A liver cancer targeting gene expression element ag and its application
A gene expression and targeting technology, applied in gene therapy, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problems of limited application, high cost of siRNA, unfavorable regulation of shRNA, etc., to block energy supply and inhibit proliferation. Effect
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Embodiment 1
[0024] Cloning of Regulatory Sequences Expressed by Recombinant AFP Positive Cells
[0025] First retrieve the mRNA sequence of the human AFP gene from GenBank (GenBank accession number: NM_001134), and then obtain the genome sequence of the No. 4 chromosome of people including the AFP gene from GenBank through online genome comparison analysis method (GenBank accession number : NT_006216.14), and based on the AFP gene mRNA sequence, its 5' end was used as the start point of AFP gene transcription, and the primers were designed and synthesized according to its upstream genome sequence: 5'-AGA TCT CAG ATT GAA TTA TTTGCC TGT CA -3', Primer 2: 5'-GGA TCC TAG GAA GTT TTC GCA ATA ATAC-3', Primer 3: 5'-AGA TCT GCC CCA AAG AGC TCT GTG T-3' and Primer 4: 5'-GGATCC AAA TCA TGC TGA AAT TCT TTT ATA CTC-3', using the polymerase chain reaction (Polymerase Chain Reaction, PCR) to use the genomic DNA of human liver cancer cell SMMC7721 as a template, carry out nucleic acid amplification with...
Embodiment 2
[0028] Cloning of artificial microRNA against human GAPDH
[0029] The mRNA sequence of the human GAPDH gene was retrieved from GenBank (GenBank accession number: NM_002046), and the appropriate site on the mRNA sequence was analyzed using online software to determine the target of the artificial microRNA, and the synthetic primer 1: 5'-TGC TGT TGA was designed CAG TGA GCGCGC TCA TTT CCT GGT ATG ACA ATA GTG AAG CCA CAG ATG TA-3', Primer 2: 5'-TCC GAG GCA GTA GGC AAG CTC ATT TCC TGG TAT GAC AAT ACATCT GTG GCT TCA CTATT-3', Primer 3 : 5′-AGATCT GAT CCA AGA AGG TATATT GCT GTT GAC AGT GAG CG-3′ and primer 4: 5′-GGA TCC ATC GTA GCCCTT GAA GTC CGA GGC AGT AGG CA-3′. Primer 1 and primer 2 were firstly subjected to overlap extension PCR to obtain amplified product C of 97 bp, and then amplified product C was used as a template to perform PCR amplification with primer 3 and primer 4 to obtain amplified product D of 142 bp, see figure 2 , wherein lane 1 is DL2000DNALadder, lane 2 is a...
Embodiment 3
[0032] Construction of liver cancer targeting gene expression element AG and preparation of adenovirus vector
[0033] First construct the pDC312-BGHpA vector: cut the adenovirus shuttle plasmid pDC312 vector (Microbix, USA) with restriction endonuclease Xba I single enzyme, fill in with Klenow enzyme, and then use restriction endonuclease HindIII single enzyme cut , Remove the part between Xba I and HindIII (from HindIII, Sac I, Ecl136 II, Acc I, Sal I to Xba I), one end of the recovered part is a blunt end, and the other end is a HindIII sticky end. The pcDNA3.1 (+) vector (Invitrogen Company, U.S.) was double-digested with restriction endonucleases PvuII and HindIII, and the recovery included HindIII, Asp718, Kpn I, BamH I, BstXI, EcoR I, EcoRV, BstXI, Not I, Fragment of the Xho I, Xba I, Dra II, Apa I, and Pme I multiple cloning sites and the bovine growth factor gene polyadenylation signal (BGHpA) with a HindIII sticky end on one end and a blunt PvuII end on the other. T...
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