Multiple anti-FMD shRNA tandem expression vector as well as construction method and application thereof
A tandem expression and foot-and-mouth disease technology, applied in the fields of virology and genetic engineering, can solve problems such as virus escape, achieve the effect of reducing economic losses, reducing immune escape, and broad-spectrum inhibition of foot-and-mouth disease virus
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Embodiment 1
[0055] Example 1: Design of anti-foot-and-mouth disease shRNA and construction of multiple anti-foot-and-mouth disease shRNA tandem expression vectors and lentiviral vectors:
[0056] According to the whole genome sequences of each serotype of foot-and-mouth disease published on NCBI (AF189157, AY304994, AY593751 and AF274010), multiple sequence alignments were performed using BLAST software, and three conserved regions were selected in the 3B and 3D genes: (1) In the 3B region 5'-CCT GTC GCT TTG AAA GTG AAA GC-3' is located at 4900-4922nt; (2) 5'-GAG ATT CCA AGC TAC AGA TCA CTT TAC CTG CGT TGG GTG AAC GCC GTG TGCGGT GAC GC-3' in the 3D region Located at 6934-6992nt; (3) 5'-GAC GAG TAC CGG CGT CTC TTTGAG CC-3' in the 3D region is located at 6892-6917nt. The double-stranded DNA sequences of the three shRNAs designed according to the 3B and 3D conserved regions in the foot-and-mouth disease virus genome are as shown in Sequence Table 1-3, wherein Cons1 is sequence 1, Cons2 is ...
Embodiment 2
[0088] Example 2: Multiple anti-foot-and-mouth disease shRNA tandem expression lentivirus titer determination at the cellular level:
[0089] Take about 2 x 10 5 293T cells were seeded in a 96-well plate at the density of cells / mL, and 100 μl of 10% FBS+DMEM medium was added to each well. During the measurement, each 8 wells was set as a group, and 10 μl of the lentivirus stock solution prepared in Example 1 was added to the first well, and then each well was diluted at a ratio of 10:1 in turn, and the last well was used as a blank control. 48 hours after inoculation, observe the number of green fluorescent cells under a fluorescent microscope. If there are more than 5 luminescent cells in the previous well, there is no fluorescent expression in this well and subsequent wells; or if there is no fluorescent expression in the subsequent wells, and this well If there are less than 5 luminescent cells, this well is used as a metering well (counted as 1 IU), and the number of lu...
Embodiment 3
[0092] Embodiment 3: by Transgenic BHK-LV cells were obtained by transforming multiple anti-foot-and-mouth disease shRNA tandem expression lentiviral vectors:
[0093] (1) BHK-21 cells were treated with a concentration of 2×10 5 Inoculate cells / mL in 35m cell culture dish, add 1.5ml double antibody-free 10% FBS+MEM medium and culture overnight;
[0094] (2) Take 500 μl lentiviral stock solution (not less than 1×10 6 IU / ml) was fully mixed with 9 μl polybrene (Polybrene) with a concentration of 1 μg / μl, and allowed to stand for 5 minutes;
[0095] (3) Add the mixed solution dropwise into the cell culture dish, shake gently to mix the medium evenly, place it in a carbon dioxide incubator at 37°C for 12-24 hours, and then replace it with 10% FBS+MEM medium;
[0096] (4) 72 hours after the lentiviral vector infected BHK-21, the fluorescence expression of the cells was observed by a fluorescence microscope; since pSicoR had no screening resistance gene, the transgenic BHK-LV ce...
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