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Recombinant plasmid capable of efficiently expressing human liver growth factor in eukaryotic cell

A high-efficiency expression technology of hepatocyte growth factor, applied in the field of biomedicine, can solve the problems of low conversion rate of recombinant plasmids and poor therapeutic effect

Inactive Publication Date: 2006-12-13
BEIJING NORTHLAND BIOTECH
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Problems solved by technology

[0006] The present invention greatly increases the expression of the target protein after the recombinant plasmid enters the cells by introducing part of the genome intron sequence of human HGF, thereby overcoming the technical problem of low conversion rate of the recombinant plasmid and poor therapeutic effect

Method used

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  • Recombinant plasmid capable of efficiently expressing human liver growth factor in eukaryotic cell
  • Recombinant plasmid capable of efficiently expressing human liver growth factor in eukaryotic cell
  • Recombinant plasmid capable of efficiently expressing human liver growth factor in eukaryotic cell

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Experimental program
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Embodiment Construction

[0014] (1), construction of recombinant plasmid pGK

[0015] First, a 0.7kb ColE1 sequence was amplified using pUC19 as a template by PCR. Using Invitragen's pcDNA3.1 as a template to amplify a 1kb DNA fragment, which contains a cell virus promoter, multiple cloning sites and bovine growth hormone terminator; using Navagen's pET-9a as a template to amplify a 0.9kb DNA fragment The phosphotransferase gene (anti-kanamycin), and then use T4 ligase to sequentially connect the three fragments to form a new recombinant eukaryotic expression plasmid pK.

[0016] Then, using the HGF cDNA reported in PubMed as a template, the full cDNA sequence of the HGF protein was amplified. Treat the HGF fragment and plasmid pK with restriction endonuclease, and then use T4 ligase to insert the HGF fragment into the pK plasmid in cis to form a recombinant plasmid pGK 0 . The detailed structure is as figure 1, The intron fragments between exons 4 and 5, 8 and 9, 15 and 16 were amplified and pro...

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Abstract

The invention relates to the naked DNA gene therapeutic category in biomedical domain. When the invention constructs the people,s hepatocyte growth factor (HGF) recombinant plasmid, it introduces partial included subsequence of HGF genomere DNA sequence, which makes the expression quantity of the said recombinant plasmid in eukaryotic cell largely increased (about 100 times) compared with the recombinant plasmid of cDNA sequence and accordingly overcomes the weak therapeutic efficacy problem caused by low therapeutic rate of present naked DNA gene.

Description

1. Technical field [0001] The invention belongs to the category of naked DNA gene therapy in the field of biomedicine, and is to construct a recombinant plasmid containing human HGF cDNA and a small amount of human HGF intron, so that human HGF can be expressed in large quantities in eukaryotic cells. 2. Background technology [0002] Arterial occlusive disease is a kind of vascular disease that seriously threatens human health. It mainly includes lower extremity arterial occlusive disease and coronary artery occlusive disease. Difficulty and other characteristics, and seriously affect the life and work of patients. At present, there is no good drug treatment method, mainly with the help of surgical treatment, but the patients after surgical treatment have many complications and poor prognosis, and may eventually develop into amputation or systemic failure. Therefore, there is an urgent need to study new methods and new drugs for the treatment of arterial occlusive disease....

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N15/12A61K48/00A61P9/10
Inventor 许松山聂李亚马素永文美玉
Owner BEIJING NORTHLAND BIOTECH
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