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Integration plasmid and cell line expressing recombinant human FVIII, and construction method and application thereof

A technology that integrates plasmids and construction methods. It can be applied to cells modified by introducing foreign genetic material, applications, and recombinant DNA technology. It can solve problems such as the failure of replacement therapy, achieve high integration rate, wide applicability, and avoid construction and purification. effect of the process

Inactive Publication Date: 2013-07-24
SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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Problems solved by technology

The expression of natural FVIII protein undergoes a very complex post-translational modification (post-translational modifications, PTMs) process, and in the expression s

Method used

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  • Integration plasmid and cell line expressing recombinant human FVIII, and construction method and application thereof
  • Integration plasmid and cell line expressing recombinant human FVIII, and construction method and application thereof
  • Integration plasmid and cell line expressing recombinant human FVIII, and construction method and application thereof

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Embodiment Construction

[0033] The invention provides a site-specific integration of AAV to construct a human 293 cell line expressing FVIII and its construction method, as well as a method for expressing active recombinant FVIII by using the human cell line.

[0034] Further speaking, the present invention utilizes the rAAV integration plasmid and the plasmid expressing the AAV Rep protein necessary for integration to co-transfect HEK 293 cells, and obtains the 293 cell line stably expressing FVIII after screening. On the one hand, the recombinant virus construction and purification processes are avoided, It is simpler and easier to operate; on the other hand, there is no molecular weight limitation of the recombinant virus genome, and it has wider applicability.

[0035] The technical solution of the present invention will be further described below in conjunction with a preferred embodiment and corresponding drawings.

[0036] This embodiment may include the following steps:

[0037] 1. Amplify t...

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Abstract

The invention relates to an integration plasmid and a cell line expressing recombinant human FVIII, and a construction method and an application thereof. The integration plasmid comprises a rAAV plasmid backbone and a B-zone-deleted FVIII gene connected between two ITR elements in the rAAV plasmid backbone. Further, the integration plasmid can comprise a GFP gene used in subsequent cell line screening and marking, and/or an AAVp5 promoter used for improving integrating efficiency. The cell line comprises HEK293 cells which are obtained by co-transfection by using the above integration plasmid or an integration plasmid constructed with the method above, and plasmid used for expressing AAVRep protein. The HEK293 cells can stably express BDD-FVIII. The cell line can be applied in producing human recombinant coagulation factor VIII. The method provided by the invention has the advantages that: (1) recombinant virus construction and purification processes are avoided; the method is simpler and easier to operate; integration rate is high; recombinant viral genome molecular weight limit is avoided, such that the method can be more widely applied; (2) human HEK293 cell is adopted as a host, and structure and function of expressed FVIII protein are more close to natural FVIII protein.

Description

technical field [0001] The present invention relates to a vector for expressing recombinant human blood coagulation factor, a cell line and a construction method thereof, in particular to a 293 cell line expressing recombinant human blood coagulation factor VIII by site-directed integration of AAV, a construction method and application thereof. Background technique [0002] Hemophilia A (hemophilia A), also known as hemophilia A, is an X-linked recessive genetic disease with an incidence of about (1~2) / 10000 in males 1 . Regular infusion of FVIII products such as plasma-derived FVIII concentrate or recombinant human coagulation factor FVIII (rh FVIII) is the main way to treat hemophilia. rh FVIII products have been used for more than 20 years, but all proteins are produced by Chinese hamster ovary cells (CHO) or baby hamster kidney cells (BHK). The expression of natural FVIII protein undergoes a very complex post-translational modification (post-translational modifications...

Claims

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Application Information

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IPC IPC(8): C12N15/864C12N15/66C12N5/10C12P21/02
Inventor 刘晓玫张春
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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