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CHO (Chinese hamster ovary) cell strain for stably expressing human clotting factor VIII and application thereof

A stable expression, human blood coagulation technology, applied in coagulation/fibrinolytic factors, artificial cell constructs, animal cells, etc., can solve problems such as cross-infection and limited plasma sources

Active Publication Date: 2015-05-20
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Domestic manufacturers of blood coagulation factor eight for the treatment of hemophilia Hualan Bio, Beijing Tiantan Biology, and Shanghai Institute of Biological Products all obtain blood coagulation factor eight through plasma extraction, but plasma-derived blood coagulation factor eight has the following defects: 1. The source of plasma is limited 2. If the plasma contains viruses, it is easy to cause cross-infection, and the eight human coagulation factors obtained by genetic recombination overcome the above defects

Method used

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  • CHO (Chinese hamster ovary) cell strain for stably expressing human clotting factor VIII and application thereof
  • CHO (Chinese hamster ovary) cell strain for stably expressing human clotting factor VIII and application thereof
  • CHO (Chinese hamster ovary) cell strain for stably expressing human clotting factor VIII and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Use the CHO cell line H9C2 to prepare the eight coagulation factors:

[0030] 1 Experimental method

[0031] 7-day continuous culture of CHO cell line H9C2

[0032] The CHO cell line H9C2 with the preservation number of CCTCC No: C2014191 was divided into 1×10 5 Individuals / ml were inoculated into a 2ml system, which was in a 6-well cell culture plate, and the culture medium was CD CHO Serum-Free Medium for CHO Cells, add glutamine at a final concentration of 8mM, and add 20μl Anti-clumping Agent. Since this cell line contains the G418 resistance tag, G-418 Disulfate with a final concentration of 800μg / ml should be added. Count immediately after inoculation to determine the actual inoculum density. The six-well plate was cultured on a shaker in a cell incubator with 37° C., relative humidity of 70-80%, and 5% carbon dioxide at a rotation speed of 125±5 rpm.

[0033]After 2 days of culture (recorded as the first day at the time of inoculation, here is the second da...

Embodiment 2

[0039] Stability detection of human coagulation factor 8 expressed in CHO cell line H9C2:

[0040] 1 Experimental method

[0041] (1) Continuous passage of cell lines

[0042] The CHO cell line H9C2 with the preservation number of CCTCC No: C2014191 was divided into 1×10 5 Individuals / ml were inoculated into a 2ml system, which was in a 6-well cell culture plate, and the culture medium was CD CHO Serum-Free Medium for CHO Cells, add glutamine at a final concentration of 8mM, and add 20μl Anti-clumping Agent. Since this cell line contains the G418 resistance tag, G-418Disulfate with a final concentration of 800μg / ml should be added. Placed at 37° C., relative humidity 70-80%, and 5% carbon dioxide cell incubator on a shaker for cultivation, with a rotation speed of 125±5 rpm.

[0043] After 2 days of culture, the cell growth was observed and counted. When counting, first take 50 μL of culture solution (that is, the liquid in which the cells are uniformly dispersed by pipe...

Embodiment 3

[0056] Detection of green fluorescent label in CHO cell line H9C2

[0057] 1 Experimental method

[0058] (1) Fluorescence microscope observation

[0059] In the logarithmic growth phase of the CHO cell line H9C2 (generally 2-3 days after inoculation), the cell suspension was taken out and added to a 10mm cell culture dish (Shanghai Wohong Biotechnology Co., Ltd.), and analyzed with an inverted fluorescence microscope (Olin) Bath, Japan) to observe the green fluorescence. The green fluorescence can be observed at 40 times magnification, and the green fluorescence photo is taken at 100 times magnification, and the white light photo of the same position is taken as a control at the same time.

[0060] Such as Figure 4 Shown is the fluorescent photo of the CHO cell line H9C2 observed under a fluorescent microscope at a magnification of 100 times, and there is a white light photo of the same position as a control. Such as Figure 4 It shows that the cell line is in the logar...

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Abstract

The invention relates to the technical field of biology, particularly a CHO (Chinese hamster ovary) cell strain for stably expressing human clotting factor VIII and application thereof. The name of the CHO cell strain for stably expressing human clotting factor VIII is Chinese hamster ovary cell CHO-S / H9C2, and the collection number of the cell strain is CCTCC No:C2014191. The CHO cell strain for stably expressing human clotting factor VIII can stably express the human clotting factor VIII. After 40 times of cell passage, the active kit detects that the human clotting factor VIII in the cell culture supernate is kept at 7IU / ml above.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a CHO cell strain stably expressing human coagulation factor VIII and application thereof. Background technique [0002] Hemophilia A is a blood coagulation disorder caused by the lack or abnormality of the eight coagulation factors in the human body. In the coagulation cascade reaction, coagulation factor 8 acts as a cofactor of activated factor 9 to activate factor 10, thereby stimulating the occurrence of endogenous coagulation reaction. Hemophilia A, a hereditary coagulation disorder, is caused by insufficient or qualitative defects in the eight coagulation factors in the patient's body. About one case of hemophilia A occurs in every 5,000 males. About 100,000 people around the world suffer from hemophilia A, and about 20,000 people in China suffer from hemophilia. However, due to the high prices of Bayer, Wyeth and other foreign products, as well as import and export restrictio...

Claims

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Application Information

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IPC IPC(8): C12N5/071C07K14/755G01N33/68C12Q1/02
Inventor 李大伟李东升杲光伟陈昊朱奇郭昊武正华邓江山马少司荣荣苏琪达
Owner SHANGHAI JIAO TONG UNIV
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