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Hepatocellular carcinoma targeting gene expression element AP and applications thereof

A gene expression and targeting technology, applied in liver cancer targeting gene expression element AP and its application field, can solve the problems of high cost of siRNA, limited application, unfavorable regulation of shRNA, etc., and achieve the effect of inhibiting proliferation

Inactive Publication Date: 2012-05-02
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cost of chemically synthesized siRNA is too high, and the expression of shRNA is not conducive to regulation, these factors greatly limit the application of this technology

Method used

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  • Hepatocellular carcinoma targeting gene expression element AP and applications thereof
  • Hepatocellular carcinoma targeting gene expression element AP and applications thereof
  • Hepatocellular carcinoma targeting gene expression element AP and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Cloning of Regulatory Sequences Expressed by Recombinant AFP Positive Cells

[0023] 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'-AGATCT 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

[0026] Cloning of artificial microRNA against human DNA polymerase

[0027]The mRNA sequence of the human DNA polymerase gene was retrieved from GenBank (GenBank accession number: NM_016937), 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 was designed. TGT TGA CAG TGA GCGACC AAT TTA GAG TTC ATC ATT ATA GTG AAG CCA CAG ATG TA-3', Primer 2: 5'-TCC GAG GCA GTA GGC ACC CAA TTT AGA GTT CAT CAT TAT ACATCT GTG GCT TCA CTA TA-3' , Primer 3: 5'-AGA TCT GAT CCAAGAAGG 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 DL2000DNAL...

Embodiment 3

[0030] Construction of liver cancer targeting gene expression element AP and preparation of adenovirus vector

[0031] 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, Ecl136II, 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, USA) was double-digested with restriction enzymes Pvu II and HindIII, and the recovery included HindIII, Asp718, Kpn I, BamH I, BstXI, EcoR I, EcoRV, BstXI, Not I , Xho I, Xba I, Dra II, Apa I, and Pme I multiple cloning sites and a fragment of the bovine growth factor gene polyadenylation signal (BGHpA), with a HindIII sticky end at one end and a blunt end of PvuII at the other end. The above...

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Abstract

The invention provides a hepatocellular carcinoma targeting gene expression element AP and applications thereof, and the gene expression element AP has a nucleotide sequence indicated in SEQ ID No.1. After the expression element AP is guided into Alpha-fetoprotein positive tumor cells, the artificial microRNA aiming at DNA polymerase can be expressed in the cell and the expression of the DNA polymerase in the cell can be effectively inhibited, the copy of the DNA in the cell is further affected; the cell cycle of is blocked finally, the multiplication of the AFP positive hepatoma cell is specially inhibited and the expression element AP can be applied to the preparation of the targeting gene curing medicine of AFP (alpha fetal protein) positive liver cancer.

Description

technical field [0001] The present invention belongs to biological technology, and relates to gene expression regulation, in particular, relates to the design and preparation of a targeted gene expression element AP specific for alpha-fetoprotein-positive liver cancer and its specificity in alpha-fetoprotein-positive liver cancer cells. Expression, and its transcription products can effectively inhibit the expression of DNA polymerase in liver cancer cells after intracellular processing, so that DNA replication in cells cannot proceed due to the lack of DNA polymerase, thereby achieving the effect of inhibiting the proliferation of liver cancer cells. The invention can be used to prepare targeted tumor gene therapy drugs. Background technique [0002] Malignant tumor is currently one of the main "killers" of human beings. With the change of people's lifestyle and environment in recent years, its incidence is on the rise. It has become the first cause of death in many cities ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11
Inventor 曹江贾振宇陈萍毛晨宇
Owner ZHEJIANG UNIV
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