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Recombinant adenovirus of target-oriented coexpressed new p53 and P53AIP1

A technology of recombinant adenovirus and co-expression, applied in the direction of p53 protein, double-stranded DNA virus, virus, etc., can solve the problem of low activity and achieve the effect of broad anti-cancer spectrum and strong anti-cancer function

Active Publication Date: 2010-05-12
广州市凯诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

More importantly, studies have proved that this tumor-specific promoter has strong activity in tumor cells, but has no or very low activity in normal cells

Method used

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  • Recombinant adenovirus of target-oriented coexpressed new p53 and P53AIP1
  • Recombinant adenovirus of target-oriented coexpressed new p53 and P53AIP1
  • Recombinant adenovirus of target-oriented coexpressed new p53 and P53AIP1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Formation of R72 tumor suppressor p53: The method is to use the cloned human p53 gene as a template and use gene mutation technology to mutate the codon ccc (P72) of the wild-type proline at position 72 into sperm. The amino acid codon cgg (R72) forms a new endonuclease smal1 site in the mutation region.

[0030] 1). Human wild-type tumor suppressor p53 gene 5'-end Nco1 to the 72nd codon fragment amplification:

[0031] Using human wild-type P72 tumor suppressor p53 gene cDNA N'-terminal -Nco1-mutant region fragment as a template, PCR amplification was performed with artificially synthesized primers.

[0032] The PCR primers are designed as follows:

[0033] Primer 1:

[0034] N terminal: 5’-gccttccgggtcactg aggagccg-3’

[0035] 30mer Nco1

[0036] Primer 2: ccg is the arginine codon

[0037] N terminal: 5’-gaatgccagaggctgctccc gtggcccctgca-3’

[0038] 35 mer

[0039] After tailing and adding adenine to the PCR amplification product, it is connected to the T-easy vector,...

Embodiment 2

[0052] Example 2: Cloning of apoptosis-inducing protein gene regulated by tumor suppressor gene p53:

[0053] 1) Transfect the expression plasmid containing the wild-type p53 gene into Hela tumor cells after 48 hours of apoptosis, collect the cells, and extract the total ribonucleic acid. After reverse transcriptase, the complementary deoxyribonucleic acid (cDNA , The same below).

[0054] 2) Amplify the p53AIP1 gene by PCR technology: use the synthesized cDNA as a template and artificially synthesized primers for PCR amplification.

[0055] The primer design is as follows:

[0056] Primer 1: (Introduce endonuclease site Sma1 at the N-terminus)

[0057] 5′-ctcccggggatgggatcttcctctgaggcgagcttcaga-3′

[0058] Primer 2: (Introduce endonuclease site Xba1 at the c-terminus)

[0059] 5′-tgagatcttcagttcccagctctgtccaatgctctg-3′

[0060] After tailing and adding adenine to the PCR amplification product, it is connected to the T-easy vector, and the bacteria is transformed, amplified, extracted, pu...

Embodiment 3

[0061] Example 3. Artificial synthesis and cloning of tumor-specific promoter DNA fragments: Based on the known gene sequence, the tumor-specific promoter DNA fragments were artificially synthesized according to conventional techniques, and PCR amplification was performed with the following primers.

[0062] Primer 1: (Introduce endonuclease sites Nru1, Kpn1 and Xba1 at the N-terminus)

[0063] 5’-tgtcgcgaggtacctctagaatttcagtgttgttttcct-3’

[0064] Primer 2: (Introduce endonuclease sites EcoR1 and Stu1 at the C-terminus)

[0065] 5′-gcgatatcaggcctgtccggttcggtttgccaaaagcg-3′

[0066] After tailing and adding adenine to the PCR amplification product, it is connected to the T-easy vector, and the bacteria is transformed, amplified, extracted, purified, and confirmed by DNA sequencing.

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Abstract

The present invention is the constituting method, use and significance of the recombinant adenovirus in expression box structure and co-expressing the new tumor inhibiting gene p53 and the p53 controlled apoptosis inducing protein gene p53AIP1. The present invention features the wild p53 cancer inhibiting gene with the 72nd amino acid mutated from praline into arginine; the internal ribosome binding site connection between p53 gene and p53AIP1 gene; the expression box promoter driven with the specific tumor promoter PEG-3; and recombinant adenovirus being blood serum A5 type adenovirus with copying fault. The recombinant adenovirus has powerful apoptosis function and no damage on normal cell, and thus possesses importance in genetic treatment of tumor, especially malignant tumor.

Description

Technical field [0001] The present invention relates to a method for preparing a recombinant adenovirus for targeted co-expression of p53 and p53AIP1. The prepared recombinant adenovirus has a new type of inhibitor that has a targeted co-expression in tumor cells and has a stronger apoptosis function than wild-type P53 cells. The oncogenes p53 and p53 regulate the apoptosis-inducing protein (P53AIP1), achieve the effect of killing malignant tumors directly, and can be used for gene therapy of various cancers. Therefore, its technical field is related to life medicine and biotechnology. Background technique [0002] The P53 gene is the main member of the tumor suppressor gene family, and is currently the gene with the highest correlation with human tumors. Wild-type p53 gene plays an important role in maintaining the normal growth of cells, preventing and inhibiting the occurrence and proliferation of malignant tumor cells. Various factors in the environment, such as ultraviolet...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/861C12N15/12C07K14/47A61K48/00A61P35/00A61K35/13
CPCC12N15/8616C12N2710/10343A61K48/00C12N2710/10345C12N2830/008C07K14/4746A61K35/13C12N15/86A61P35/00
Inventor 王尚武朱雅南
Owner 广州市凯诺生物科技有限公司
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