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Host cell containing vector for expressing functional recombinant human coagulation factor VII and high-level expression method of functional recombinant human coagulation factor VII

A technology of human coagulation factor and host cell, which is applied to the host cell and the high-level expression field of the vector expressing functional recombinant human coagulation factor VII, can solve the problems of excessive exogenous gene fragment and low productivity of functional FVII, Achieve the effect of improving expression level, increasing synergy, and avoiding mutual influence

Active Publication Date: 2015-01-21
山西省博奥特医学检验有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the overall productivity of functional FVII is still low due to the difference in promoter strength between different promoters, as well as the excessive size of foreign gene fragments and the interaction between co-expressed genes.

Method used

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  • Host cell containing vector for expressing functional recombinant human coagulation factor VII and high-level expression method of functional recombinant human coagulation factor VII
  • Host cell containing vector for expressing functional recombinant human coagulation factor VII and high-level expression method of functional recombinant human coagulation factor VII
  • Host cell containing vector for expressing functional recombinant human coagulation factor VII and high-level expression method of functional recombinant human coagulation factor VII

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Functional recombinant human coagulation factor VII expression vector pInsulator4x-CAGGS-VKORC1-GGCX-

[0030] Construction of FVII;

[0031] (1) Gene cloning of human coagulation factor VII (FVII), mouse vitamin K epoxide reductase complex subunit 1 (VKORCl) and mouse γ-glutamyl carboxylase (GGCX).

[0032] Human fetal liver tissue and mouse liver tissue were taken respectively, and total RNA was extracted and prepared using TRIZOLLS kit, and the integrity of total RNA was detected by 1% formaldehyde-denatured agarose gel electrophoresis; total RNA was used as a template, and Oligo (dT) was used as a primer According to the instructions of the kit, the first strand of cDNA was synthesized; using the respective reverse transcription reaction products as templates, the target fragments were amplified with the designed and synthesized FVII, GGCX and VKORC primers. The PCR reaction system is as follows: cDNA template 10 ul, 25mmol / L 10×PCR Buffer 5 ul, 25mmol / L...

Embodiment 2

[0073] Example 2: Transfection, screening and cloning of CHO mammalian cells

[0074] (1) Linearization of the pIsulator4x-VOKRC1-GGCX-FVII plasmid constructed in Example 1

[0075] The plasmid was digested with Swa I, digested at 25 °C for 8 h, then added 1 / 10 volume of 3 M sodium acetate and 2.5 volumes of absolute ethanol, incubated at -20 °C for 2 h, and centrifuged at 14000 rpm for 10 min at low temperature. Discard the supernatant, add 500 uL of 70% ethanol, and centrifuge at 14000 rpm for 1 min at low temperature. Repeat the above steps, discard the supernatant, and add 10 uL sterile water.

[0076] (2) Plasmid transfection

[0077] CHO cells were transfected with lipofectin liposomes. Routinely culture DHFR-deficient CHO cells in DMEM medium (HT selection medium) containing 4 g / L hypoxanthine (H) and thymine (T). 5 / L inoculated into a 6-well plate at 2 ml / L, and cultured until the cells grew into a 50%-70% monolayer, the culture medium was discarded, the cells wer...

Embodiment 3

[0080] Example 3: Expression, purification and identification of human recombinant coagulation factor VII

[0081] (1) Expression and purification of FVII

[0082] The high-expressing cell lines obtained by limiting dilution were acclimatized in the absence of serum, expanded and cultured under MTX pressure, and the culture supernatant was collected. After centrifugation at 1000rpm at 4°C, the supernatant was filtered through a 0.22 μM filter membrane and Ni sepharose TM 6 Fast Flow separation and purification. Equilibration buffer (20 mM Tris, 500 mM NaCl, 10 mM imidazole pH 7.8) to equilibrate Ni sepharose TM 6 Fast Flow column, and then put the filtered supernatant on the column. After sample loading, wash with equilibration buffer to baseline, then use elution buffer (20 mM Tris, 500 mM NaCl, 50 mM imidazole pH 7.8) to elute impurities, and finally wash with elution buffer (20 , 500 mM sodium chloride, 250 mM imidazole (pH 7.8) to elute the target protein peak.

[0...

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Abstract

The invention provides a host cell containing a vector for expressing a functional recombinant human coagulation factor VII and a high-level expression method of the functional recombinant human coagulation factor VII, and aims at establishing an expression vector containing F VII, GGCX and VKORC1 recombinant nucleic acids and improving the g-carboxylation modification of the recombinant F VII by virtue of coordinated expression of the GGCX and the VKORC1. The host cell contains the F VII recombinant nucleic acid (human coagulation factor VII), depending on which the vitamin K is coded, a VKORC1 (mouse vitamin K epoxide reductase complex subunit 1) recombinant nucleic acid and a GGCX (mouse g glutamyl carboxylase) recombinant nucleic acid, as well as an insulator (4X) recombinant nucleic acid; the high-level expression method of the functional recombinant human coagulation factor VII comprises the steps of establishing an expression vector, mediating the expression vector into a DHFR deficient CHO animal cell by use of a lipidosome method and screening out positive clones by virtue of a DMEM culture medium, performing serum-free acclimation and culture on the host cell strain, purifying the h FVII recombinant protein by virtue of nickel ion affinity chromatography and performing SDS-PAGE and Westernblot detection, and finally, determining the procoagulant activity of the FVII recombinant protein according to the prothrombin time (PT).

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a host cell containing an expression vector, and the expression vector contains: recombinant nucleic acid encoding vitamin K-dependent human blood coagulation factor VII (FVII), mouse vitamin K reductase complex subunit 1 ( VKORC1) recombinant nucleic acid and mouse g amino acid carboxylase (GGCX) recombinant nucleic acid, and insulator (4X) recombinant nucleic acid. Among them, mouse vitamin K reductase complex subunit 1 (VKORC1), mouse g amino acid carboxylase (GGCX), and human blood coagulation factor VII (FVII) are stably expressed in the host cells. The present invention also relates to a method capable of expressing functional human blood coagulation factor VII at a high level, and the host cell can significantly increase the yield of functional human blood coagulation factor VII when cultured under specific conditions and scale. Background technique [0002] Coagulation factor ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N9/64
Inventor 张全爱赵邑张禄卿郑耀武何永吉潘薇薇赵峰梅梁雅丽曹鹏程赵亚蕊
Owner 山西省博奥特医学检验有限公司
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