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Non-blood serum preparation of recombinant human blood coagulation factor IX

A serum-free, human technology, applied in the field of preparation of recombinant hFIX, achieves good application effect, stable treatment, and improved production yield

Active Publication Date: 2009-07-15
浙江耶大生物医药有限公司
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  • Summary
  • Abstract
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Problems solved by technology

Use in mouse model of hemophilia B yields remission

Method used

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  • Non-blood serum preparation of recombinant human blood coagulation factor IX
  • Non-blood serum preparation of recombinant human blood coagulation factor IX
  • Non-blood serum preparation of recombinant human blood coagulation factor IX

Examples

Experimental program
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Embodiment Construction

[0031] 1. Serum-free acclimatization of CHO-K1

[0032] CHO-K1 cells (Chinese hamster ovary cell K1 strain) were purchased from the American Type Culture Collection (CCL-61 TM , CHO-K1). Frozen cells were thawed in a water bath at 42°C, and the cells were washed with serum-free, and directly washed with 5ml of serum-free medium (serum-free medium was purchased from Gibco, CHO-S-SFM) at 37°C, 5% CO 2Cultivate under the same conditions, change the same medium every other day, digest with recombinant trypsin (Promga) when there are obvious clonal colonies, dilute the cells to 103 / ml, and inoculate them into 96-well cell culture plates respectively. Screen the cells with fast growth, large cell volume and no toxic particles as CHO-K1 serum-free suitable cells, and expand and save.

[0033] 2. Gene modification of CHO-K1 transfected with hFIX

[0034] The plasmid (pRBS-CMVhFIX) expressing hFIX was constructed by our laboratory, and its construction method is a conventional vecto...

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Abstract

The invention relates to the preparation of recombinant human coagulation factor IX and an application thereof. The recombinant hFIX protein and hFIX of human blood source have the same molecular composition and biological activity and can be eased by being used for hemophilia B mouse model. The recombinant hFIX protein can be used as a standard in the tests for clinically detecting hFIX and the like, and can also cure diseases requiring supplementing hFIX, such as hemophilia B, etc.

Description

field of invention [0001] The invention belongs to the technical field of gene therapy, and specifically relates to the preparation and application of a recombinant hFIX (human blood coagulation factor IX) used as a detection standard sample or a drug for treating hemophilia B. technical background [0002] Hemophilia is a group of hereditary bleeding disorders, which is a severe coagulation disorder caused by the lack of certain coagulation factors in the blood. These include hemophilia A (factor VIII, AHG deficiency), hemophilia B (factor IX, PTC deficiency) and hemophilia C (factor XI, PTA deficiency). [0003] Hemophilia B (hemophilia B) is a severe coagulation disorder caused by the deficiency of human coagulation factor IX (human clotting factor IX, hFIX). The gene encoding hFIX (hFIX) is located at the far end of the long arm of the human X chromosome and is located in the Xq27.1 region. It is 34kb in length and consists of 8 exons and 7 introns. The full length of t...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/85C07K14/745A61K38/36A61P7/04G01N33/68
Inventor 陈金中万里明薛京伦
Owner 浙江耶大生物医药有限公司
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