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Vector for expressing recombinant blood coagulation factor FIX and construction method and applications thereof

A blood coagulation factor and carrier technology, applied in the field of genetic engineering, can solve the problems of unsafe packaging and complexity of viruses, and achieve the effect of alleviating the bleeding symptoms of hemophilia

Active Publication Date: 2019-11-08
广州辉园苑医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of this, it is necessary to provide a safe plasmid vector for hemophilia B gene therapy and its application that is not integrated into the host genome and can replicate in the cell for the problems of virus insecurity and packaging complexity

Method used

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  • Vector for expressing recombinant blood coagulation factor FIX and construction method and applications thereof
  • Vector for expressing recombinant blood coagulation factor FIX and construction method and applications thereof
  • Vector for expressing recombinant blood coagulation factor FIX and construction method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1, construction and separation and purification of vector

[0027] The plasmid packaged into recombinant FIX is named pCEP4-HSA-FIX-TripleL, and the construction method is as follows figure 1 As shown, the replicon of the expression vector has a human herpes virus replicon (replication origin, OriP), and an EBNA-1 expression cassette, and the cDNA (SEQ ID NO: 1) of the human FIX gene is cloned into the downstream of the CMV promoter , In addition, the plasmid also includes a CMV enhancer and a CMV promoter, both of which can enhance the long-term expression of the transgene FIX. Human coagulation factor genes are synthesized by the company, and the front end of FIX cDNA is the secretion signal peptide coding sequence of HSA (SEQ ID NO: 2).

[0028] The secretion efficiency of recombinant protein varies greatly due to different signal peptides. In order to improve the secretion efficiency of coagulation factors, the present invention replaces the signal pepti...

Embodiment 2

[0036] Embodiment 2, construction required umbilical cord mesenchymal stem cells

[0037] The human umbilical cord mesenchymal stem cells required for the construction of the present invention are prepared and obtained by the following steps:

[0038] Take the 25cm2 culture flask for culturing the 3rd-5th generation human umbilical cord mesenchymal stem cells;

[0039] Wipe around the cap of the culture bottle with an alcohol cotton ball, and then unscrew the cap;

[0040] Aspirate and discard the original culture medium in the culture bottle, wash each bottle with 5ml of PBS buffer twice, and aspirate and discard the washing solution;

[0041] Add 0.5ml of trypsin-EDTA to each bottle and put it in a 37°C incubator for 5 minutes;

[0042] Observe the cells under an inverted microscope, and gently tap the culture bottle with the palm of your hand to ensure that the human umbilical cord mesenchymal stem cells are completely digested;

[0043]Add 2ml of complete medium to each...

Embodiment 3

[0045] Example 3, Vector pCEP4-HSA-FIX-TripleL introduced into cells and expressed

[0046] The cells obtained in Example 2 were resuspended with the electroporation solution, and centrifuged and washed once; the cell suspension was counted and resuspended to a density of 1.0-1.2×10 6 / ml cells; get 0.4ml electroporation cup, add cell suspension 100 μ l to each cup, add the gene carrier pCEP4-HSA-FIX-TripleL plasmid DNA 10 μ g that embodiment 1 obtains; Instrument, select eukaryotic cell mode, voltage 350V, electric shock 60μs, electric shock 1 time. Transfer the electroporated cells into a 25cm2 culture flask, add 5ml of complete medium, and culture at 37°C.

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Abstract

The invention belongs to the field of gene engineering, and particularly relates to a vector for expressing a recombinant blood coagulation factor FIX and a construction method and applications thereof. Aiming at the problems of insecurity of viruses and packaging complexity of adeno-associated viruses, the invention provides a safe cell which is not integrated into a host genome, can be copied incells and is used for hemophilia gene therapy and a construction method and applications of the cell. The plasmid vector comprises an EBV gene for encoding EBV nucleoprotein EBNA-1, an oriP replication starting point of EBV, a blood coagulation factor hFIX encoding sequence and the like. The recombinant vector disclosed by the invention is a gene therapy product which introduces the blood coagulation factor into cells to treat hemophilia for the first time, and lays a foundation for the vector to further safely treat hemophilia B.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a vector expressing recombinant blood coagulation factor FIX and its construction method and application. The vector can be used for treating hemophilia B. Background technique [0002] Hemophilia B is an inherited bleeding disorder caused by deficiency or dysfunction of clotting factor FIX, which is caused by a recessively inherited defect in the gene for clotting factor FIX on the X chromosome. The clinical feature of hemophilia is spontaneous joint and muscle bleeding, and wound bleeding is difficult to heal itself. Long-term repeated attacks can lead to joint deformation and seriously affect the quality of life of patients. The existing technology mainly adopts preventive coagulation factor replacement therapy for treatment. Regular infusion of purified FIX factor can significantly reduce the severity and frequency of bleeding in patients with hemophilia B, but...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10A61K38/37A61K48/00A61P7/04
CPCC12N15/85C12N5/0665A61K38/37A61K48/0025A61K48/005A61P7/04C12N2510/02
Inventor 江福能邹翠云钟惟德朱建国张鑫
Owner 广州辉园苑医药科技有限公司
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