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Transferring carrier of liver cancer target to ward gene introduced by acceptor of alphafetoprotein enhancement type epidermal growth factor dual controlled by transcription and transfer

A technology of epidermal growth factor and alpha-fetoprotein, which is used in gene therapy, introduction of foreign genetic material using vectors, recombinant DNA technology, etc.

Inactive Publication Date: 2003-07-23
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These results suggest that the targeting of quaternary complex-mediated gene transfection is limited by the non-specificity of EGFR expression

Method used

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  • Transferring carrier of liver cancer target to ward gene introduced by acceptor of alphafetoprotein enhancement type epidermal growth factor dual controlled by transcription and transfer
  • Transferring carrier of liver cancer target to ward gene introduced by acceptor of alphafetoprotein enhancement type epidermal growth factor dual controlled by transcription and transfer
  • Transferring carrier of liver cancer target to ward gene introduced by acceptor of alphafetoprotein enhancement type epidermal growth factor dual controlled by transcription and transfer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: PCR amplification preS2 gene:

[0021] Amplify and purify the pBR322-2HBV plasmid DNA, and use it as a template to amplify the preS2 gene by PCR. The 25μL PCR reaction system contains plasmid DNAlng, 10×PCR buffer 2.5μL, Mg 2+ 0.5mmol / L, dNTP 5nmol / L, Taq enzyme 3U and upstream and downstream primers 10nmol / L. The reaction conditions were as follows: 35 rounds of denaturation at 94°C for 5 min, denaturation at 94°C for 40 sec, annealing at 50°C for 40 sec, extension at 72°C for 50 sec, and a final extension at 72°C for 7 min.

Embodiment 2

[0022] Example 2: Construction of antisense RNA liver cancer targeting expression vector for preS2:

[0023] After the preS2 PCR amplification product was subjected to 2% agarose gel electrophoresis, the gel containing the target gene fragment was excised, and the amplification product was recovered according to the operation steps of the DNA purification and recovery kit. The PCR product and pEBAF plasmid DNA were respectively digested with Xbal, and ligated overnight at room temperature with T4 ligase. The next day, 10 μl of the ligation product was taken to transform into DH5α, and recombinant positive single clones were obtained.

Embodiment 3

[0024] Example 3: Preparation of expression system for liver cancer targeted gene transfer mediated by AFP-enhanced EGFR

[0025] The 16-peptide GE7 for the epidermal growth factor receptor (EGFR) and the 20-peptide HA20 of the influenza virus hemagglutinin functional domain were completed on the ABI peptide synthesizer in the United States according to the operating instructions. Combine GE7 and HA20 with polycationic polypeptides respectively. Covalently linked, prepared as GE7-PL, HA20-PL covalently linked. The liver cancer-targeting plasmid DNA prepared in Example 2 and the covalent linker were uniformly mixed in 10 μl of sterilized three-distilled water in different proportions, and after standing at 25°C for 30 minutes, 10 μl of the mixture was identified by 1% agarose gel electrophoresis For DNA blockage, the concentration ratio at which DNA is completely blocked in the sample well is the optimal ratio. According to the optimal ratio, slowly drop the plasmid DNA into th...

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Abstract

A both transcrption and transfer controlled, alpha-fetoprotein enhanced, and epidermal growth factor receptor mediated liver cancer target gene transfer carrier is composed of alpha-fetoprotein mediated exogenous DNA, ligand oligopeptide, polycation polypeptide and endocytic glubule release oligopeptide. It can suppress the growth of liver cancer cells.

Description

(1) Technical field [0001] The invention relates to a hepatitis B virus-related hepatocellular cancer gene, in particular to a liver cancer-targeted gene transfer carrier mediated by epidermal growth factor receptors and liver cancer-specific transcription regulation sequences that can be dually controlled at the transfer and transcription levels. (2) Background technology [0002] The key to gene therapy is how to specifically express the target gene in the target cells, that is, the problem of targeting. Establishing a good gene transfer vector is the key. Currently, there are two types of vectors commonly used in gene therapy: viral vectors and plasmid vectors. Both types of carriers have their own characteristics. Commonly used viral vectors include retrovirus, adenovirus, and adeno-associated virus, among which the former two are the most widely used in gene therapy. Retroviruses mainly act on dividing and proliferating cells, so they are relatively targeted to liver...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61P35/00C12N15/11C12N15/63C12N15/79
Inventor 孙汶生马春红刘素侠曹英林张利宁王晓燕高立芬
Owner SHANDONG UNIV
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