Method for cloning segment PEXcDNA at 2C end of ground substance metal protease of human from placenta tissue

A placental tissue and matrix metal technology, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, genetic engineering, etc., to achieve the effect of simple method and good application prospect.

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

All of the above methods require cell culture or tissue culture, and require special experimental reagents, equipment and other conditions.

Method used

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  • Method for cloning segment PEXcDNA at 2C end of ground substance metal protease of human from placenta tissue
  • Method for cloning segment PEXcDNA at 2C end of ground substance metal protease of human from placenta tissue
  • Method for cloning segment PEXcDNA at 2C end of ground substance metal protease of human from placenta tissue

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Experimental program
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Embodiment

[0017] 1. Extract total RNA from placenta tissue with Qiagen Rneasy Mini Kit.

[0018] 1) Take fresh placental tissue, wash it with normal saline, blot it dry with filter paper, cut it into pieces, put it into a clean pre-cooled mortar, add liquid nitrogen, and grind the tissue block into powder.

[0019] 2) Transfer about 30 mg of tissue into a RNase-free 2 ml Ependorf tube.

[0020] 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.

[0021] 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.

[0022] 5) Add 600 μl of 70% ethanol, and mix well by pipetting with a pipette tip.

[0023] 6) Put the Rneasy column into a 2ml collection tube, add 700μl of the sample in 5), centrifuge at room temperature at 12000rpm for 15s, and discard the column liquid.

[0024] 7) Put 700...

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Abstract

This invention discloses a method for cloning C-terminal fragment of human matrix metalloproteinase 2 from placenta tissues. The recombinant adenovirus is constructed by: extracting total RNA from fresh placenta, performing reverse transcription to obtain the first cDNA strand of hemopexin (PEX) domain, amplifying the cDNA strand with primers (PEX446S and PEX660A) to obtain PEX215 fragment (676bp), amplifying the cDNA strand with primers (PEX462S and PEX660A) to obtain PEX198 fragment (629bp), and ligating fragments PEX215 and PEX198 with GST fusion protein vector pGEX-4T-3 and pGEX-5X-1 respectively to obtain recombinant plasmid pGEX-4T-3 / PEX215 and pGEX-5X-1 / PEX198.

Description

technical field [0001] The invention relates to a method for cloning human matrix metalloproteinase 2 C-terminal fragment PEX cDNA from placental tissue. Background technique [0002] Human matrix metalloproteinase 2 (Matrix metalloproteinase-2, MMP2) is also known as gelatinase A. The human MMP2 precursor is 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 angiogenic blood vessels through the bin...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/57
Inventor 吕文清
Owner XIAMEN UNIV
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