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Method for constructing straight nucleus expressed plasmid in PEX secretion type of human and application

A technology for expressing plasmids and construction methods, which is applied in the fields of application, botany equipment and methods, biochemical equipment and methods, etc. It can solve the problems of not being able to secrete extracellularly, no detection labels, etc., and achieve the effect of simple and convenient construction methods

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

AI Technical Summary

Problems solved by technology

The existing PEX eukaryotic expression vectors either cannot be secreted extracellularly or have no detection label or have the above two defects

Method used

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  • Method for constructing straight nucleus expressed plasmid in PEX secretion type of human and application
  • Method for constructing straight nucleus expressed plasmid in PEX secretion type of human and application

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Embodiment Construction

[0021] Below by embodiment the present invention will be further described.

[0022] 1. Extract total RNA from placenta tissue with Qiagen Rneasy Mini Kit.

[0023] 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.

[0024] 2) After the liquid nitrogen evaporates, transfer about 30 mg of the tissue into a RNase-free 2 ml Ependorf tube.

[0025] 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.

[0026] 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.

[0027]5) Add 600 μl of 70% ethanol, and mix well by pipetting with a pipette tip.

[0028] 6) Put the Rneasy column into a 2ml collection tube, add 700μl of the sample in 5), centrifuge at room...

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Abstract

This invention relates to a method for constructing eukaryotic expression plasmid capable of excreting human hemopexin, and its application. The method comprises: designing two PCR primers PEX446sense and PEX660antisense according to human MMP2 sequence, its coding frame, and multi-coloning sites of pSecTag A vector, amplifying human MMP2 C-terminal segment PEX cDNA by using pGEX-4T-3 / PEX215 plasmid as the template, and constructing prokaryotic expression plasmid pSecTag A / PEX. The method is simple, and prokaryotic expression vector condtructed by using human PEX cDNA as the inserting segment is advantageous for research on human PEX cell function and application. The constructed plasmid can be used as a genetic therapy drug for treating tumors.

Description

technical field [0001] The invention relates to a method for constructing human PEX secreted eukaryotic expression plasmid. Background technique [0002] Human matrix metalloproteinase 2 (Matrix metalloproteinase-2, MMP2) is also known as gelatinase A. The precursor of human MMP2 is 660 amino acids, the first 29 amino acids are the signal peptide sequence, and the latter 80 amino acids are the propeptide domain, which can maintain the zymogen form. The catalytic domain follows 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 angiogenic blood vessels through the binding of the PEX fragment to the i...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/12C12N15/66A61K48/00A61P35/00
Inventor 吕文清
Owner XIAMEN UNIV
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