Blood coagulation factor FVIII protein variant, expression vector and use

Abstract

This invention provides a blood coagulation factor FVIII protein variant, an expression vector, and its use. The coding nucleic acid sequence of the blood coagulation factor FVIII protein variant of this invention is shorter than that of the wild-type blood coagulation factor FVIII protein, making it easier to achieve conventional viral vector packaging, exhibiting enhanced protein expression activity, and resulting in relatively high expression levels of blood coagulation factor FVIII protein upon transfer to cells.

Claims

1. A blood coagulation factor FVIII protein variant characterized in that its amino acid sequence is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. A nucleic acid encoding a blood coagulation factor FVIII protein variant, characterized in that its nucleotide sequence is represented by one of the sequences SEQ ID NO: 3-15 or SEQ ID NO: 60-68.

3. An expression vector characterized by comprising the nucleic acid described in claim 2.

4. The expression vector is selected from one of the following groups: adeno-associated virus vectors, reverse transcription virus vectors, adenoid virus vectors, plasmids, or slow virus vectors. Preferably, the expression vector according to claim 3 is characterized in that the adeno-associated virus vector includes the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 serotype.

5. The expression vector further comprises a promoter, enhancer, intron, one or more terminally inverted repeat sequences, Kozak sequence, and / or polyA tail, which are operably connected to the nucleic acid. The expression vector according to claim 3 or 4, characterized in that, optionally, the enhancer and the promoter are sequentially operablely connected to the nucleic acid sequence, or the intron is located inside or on both sides of the nucleic acid sequence, or the terminal inverted repeat is located at the 5' or 3' end of the nucleic acid sequence, or the Kozak sequence is located between the 5' end of the nucleic acid sequence and the intron, or the polyA tail is located between the 3' end of the nucleic acid sequence and the terminal inverted repeat.

6. The nucleotide sequence of the promoter is shown in one of the sequences with SEQ ID NO: 16 to 18, the nucleotide sequence of the enhancer is shown in one of the sequences with SEQ ID NO: 19 to 21, the nucleotide of the intron is shown in one of the sequences with SEQ ID NO: 22 to 24, the nucleotide sequence of the terminal inverted repeat sequence is shown in SEQ ID NO: 25 or SEQ ID NO: 26, the Kozak sequence is gccacc, and the nucleotide sequence of the polyA tail is shown in SEQ ID NO:

27. Preferably, the expression vector is characterized in that it contains any of the sequences shown in SEQ ID NO: 28 to 43.

7. A host cell characterized by comprising the nucleic acid described in claim 2 or the expression vector described in any one of claims 3 to 6.

8. A pharmaceutical composition comprising a blood coagulation factor FVIII protein variant as described in claim 1, a nucleic acid as described in claim 2 and / or an expression vector as described in any one of claims 3 to 6, and a pharmaceutically acceptable additive.

9. Use of a variant of the blood coagulation factor FVIII protein according to claim 1, the nucleic acid according to claim 2, and / or an expression vector according to any one of claims 3 to 6, in the expression of the blood coagulation factor FVIII protein or in the preparation of a drug for the treatment of a disease requiring the blood coagulation factor FVIII protein.

10. A method for producing the blood coagulation factor FVIII protein, Expressing the nucleic acid described in claim 2 and / or the expression vector described in any one of claims 3 to 6 within a cell, This includes collecting the blood coagulation factor FVIII protein produced from the aforementioned cells, A method for producing the blood coagulation factor FVIII protein, characterized by the features described herein.

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