Preparation and application of high-activity blood coagulation factor IX mutant, recombinant protein and fusion protein
A blood coagulation factor and mutant protein technology, applied in the field of hemophilia B, can solve the problem of not showing high activity, and achieve the effect of good gene therapy and recombinant protein replacement therapy
Patent Information
- Authority / Receiving Office
- CN · China
- Current Assignee / Owner
- Publication Date
- 2017-03-15
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Abstract
Description
technical field
[0001] The invention belongs to the field of hemophilia B, and in particular relates to the preparation and application of a highly active coagulation factor IX mutant, recombinant protein and fusion protein. Background technique
[0002] Hemophilia B is a bleeding disorder caused by the deficiency of highly active coagulation factor IX. The activity of factor IX in severe patients is often lower than 1% of normal, and spontaneous bleeding often occurs, leading to muscle hematoma or joint deformity. Infusion of factor IX preparations (currently usually recombinantly expressed factor IX protein in vitro) to replenish the level of factor IX in patients is currently the only effective treatment, but requires frequent administration. Gene therapy is a treatment method currently undergoing clinical trials. The normal factor IX gene is introduced into the patient's body for long-term expression through a viral vector, so as to increase the level of factor IX and pr...
Examples
Embodiment 1
[0033] The amino acid sequence of the mutant protein of highly active blood coagulation factor IX is shown in SEQ ID NO:2.
[0034] The preparation method of the mutant protein of highly active blood coagulation factor IX comprises the steps:
[0035] (1) connecting the human coagulation factor IX gene of human wild type or factor IXArg384Gln mutation into the vector to obtain a recombinant vector;
[0036] (2) Transforming the above-mentioned recombinant vector into a host cell to obtain a recombinant strain;
[0037] (3) continuous perfusion culture of the above-mentioned recombinant bacterial strain in serum-free medium to induce the expression of the mutant protein of recombinant highly active blood coagulation factor IX;
[0038] The serum-free medium is "SAFC Biosciences EX-CELL TM 302” (commercialized reagent). In order to ensure product safety and prevent blood-derived preparations from spreading infectious diseases, serum-free medium is used for mammalian cell cult...
Embodiment 2
[0042] The preparation method of the gene therapy AAV vector of highly active blood coagulation factor IX comprises the following steps:
[0043] (1) AAV vectors were prepared using the three-plasmid method. The rAAV-hFIXcoR384Q vector plasmid, the helper plasmid containing AAV Rep / Cap and the helper plasmid containing the adenovirus helper gene were transfected into 293 cells, and then the rAAV-hFIXcoR384Q vector was purified by cesium chloride ultracentrifugation and passed silver staining and quantitative PCR Check the titer. Other vectors rAAV-hFIXcoR384L and rAAV-hFIXco were prepared by the same method.
[0044] (2) The purified rAAV-hFIXR-384Q, rAAV-hFIXco-R384L and rAAV-hFIXco vectors were injected into hemophilia B mice. Select 4-8 week old hemophilic mice, and inject the three vectors into 4x10 via the tail vein 11 Virus particles / ml were injected into 6-7 mice respectively. Three mice were injected with PBS as negative control.
[0045] (3) aPTT detects factor I...