Duck Tembusu virus infectious clone strain, and preparation method and application thereof
A duck Tembusu virus, infectious cloning technology, applied in the field of bioengineering, can solve problems such as lack of vaccines
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Embodiment 1
[0038] Example 1 Construction of full-length cDNA clone of duck Tembusu virus FX2010 strain genome
[0039] 1. Materials and methods
[0040] 1.1 Materials
[0041] 1.1.1 Viruses and Cells
[0042] Duck Tembusu virus FX-2010 strain and DF-1 cells were preserved by Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences.
[0043] 1.2 Method
[0044] 1.2.1 Nucleic acid extraction of FX2010 duck Tembusu virus
[0045]Use Takara’s RNAiso Plus reagent to extract viral RNA, and operate according to the instructions on the product manual. The specific method is as follows: pipette 300 μl of virus liquid into a 1.5ml RNase-free centrifuge tube, add 700 μl RNAiso plus Reagent, pipette and mix well, and let stand at room temperature for 5 minutes; then add 200 μl chloroform to the above mixture, vibrate vigorously for 15 s, let stand at room temperature for 2 min, and then centrifuge at 12000 rpm for 10 min at 4°C. Take the supernatant and transfer it to an...
Embodiment 2
[0111] Example 2 Rescue and Identification of Duck Tembusu Virus FX2010 Strain
[0112] 1. In vitro transcription
[0113] Using the above viral cDNA as a template, using T7Kit in vitro transcription kit for in vitro transcription to obtain infectious RNA.
[0114] 2. Purification of in vitro transcription products
[0115]Lithium chloride precipitation was used to purify the transcription product. Lithium chloride (LiCl) was included in the in vitro transcription kit. The specific method is as follows:
[0116] Add 30ul Nuclease-free Water and 30ul LiCl to the transcription termination product, mix gently, freeze at -20°C for at least 30 minutes, centrifuge at 14000xg, 4°C for 15 minutes, remove the supernatant carefully, then add 1ml of 70% ethanol (use DEPC water Preparation), 14000xg, centrifuge at 4°C for 15 minutes, discard the supernatant, let the ethanol evaporate completely, add 20ul Nuclease-free Water to dissolve the RNA precipitate, store it at -70°C for later...
Embodiment 3
[0156] Example 3 Rescue of E Gene Replacement Recombinant Duck Tembusu Virus
[0157] 1. Materials and methods
[0158] 1.1 Materials
[0159] 1.1.1 Viruses and Cells
[0160] The segmented amplified fragments of duck Tembusu virus FX-2010 and FX2010-180P and the plasmids constructed by segments, and DF-1 cells were all preserved by the Shanghai Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.
[0161] 1.2 Method
[0162] 1.2.1 Construction of recombinant viral infectious cDNA
[0163] 1.2.1.1 Recombinant virus genome construction strategy
[0164] The FX2010-180P attenuated strain was artificially attenuated from the FX-2010 strain. The recombinant virus in this study replaced the E gene of the FX-2010 strain with the E gene of the FX2010-180P attenuated strain, while retaining other regions of the virulent genome. segment strategy ( Figure 9). By comparing the E gene sequences (table 4) of the two strains, find different sites, then desi...
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