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Recombinant adenovirus for expression of novel tumour suppressor gene p53

A recombinant adenovirus, tumor suppression technology, applied in gene therapy, antitumor drugs, genetic engineering, etc., can solve the problem of low promoter activity

Inactive 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

However, it has been proved that the activity of this promoter is low, and further research is needed

Method used

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  • Recombinant adenovirus for expression of novel tumour suppressor gene p53
  • Recombinant adenovirus for expression of novel tumour suppressor gene p53
  • Recombinant adenovirus for expression of novel tumour suppressor gene p53

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment one: the formation of R72 type tumor suppressor p53: its method is to adopt gene mutation technology, make wild-type 72nd proline codon ccc (P72) be mutated into arginine codon cgg (R72), in The mutated region forms a new endonuclease Small site.

[0018] 1). Nco1 to the 72nd codon fragment amplification of the 5'-end of the human wild-type tumor suppressor p53 gene:

[0019] The human wild-type P72 tumor suppressor p53 gene's 5'-end Nco1 to mutant region fragment was used as a template, and artificially synthesized primers were used for PCR amplification. The PCR primers were designed as follows:

[0020] Primer 1:

[0021] N-terminal: 5'-gccttccgggtcactg aggagccg-3'

[0022] 30mer Nco1

[0023] Primer 2: ccg is the arginine codon

[0024] N-terminal: 5'-gaatgccagaggctgctccc gtggcccctgca-3'

[0025] 35mer

[0026] After tailing reaction and addition of adenine, the PCR amplified product was connected to the T-easy vector, transformed into bacteria, ...

Embodiment 2

[0039] Example 2: Construction of an adenovirus shuttle plasmid containing the R72-type p53 tumor suppressor gene

[0040] 1). PCR amplification of R72-type p53 full-length DNA fragments: the R72-type wild-type p53 gene expression plasmid pCMV-neo-p53 was used as a template, and artificially synthesized primers were used for PCR reaction.

[0041] Primers were designed as follows:

[0042] Primer 5 (introducing endonuclease site Nhe1 at the N-terminus):

[0043] N-terminus: 5'-ta gctagc tcactgccatggaggagccg-3'

[0044] 28mer Nhe1

[0045] Primer 6 (introducing endonuclease site BamH1 at the N-terminus):

[0046] N-terminal: 5'-ggc ggatcc tgtcagtgtgagtgagg-3'

[0047] 26mer BamH1

[0048] After tailing and adding adenine, the PCR amplified product was connected to the T-easy vector; after transforming the bacteria, it was amplified, extracted, purified, and confirmed by DNA sequencing.

[0049] 2). Construction of an adenovirus shuttle plasmid containing the R72 tumor ...

Embodiment 3

[0051] Example 3 Recombinant adenovirus expressing R72-type tumor suppressor gene p53

[0052] 1). Preparation of linearized R72-containing recombinant shuttle plasmid pDC315-p53: Take an appropriate amount of plasmid DNA, digest it with endonuclease LoxP, separate by electrophoresis, purify the linearized plasmid DNA with LoxP, and set aside;

[0053] 2). Embryonic kidney 293 cells were cultured, and the embryonic kidney 293 cells expressing the E1 region gene of the adenovirus were co-transfected with the premixed adenovirus vector and the linearized shuttle plasmid pDC315-p53.

[0054] 3). Screen positive clones, isolate, purify, and identify recombinant adenovirus.

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Abstract

The invention discloses the expression structure of a recombinant adenovirus p53 for expressing anti-tumor gene, characterized in: (1) the 72nd amino acid of the wild p53 anti-tumor gene changes frompraline to arginine, other amino acids components and their arranging sequences do not change, (2) splicing the p53 anti-cancer gene to the shuttle vector pDC315 strong promoter mMCV downstream at itsN-terminal with incision enzyme Nhe1 and C-terminal with incision enzyme BamH1 fragment, and (3) the recombinant adenovirus is replication defective adenovirus displacing junction of adenovirus Ad5 with Ad35 junction. The new p53 anti-cancer gene adenovirus has strong infectivity, broad infection tissue / cell, strong expression of p53 anti-cancer gene, and product with strong apoptosis.

Description

technical field [0001] The invention relates to a method for preparing a recombinant adenovirus of a new human tumor suppressor gene p53. The prepared recombinant adenovirus expresses a novel tumor suppressor gene p53 protein with a stronger apoptosis function of wild-type p53, and is used for various Cancer treatment. 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 it is the gene most closely related to the occurrence of human tumors. The wild-type p53 gene plays an important role in maintaining normal cell growth and inhibiting the proliferation of malignant tumor cells. Various factors in the environment, such as ultraviolet rays, radioactive and chemical substances, and some metabolites produced by the body itself, can cause cellular DNA damage. Under normal physiological conditions, the DNA of these cells can be repaired or degraded and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/861C07K14/435A61K48/00A61K38/17A61P35/00
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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