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Method for mass producing human blood coagulation factor vii derivative

A technology for mass production of human blood coagulation factors, applied in blood coagulation/fibrinolytic factors, biochemical equipment and methods, botany equipment and methods, etc., can solve the problems of low expression and low productivity, and achieve large-scale and high-efficiency effects

Inactive Publication Date: 2016-07-06
HANMI PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the production of FVII in animal cells using gene recombination technology has a problem of low productivity due to its low expression in most cases

Method used

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  • Method for mass producing human blood coagulation factor vii derivative
  • Method for mass producing human blood coagulation factor vii derivative
  • Method for mass producing human blood coagulation factor vii derivative

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Embodiment 1: construct the expression vector (pX0GC-hFVII) that is used for recombinant FVII expression

[0076] 1-1. Obtaining human FVII gene

[0077] First, the human FVII gene including the signal sequence was obtained by polymerase chain reaction (PCR). For amplification of the FVII gene, PCR was performed using a human fetal liver cDNA library (TAKARABIO Inc., USA) as a template and forward and reverse primers of SEQ ID NO: 1 and 2 shown below.

[0078] After denaturing the reaction mixture containing the polymerase, primers, cDNA library and dNTPs at 95°C for 1 min, 30 cycles of reactions (30 s at 95°C, 30 s at 60°C, and 30 s at 68°C 90 seconds), and then reacted at 68° C. for 5 minutes.

[0079] Specifically, for the convenience of cloning, a BamHI recognition site was inserted into the primer shown in SEQ ID NO: 1, and an XhoI recognition site was inserted into the primer shown in SEQ ID NO: 2. Primers are shown in Table 1.

[0080] 【Table 1】Primers us...

Embodiment 2

[0086] Embodiment 2: construct the expression vector of recombinant FVII derivative

[0087] A polynucleotide encoding a FVII derivative was obtained using an expression vector (pX0GC-FVII) containing a FVII gene, in which part of SOD1 was conjugated to the C-terminus of FVII, and an expression vector capable of expressing the derivative was constructed.

[0088] 2-1. Obtaining Human FVII Derivative Genes

[0089] Construction of an expression vector (pX0GC-FVII-ATKAVC) for expressing a recombinant FVII derivative comprising a polynucleotide encoding SOD1 SEQ ID NO: 1 to 6 (ATKAVC, SEQ ID NO: 5) - the expression vector pX0GC prepared in Example 1 - the 3' end of the FVII gene added in FVII. Specifically, using the expression vector pX0GC-FVII as a template and the forward and reverse primers shown in SEQ ID NO: 6 and 7 shown below, a PCR reaction (denaturation at 95° C. for 1 minute; 30 cycles of reaction (at 95 60 seconds at 60°C, 60 seconds at 60°C, and 90 seconds at 6...

Embodiment 3

[0095] Example 3: Construction of a cell line expressing a human FVII derivative (hFVII-SOD1(ATKAVC))

[0096] The hFVII derivative was expressed using the expression vector constructed in Example 2.

[0097] 3-1. Transformation of FVII derivative (pX0GC-FVII-ATKAVC) using CHO cell line

[0098] The recombinant expression vector pX0GC-FVII-ATKAVC prepared in Example 2-2 was introduced into a DG44 / CHO cell line (CHO / dhfr-) (Urlaub et al., Somat. Cell. Mol. Genet., 12, 555-566, 1986) to obtain transformants, and express FVII-ATKAVC derivatives in the transfectants.

[0099] Specifically, the DG44 / CHO cell line was cultured enough to cover 80% to 90% of the bottom of the culture vessel, and then, the cells were washed three times with Opti-MEM (Gibco, catalog number 51985034).

[0100] Meanwhile, a mixture of 3 mL of Opti-MEM and 5 μg of the expression vector (pX0GC-FVII-ATKAVC) and a mixture of 3 mL of Opti-MEM and 20 μL of lipofectamin (Gibco, catalog number 18324-012) we...

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Abstract

The present invention relates to a method for mass producing human blood coagulation factor VII, and more specifically, to a method for mass producing human blood coagulation factor VII, and a cell line for mass production of a human blood coagulation factor VII derivative, the method comprising the steps of: a) preparing an expression vector comprising i) a base sequence of dihydrofolate reductase (DHFR) promoter in which at least one CCGCCC is removed in a GC-rich region and a base sequence encoding DHFR operably linked thereto, and ii) a base sequence of an early gene of cytomegalovirus (CMV) and a base sequence encoding a human blood coagulation factor VII derivative operably linked thereto; b) transforming an animal cell line by using the expression vector of step; c) selecting a cell line expressing a human blood coagulation factor VII derivative with high efficiency by culturing the transformed animal cell line of step b) in the presence of a DHFR inhibitor; and d) culturing the selected animal cell line of step c) by adding at least one selected from the group consisting of sodium butyrate, vitamin K and a culture medium additive. The present invention can express a human blood coagulation factor VII derivative with high efficiency and in a large quantity by using a vector in which GC-rich repeating sequences are deleted in a DHFR promoter region, and thus can be usefully applied to the preparation of a therapeutic agent for hemophilia.

Description

technical field [0001] The present invention relates to a method for mass production of human coagulation factor VII derivatives and a cell line for mass production of human coagulation factor VII derivatives, said method comprising: a) constructing an expression vector comprising i) its GC The nucleotide sequence of the dihydrofolate reductase (hereinafter DHFR) promoter lacking at least one CCGCCC repeat in the enriched region, and the nucleotide sequence encoding DHFR operably linked thereto, and ii) cytomegalovirus (CMV) the nucleotide sequence of the early gene promoter, and the nucleotide sequence of the human blood coagulation factor VII derivative that is operably linked to it; b) transfect the animal cell line with the expression vector of step a); c) Cultivate the transfected animal cell line of step b) in the presence of a DHFR inhibitor to select a cell line capable of highly expressing a derivative of human coagulation factor VII; At least one of the additional a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/12C12P21/02
CPCC07K14/745C12N9/003C12N9/6437C12N15/85C12P21/02C12Y105/01003C12Y304/21021C12N15/11
Inventor 康喜哲金真荣李炳善金贤煜崔仁荣权世昌
Owner HANMI PHARMA
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