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Targeted coexpression p53 and Mda-7 recombination adenovirus

A recombinant adenovirus, co-expression technology, applied in the biological field, can solve problems such as inactivity

Inactive Publication Date: 2008-03-26
王尚武
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

More importantly, this tumor-specific promoter is not active in normal cells

Method used

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  • Targeted coexpression p53 and Mda-7 recombination adenovirus
  • Targeted coexpression p53 and Mda-7 recombination adenovirus
  • Targeted coexpression p53 and Mda-7 recombination adenovirus

Examples

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Embodiment 1

[0022]Embodiment 1, the formation of R72 type tumor suppressor p53: Its method is to take the human p53 gene of clone as template, adopt gene mutation technology, make the codon ccc (P72) of wild-type 72nd proline be mutated into sperm The amino acid codon cgg (R72) forms a new endonuclease smal1 site in the mutation region.

[0023] 1). Amplification of the fragment from the 5'-end Nco1 to the 72nd codon of the human wild-type tumor suppressor p53 gene:

[0024] Take the N'-terminal-Nco1-mutated region in the cDNA of human wild-type P72 tumor suppressor p53 gene

[0025] The segment was used as a template for PCR amplification with artificially synthesized primers.

[0026] The PCR primers were designed as follows:

[0027] Primer 1:

[0028] N-terminal: 5'-gccttccgggtcactg aggagccg-3'

[0029] 30mer Nco1

[0030] Primer 2: ccg is the arginine codon

[0031] N-terminal: 5'-gaatgccagaggctgctccc gtggcccctgca-3'

[0032] 35mer

[0033] After tailing and adding adenin...

Embodiment 2

[0046] Example 2. Artificial synthesis and cloning of Mda-7 DNA fragment: According to the known gene sequence, the Mda-7 coding DNA fragment was artificially synthesized according to conventional techniques, and PCR amplification was performed with the following primers. Primer 1 (43mer):

[0047] 5'-agc tatgaattttcaacagaggctgcaaagcctgtgg-3'

[0048] Smal1

[0049] Primer 2 (37mer):

[0050] 5'-cgac atcagagcttgtagaatttctgcatcc-3'

[0051] Xbal

[0052] After tailing and adding adenine, the PCR amplification product was connected to the T-easy vector, transformed into bacteria, amplified, extracted, purified, and confirmed by DNA sequencing.

Embodiment 3

[0053] Example 3. Artificial synthesis and cloning of tumor-specific promoter DNA fragments: According to the known gene sequence, a tumor-specific promoter DNA fragment was artificially synthesized according to conventional techniques, and PCR amplification was performed with the following primers.

[0054] Primer 1: (introduce endonuclease sites Nru1, Kpn1 and Xba1 at the N-terminus)

[0055] 5'-tgtcgcgaggtacctctagaatttcagtgttgttttcct-3'

[0056] Primer 2: (introduce endonuclease sites EcoR1 and Stu1 at the C-terminus)

[0057] 5′-gcgatatcaggcctgtccggttcggtttgccaaaagcg-3′

[0058] After tailing and adding adenine, the PCR amplification product was connected to the T-easy vector, transformed into bacteria, amplified, extracted, purified, and confirmed by DNA sequencing.

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Abstract

The present invention is recombinant adenovirus for the expression box of targeting co-expressed tumor suppressor genes p53 and Mda-7 and its construction process, use and significance. The present invention features the mutant wild type tumor suppressor gene P53 with the No. 72 place amino acid changed from proline into arginine, the connection between the tumor suppressor gene P53 and the gene Mda-7 by means of the internal ribosome binding site, the tumor specific promoter PEG-3 to drive the expression box, and the recombinant serum A5 type adenovirus with copy defect. The new type of adenovirus has two kinds of targeting co-expressed anticancer genes, p53 and Mda-7, powerful biological function of apoptosis and no damage on health cell. The present invention is significant in the gene therapy of tumor, especially malignant tumor.

Description

technical field [0001] The invention belongs to the field of biotechnology. The invention describes a method for constructing a novel recombinant adenovirus that specifically replicates in tumor cells and expresses genes related to melanocyte differentiation and can be applied to tumor gene therapy. Background technique [0002] Melanocyte differentiation-related genes (English name melanoma differentiation associated (mda) genes), also known as interleukin-24 (abbreviated IL-24, the same below), are a member of the interleukin-10 family. Mda-7 / IL-24 gene is a structurally conserved gene, and its expression product is a glycoprotein consisting of 206 amino acids. [0003] Mda-7 / IL-24 is a gene with specific tumor cell apoptosis discovered by differential hybridization after melanocytes were treated with interferon-β and mezerein (MEZ). Its anti-tumor mechanism is considered to be mainly related to the activation of the immune system. Its expression is limited to melanocyt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N15/12C12N15/24C12N15/28A61K48/00A61P35/00
Inventor 王尚武
Owner 王尚武
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