमाल किया जा अनुमति देता है.
Abstract
इन्फ्लूएंजा अनुसंधान समूहों की एक संख्या से प्रयास और एक इन्फ्लूएंजा वायरस रिवर्स आनुवंशिकी के विकास में सुधार में निर्णायक किया गया है. मूलतः 1999 में स्थापित<sup1,2></sup> प्लाज्मिड आधारित रिवर्स आनुवंशिक पुनः संयोजक वायरस उत्पन्न तकनीक इन्फ्लूएंजा अनुसंधान के क्षेत्र में क्र ांतिकारी परिवर्तन किया है क्योंकि विशिष्ट सवालों के अनुवांशिक इंजीनियर, संक्रामक, पुनः संयोजक इन्फ्लूएंजा वायरस से जवाब दिया गया है. इस तरह के अध्ययन वायरस प्रतिकृति, वायरल प्रोटीन के समारोह, वायरल प्रतिकृति और / या रोगजनन में वायरल प्रोटीन में विशिष्ट परिवर्तन के योगदान और, भी, वायरल vectors रीकॉम्बीनैंट इन्फ्लूएंजा विदेशी प्रोटीन व्यक्त वायरस का उपयोग शामिल<sup> 3</sup>.
Protocol
Discussion
प्लास्मिड डीएनए से पुनः संयोजक इन्फ्लूएंजा वायरस के बचाव एक बार प्रोटोकॉल नियमित रूप से प्रयोगशाला में किया जाता है एक सरल और सीधा प्रक्रिया है, लेकिन शुरुआत में, कई बातें गलत जा सकते हैं. यह आवश्यक है ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
लेखकों गार्सिया Sastre Adolfo और पीटर Palese प्रयोगशालाओं में इन्फ्लूएंजा रिवर्स आनुवंशिकी तकनीक और plasmids के विकास के लिए अतीत और वर्तमान सदस्यों को शुक्रिया अदा करना चाहता हूँ. एजी-एस प्रयोगशालाओं में अनुसंधान CRIP, इन्फ्लुएंजा अनुसंधान और निगरानी के लिए एक NIAID वित्त पोषित की उत्कृष्टता केंद्र (HHSN266200700010C) के द्वारा आंशिक रूप से और NIAD R01AI046954 अनुदान, U01AI070469 और P01AI058113 द्वारा वित्त पोषित है. LM-S प्रयोगशाला में अनुसंधान आंशिक रूप से NIAID RO1AI077719 अनुदान द्वारा वित्त पोषित है.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| DMEM | Invitrogen | 11995-065 | Store at 4°C | |
| OptiMEM | Invitrogen | 51985-034 | Store at 4°C | |
| Lipofectamine 2000 (LPF2000) | Invitrogen | 11668-019 | Store at 4°C | |
| TPCK-trypsin | Sigma | T-8802 | Store at -20°C | |
| Bovine Albumin (BA) | Sigma | A7979 | Store at 4°C | |
| Trypsin-EDTA | Invitrogen | 25300-054 | Store at -20°C | |
| Penicillin/Streptomycin (PS) 100X | Invitrogen | 15140-122 | Store at -20°C | |
| Fetal Bovine Serum (FBS) | Hyclone | SH30070.03 | Store at -20°C | |
| V-bottom 96-weel plates | Nunc | 249570 |
Cell lines
293T (catalogue number CRL-11268) and MDCK (catalogue number CCL-34) cell lines are maintained in a 37°C incubator with 5% CO2 in DMEM 10% FBS, 1% PS. Cells are available form the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA. 20110-2209 USA).
Embryonated chicken eggs
Embryonated 10-day-old chicken eggs can be obtained from Charles River Laboratories, Specific Pathogen Fee Avian Supply (SPAFAS) Avian Products and Services. Franklin Commons, 106 Route 32, North Franklin, CT 06254 USA. Eggs are incubated at 37°C preceding and after viral infection. Before and after viral infection, eggs are candled to determine viability of the embryos. It is very important to look for dead eggs before and after viral infection. Before infection a dead egg can be easily spotted by the absence of blood vessels as well as the absence of embryo mobility. When candled, live embryos move. After viral infection a dead egg (probably related to influenza virus infection) will be easily spotted by the bad appearance of the egg as seen by the smaller and bloody volume of allantoic fluid. Infected-eggs are discarded in double autoclavable bags and autoclaved following standard procedures.
Chicken red blood cells (RBC)
Chicken RBC can be purchased from Truslow Farms, 201 Valley Road, Chestertown, Md 21620. Store at 4°C. For HA assays, wash 5 ml of the chicken RBC with 45 ml of PBS 1X in a 50 ml centrifuge tube. Centrifuge for 5 minutes at 1000 rpms, RT. Discard carefully the supernatant and use a 1:1000 dilution of the pelleted RBC in PBS 1X (final concentration of 0.5-1.0% RBC).
Tissue culture supernatants and allantoic fluids
Both, tissue culture supernatants and allantoic fluids can be stored at 4°C for a short period of time. After confirming virus rescue, viruses from cell supernatants or allantoic fluid are stored at -80°C.
Plasmids
All plasmids are prepared using a plasmid maxi kit following manufacturer’s recommendations. All plasmids are aliquot at concentrations of 1 μg/ml in ddH2O and stored at -20°C. For short-term storage, the plasmid can be keep at 4°C. The concentration of the purified DNA plasmid is determined by spectrophotometry at 260 nm, with purity being estimated using the 260:280 nm ratio. Preparations with 1.8-2.0 260:280 nm ratios are considered appropriated for virus rescue purposes. Additionally, plasmid concentration and purity should be confirmed with agarose gel chromatography. Ambisense pDZ plasmids (6) containing the eight influenza A/PR/8/34 viral genes (7) are illustrated in Figure 2.
Viruses
The described protocol for rescuing influenza A/PR/8/34 can be performed under biosafety level (BSL) 2 conditions. Contaminated material, including tissue culture supernatants and embryonated eggs, should be sterilized before disposal. Rescue of other influenza virus may require higher BSL conditions and, therefore, special conditions/security measurements will need to be followed.
Tissue culture media and solutions
DMEM 10%FBS 1%PS: 445 ml Dulbecco’s modified Eagle’s medium (DMEM), 50 ml of Fetal Bovine Serum (FBS), and 5 ml of 100X Penicillin/Streptomycin (PS). Store at 4°C. This media will be used to maintain 293T and MDCK cells as well as for the transfections. DMEM 0.3%BA 1%PS: 495.7 ml of DMEM, 4.3 ml of 35% Bovine Albumin (BA). Store at 4°C. Just before use, add TPCK treated trypsin to a final concentration of 1 μg/ml. Infectious media.
10X Phosphate buffered saline (PBS): 80 g of NaCl, 2 g of KCl, 11.5 g of Na2HPO4.7H2O, 2 g of KH2PO4. Add ddH2O up to 1 liter. Adjust pH to 7.3. Sterilize by autoclave. Store at room temperature.
1X PBS: Dilute 10X PBS 1:10 with ddH2O. Sterilize by autoclave and store at room temperature.
References
- Neumann, G., Watanabe, T., Ito, H., Watanabe, S., Goto, H., Gao, P., Hughes, M., Perez, D. R., Donis, R., Hoffmann, E., Hobom, G., Kawaoka, Y. Generation of influenza A viruses entirely from cloned cDNAs. Proc Natl Acad Sci U S A. 96, 9345-9350 (1999).
- Fodor, E., Devenish, L., Engelhardt, O. G., Palese, P., Brownlee, G. G., Garcia-Sastre, A. Rescue of influenza A virus from recombinant DNA. J Virol. 73, 9679-9682 (1999).
- Martinez-Sobrido, L., Garcia-Sastre, A. Recombinant influenza virus vectors. Future Virology. 2, 401-416 (2007).
- Palese, P., Shaw, M. L., Knipe, D. M., Howley, P. H. Orthomyxoviridae. The viruses and their replication. Fields Virology. , 1647-1689 (2006).
- Schickli, J. H., Flandorfer, A., Nakaya, T., Martinez-Sobrido, L., Garcia-Sastre, A., Palese, P. Plasmid-only rescue of influenza A virus vaccine candidates. Philos Trans R Soc Lond B Biol Sci. 356, 1965-1973 (2001).
- Quinlivan, M., Zamarin, D., Garcia-Sastre, A., Cullinane, A., Chambers, T., Palese, P. Attenuation of equine influenza viruses through truncations of the NS1 protein. J Virol. 79, 8431-8439 (2005).
- Niwa, H., Yamamura, K., Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 108, 193-199 (1991).
