Application of mthfd1 inhibitors in inhibiting and killing viruses
A technology of inhibitors, viruses, applied in the field of medicine
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Embodiment 1
[0124] Materials and methods
[0125] 1. Construction of MTHFD1 knockout cell lines
[0126] 1.1. Construction of sgRNA expression vector
[0127] sgRNA primer sequence:
[0128] sgMTHFD1-1 forward primer: 5′-caccgAAGGGGAGTGGATCAAACCT-3′;
[0129] sgMTHFD1-1 reverse primer: 5′-aaac AGGTTTGATCCACTCCCCTT c-3′;
[0130] sgMTHFD1-2 forward primer: 5′-caccgGAGGTTATTAGCTGCAGTGA-3′;
[0131] sgMTHFD1-2 reverse primer: 5'-aaac TCACTGCAGCTAATAACCTC c-3'.
[0132] The synthetic sgRNA primer sequence was treated with T4 polynucleotide kinase at 37°C for 30 minutes to phosphorylate it, denatured at 95°C for 5 minutes, and annealed at 1.5°C / min to 25°C to obtain BsmBI (or BbsI) Double-stranded DNA fragments with cohesive ends, as follows:
[0133] Forward: 5′-CACCGNNNNNNNNNNNNNNNNNNNNN;
[0134] Reverse: CNNNNNNNNNNNNNNNNNNNNCAAA-5′.
[0135] The phosphorylation, denaturation and annealing system is:
[0136] 1 μl 100 μM forward oligonucleotide strand;
[0137] 1 μl 100 μM revers...
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