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Cell strain of expression mutation type human tissue plasminogen activator imethod for construction and preparation method of expressing protein

A plasminogen, mutant technology, applied in animal/human proteins, cells modified by introducing foreign genetic material, extracellular fluid diseases, etc., can solve the problems of high price, large dosage, short half-life, etc. Achieve the effects of reducing production costs, stabilizing expression levels, and increasing expression levels

Inactive Publication Date: 2005-08-24
夏家辉
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it also has certain disadvantages
One is that its price is high, the international market price is 1375 US dollars per bottle; the other is that there are still some defects in the function of the drug, mainly in the following three points: 1), the half-life of rt-PA is too short, only 3-5 minutes; 2), The specific binding efficiency of rt-PA to fibrin in blood clot is not high; 3), the activity and effectiveness of rt-PA still need to be further improved
Due to the short half-life, the dosage is very large, and because the specificity and activity are not high enough, on the one hand, there are still 10-20% of patients whose thrombus cannot be completely dissolved by rt-PA; on the other hand, it will cause systemic fibrosis. Dissolved state, affecting the normal coagulation mechanism, resulting in bleeding, especially intracranial and gastrointestinal bleeding, these serious complications

Method used

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  • Cell strain of expression mutation type human tissue plasminogen activator imethod for construction and preparation method of expressing protein
  • Cell strain of expression mutation type human tissue plasminogen activator imethod for construction and preparation method of expressing protein
  • Cell strain of expression mutation type human tissue plasminogen activator imethod for construction and preparation method of expressing protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Preparation of the gene guide sequence used in the present invention:

[0018] 1. Acquisition of gene guide sequence PAC clone

[0019] 1.1 Microdissection, PCR, and microcloning to construct BM-specific pUC19 library (Deng H-X, Yoshiura K, Dirks RW, et al. Hum Genet 1992, 89: 13.)

[0020] 1.2 Obtaining and identification of BM-specific single-copy DNA

[0021] (1) Preparation of colony array membrane: draw 14×14 squares on two nylon membranes, mark them as A and B, place them in two plates with solid LB respectively, and pick them randomly from the library plate Spot the white clones on two grids with the same membrane coordinates, pick 14×12 cells in total, spot 100ng single-copy DNA on line 13 as a positive control, spot 100ng gDNA on line 14 as a negative control, and place the two plates separately Incubate at 37°C for 10-12 hours, and store B membrane at 4°C, take A membrane out of the dish, and treat it on filter paper soaked with the following solution, 10% S...

Embodiment 2

[0045] Preparation of the gene carrier used in the present invention:

[0046] 1. Construction of gene vector and introduction of target gene

[0047] 1.1 Construction of the carrier

[0048] 1.1.1 Digest PAC DNA with NsiI and StuI (blunt endase), recover a 3.8kb DNA fragment from ordinary agarose gel, and purify by electroelution;

[0049] 1.1.2 Digest the pGEM-TK vector DNA with HindIII, fill in with Klenow enzyme, and generate a blunt end;

[0050] 1.1.3 The pGEM-TK / HindIII fill-in product was further digested with NsiI;

[0051] 1.1.4 Ligate the 3.8kb / NsiI+StuI purified product with the pGEM-TK / HK+NsiI digested product at 16°C for 17 hours;

[0052] 1.1.5 The ligation product was transformed into JM109 competent bacteria, and cultured in an ampicillary dish at 37°C for 18 hours;

[0053] 1.1.6 Randomly pick single clones, and identify positive clones by double digestion with NsiI and NheI.

[0054] 1.1.7 Digest pCDN-GPR plasmid with XbaI and NheI to obtain Neo / XbaI+Nh...

Embodiment 3

[0094] The carrier-TNK-TPA recombinant was obtained by using Example 2, and the target gene was introduced into the host cell HT1080, and the desired cell line was obtained after screening, and expressed in vitro to confirm its site-specific introduction and high-efficiency expression.

[0095] 1. Materials:

[0096] 1.1 Cell: HT1080

[0097] Medium: high sugar DMEM+10% FBS (HT1080) EMEM+10% FBS

[0098] 1.2 Electroporator: Bio-Rad

[0099] 2. Method:

[0100] 1) Cells at 75cm 2 After passage in the culture bottle, grow to 70%~80% full bottom

[0101] 2) Harvest cells, wash 2 times with HeBs buffer, count cells

[0102] 3) Centrifuge at 15000rpm at 4°C for 10 minutes

[0103] 4) An appropriate amount of HeBS was resuspended to make the cell concentration 10 6 ~10 7 / ml

[0104] 5) Take 0.4ml electric shock cup, add 0.8ml cell suspension and about 10μg carrier DNA to each cup

[0105] 6) Electric shock was performed with an electroporator, and the electric shock condi...

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Abstract

The invention involves a cell line which can express human mutated tissue-type plasminogen activator (TNK-TPA), and its preparation methods. The collection No. of cell line in this invention is CCTCC C200006. We firstly use a DNA fragment from the short arms of human D, G group chromosomes or its homologous to construct a recombinant human source gene vector-TNK-TPA (collection number is CCTC m200032); TNK-TPA gene is then transferred into the target site of nucleolus organizing region (NOR) of human D, G group chromosomes in host cell HT1080 by the recombinant and the cell line is attained after screening, which can be used for manufacturing protein.

Description

technical field [0001] The invention relates to a human cell strain, in particular to a human cell strain capable of expressing mutant human tissue plasminogen activator (TNK-TPA). The present invention also relates to the construction method of the cell line and its application in the production method of human tissue plasminogen activin for the preparation of gene medicine. Background technique [0002] Human tissue plasminogen activator can locally dissolve intravascular thrombus by activating plasminogen, and recanalize blocked blood vessels. At present, the only company in the world that can use genetic engineering technology to carry out large-scale recombination and development of human t-PA gene and successfully develop rt-PA drug is American Genetech Company. The first-generation product developed by the company—rt-PA has become the drug of choice for the treatment of thrombotic diseases in European and American countries. However, it also has certain disadvantage...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09A61K38/48A61P7/02C07K14/745C12N5/10C12N9/72C12N15/57C12P21/02
CPCC12Y304/21069G01N2291/0256C12N9/6459A61P7/02
Inventor 夏家辉
Owner 夏家辉
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