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Method for constructing recombined adenovirus in PEX outer secretion type of human and application

A technology of recombinant adenovirus and construction method, applied in application, virus/bacteriophage, recombinant DNA technology, etc.

Inactive Publication Date: 2007-07-25
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are no research reports on the treatment of angiogenesis-related diseases such as tumors using adenoviral vector-mediated PEX at home and abroad.

Method used

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  • Method for constructing recombined adenovirus in PEX outer secretion type of human and application
  • Method for constructing recombined adenovirus in PEX outer secretion type of human and application
  • Method for constructing recombined adenovirus in PEX outer secretion type of human and application

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Embodiment Construction

[0027] Below by embodiment the present invention will be further described.

[0028] 1. Extract total RNA from placenta tissue with Qiagen Rneasy Mini Kit.

[0029] 1) Take fresh placental tissue, wash it with physiological saline, put it into a clean mortar, cut it into pieces, add liquid nitrogen, and grind the tissue block into powder.

[0030] 2) After the liquid nitrogen evaporates, transfer about 30 mg of the tissue into a RNase-free 2 ml Ependorf tube.

[0031] 3) Add 600 μl of RLT buffer solution pre-added with β-mercaptoethanol (β-ME, 10 μl / ml RLT buffer solution), and vortex to lyse the tissue.

[0032] 4) The lysate was centrifuged at room temperature for 3 minutes at the maximum speed of a desktop centrifuge, and the supernatant was transferred to another Ependorf tube.

[0033] 5) Add 600 μl of 70% ethanol, and mix well by pipetting with a pipette tip.

[0034] 6) Put an Rneasy small column into a 2ml collection tube, add 700 μl of the sample in 5), centrifuge ...

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Abstract

This invention relates to a method for constructing recombinant adenovirus capable of secreting human PEX. The method comprises: constructing plasmid pSecTag A / PEX, constructing plasmid pGEX-4T-3 / PEX 215, amplifying PEX cDNA fragment of human MMP2 by PCR, constructing eukaryon expression plasmid pSecTag A / PEX, contructing pAdEasy-pShuttle-CMV-PEX (Ad-PEX) carrier, recombining Eschericia coli adenovirus, amplifying recombinant pAdEasy-pShuttle-CMV-PEX (Ad-PEX), and packaging to obtain intact recombinant adenovirus, which can be used in gene drugs for treating angiogenesis-related diseases, such as tumors.

Description

technical field [0001] The invention relates to a virus, in particular to a method for constructing a human PEX exocrine recombinant adenovirus. Background technique [0002] Human matrix metalloproteinase 2 (Matrix metalloproteinase-2, MMP2) is also known as gelatinase A. The human MMP2 precursor has 660 amino acids, the first 29 amino acids are the signal peptide sequence, the next 80 amino acids are the propeptide domain, which can maintain the form of the zymogen, and the catalytic domain is behind the propeptide domain. After the catalytic domain is the C-terminal hemopexin-like domain, namely the PEX fragment. On the cell surface, after MT1-MMP (memberance type 1-Matrix metalloproteinase) binds to TIMP-2 (tissue inhibitor of metalloproteinase-2), the MMP2 zymogen specifically binds to the C-terminus of TIMP-2 through the C-terminal PEX fragment to activate MMP2. MMP2 localizes to the surface of tumor-invasive cells and blood vessels that form by binding to integrin α...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/861C12N15/12C12N15/66A61K35/76A61P35/00A61P9/00A61K35/761
CPCY02A50/30
Inventor 吕文清
Owner XIAMEN UNIV
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