Porcine reproductive and respiratory syndrome mutant virus as well as construction method and application thereof
A respiratory syndrome and mutant virus technology, applied in the direction of viruses, viral peptides, antiviral agents, etc., can solve the problems of persistent virus infection, inability of vaccines to produce scavenging immunity, and induction of secondary infections.
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Embodiment 1
[0039] Example 1 Construction of mutant virus RvJXwn-nsp1β-VS19GG full-length cDNA clone plasmid
[0040] Referring to the complete genome sequence of PRRSV JXwn06 (GenBank accession number EF641008), the following primers were designed to construct the full-length cDNA of PRRSV mutant strain RvJXwn-nsp1β-VS19GG (as shown in Table 1 below). The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.
[0041] Table 1 Primers for RvJXwn-nsp1β-VS19GG full-length cDNA clone construction
[0042]
[0043]
[0044] Note: The sequence in bold italic is the recognition sequence of restriction endonuclease
[0045] The construction strategy of the mutant strain RvJXwn-nsp1β-VS19GG full-length cDNA cloning plasmid is as follows: figure 1 shown, and operated using a one-step mutagenesis kit (Novizym, Nanjing, China). First with Max Super-FidelityDNA Polymerase to amplify the target fragment, follow the steps in the instruction manual for a 50 μL reaction system, use...
Embodiment 2
[0049] Example 2 Rescue of mutant virus RvJXwn-nsp1β-VS19GG
[0050] Inoculate well-growing MARC-145 cells in 6-well cell plates at a ratio of 1:3, and place at 37°C with 5% CO 2 Cultivate overnight in an incubator, and transfect when the cell density reaches about 90%.
[0051] According to the instructions of Lipofectamine LTX (Invitrogen, CA, USA) transfection reagent, take a sterile 1.5ml EP tube and add 200μL of Opti-MEM (Invitrogen, CA, USA), and then add 2.5μg of full-length infectious clones Plasmid and PLUS Reagents 2.5 μL, after gently pipetting and mixing, let stand at room temperature for 5 minutes. Then add Lipofectamine LTX 7.5μL, mix gently by pipetting, and let stand at room temperature for 20min. Then the mixture was slowly added dropwise to the cell culture plate, and after 24 hours of transfection, it was replaced with DMEM medium containing 2% FBS (fetal bovine serum), and the culture was continued for 4 days. After 4 days, typical PRRSV cell lesions app...
Embodiment 3
[0052] Identification of embodiment 3 mutant virus RvJXwn-nsp1β-VS19GG
[0053] RT-PCR Identification of Rescued Viruses
[0054] The mutant strains were continuously passaged on MARC-145 cells, and 500 μL of the rescued P3-P9 generation virus solutions were added to 1.5 mL EP tubes, and 1 mL of Trizol was added, mixed well, and then allowed to stand at room temperature for 10 minutes; then 200 μL of chloroform was added , shake vigorously for 15s, then set aside at room temperature for 10 minutes; centrifuge at 12000rpm at 4°C for 15 minutes, pipette 500 μL of the supernatant into a new 1.5mL EP tube; add 600 μL of isopropanol, stand at -20°C for 60 minutes, , centrifuge at 12000rpm for 15min, discard the supernatant; then wash the precipitate with 1000μL of 75% ethanol, continue to centrifuge at 4℃, 12000rpm for 5min, discard the supernatant, and wash twice; finally dry the precipitate at room temperature for 5min, add 30μL DEPC water to dissolve RNA, RNA concentration was ...
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