Reverse genetics of influenza viruses
Reverse genetics of influenza viruses
It mainly describes the methods used to recover infectious influenza viruses from cDNA clones. These techniques are widely used in the integration and expression of exogenous genes, the development of candidate vaccines, and the study of influenza virus reconstitution. One of the advantages of using influenza viruses as antigenic carriers is the availability of a large pool of viruses with different antigens. This means that the previously existing immunity to one virus strain does not prevent the use of another virus strain with a different antigen.
Author: T. Friedman et al., Translated by W. Jing et al. This experiment is from "Gene Transfer".
Operation method
Application of reverse genetics to obtain influenza viruses Move Application of reverse genetics to obtain influenza viruses material reagents Bovine serum albumin Cells 239T cells (ATCC, CRL-11268) Canine Kidney Cells (MDCK, ATCC, CCL-34) Prior to transfection, cells should be grown to confluence in 75cm2 cell culture flasks. D M EM Fetal Cow Serum (F CS) Lipofectam ine 2000 reagent (Invitrogen 11668-027) O pti-M emi low serum medium (GIBCO 31985) Phosphate Buffer Solution (PBS) Plasmid (0.5ug/ml), transfection purity Protein Expression Carrier Prepare a separate vector for each of the following proteins: PA, PB1, PB2, NP. R N A Expression Carrier Prepare a separate vector for each of the following influenza virus fragments: v R N A H A , M, N A , N A , N P , N S , P A , PBl, PB2" T P C K - Insulin (Sigm a-A ldrich T -8802) Instrumentation Cell culture dish (35m m 2) Cell culture bottle (75cm2) Incubator (37°C, 5 % CO2) 6-well cell culture plate Methods 1 . Mix 0.5 tons of each of the 8 RNA expression plasmids and 4 protein expression plasmids. At room temperature, dilute with 0pti_M E M medium to a final volume of l0 Ml . 2- Add 6ul Lipofectamine and 9t1 Opti-M EM medium to the second tube. Mix well and incubate for 5min0 at room temperature. 3 . Add IOOfJ Transfection Reagent Master Mix (Step 2) to the DNA Mix (Step 1). Tap the tube to mix. Incubate at room temperature for 20 min. 4 . Trypsinize confluent 239T and M D C K cells. Isolated cells were resuspended in IOml room temperature DMEM [containing 10 % FCS (no antibiotics)]. Centrifuge at 5.300 g for 5 m in. The cells were resuspended in IOml room temperature DME [10 % FCS (without antibiotics)]. 6. I : 1 mixing of 293T and MDCK cells. Add IOml of mixed cells per 35mm2 dish. 7 . Add DNA/Lipofectamine mixture to the dishes. Shake the dish to mix and distribute the cells evenly. incubate at 37°C for 6-24h. 8 . Change the medium to O pti-M E M (containing 0. 0 1 % FCS, 0 -3 % BSA and l Mg/m l of TPCK-insulin). The culture was continued. 9 - One day before viral transfection (e.g., post-transfection period at approximately 2), inoculate MDCK into 6-well cell culture plates. 37°C. 10.48 h later, collect the transfected mixed cells floating on the surface and centrifuge at 7000 g to remove the supernatant. 11. Remove the medium from the MDCK cells in 6-well plates. The MDCK cells should reach 80% to 90% confluence at the time of transfection (48h post-transfection). 12. Wash cells twice with PBS. Add 200/xl of the supernatant obtained in step 10. Incubate at 37°C with shaking for 1 h. 1 3 . Add 2 m l O p t i - M E M (containing 0- 0 1 % F C S , 0. 3 % B S A and 1/zg/m l of T P C K insulin). 37°(;, 5 % (: 0 2 incubator.). 1 4 . Cells infected by transfected supernatants were examined daily for changes in their C P E appearance and H A levels in the culture supernatant. For more product details, please visit Aladdin Scientific website.