A host cell comprising a vector expressing functional recombinant human blood coagulation factor ⅶ and its high-level expression method
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
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Embodiment 1
[0029] Example 1: Functional recombinant human coagulation factor VII expression vector pInsulator4x-CAGGS-VKORC1-GGCX-
[0030] Construction of FVII;
[0031] (1) Human clotting factor VII (FVII), mouse vitamin K epoxide reductase complex subunit 1 (VKORCl) and mouse γ-glutamyl carboxylase (GGCX) gene clone.
[0032] Human fetal liver tissue and mouse liver tissue were taken respectively, and total RNA was extracted and prepared by TRIZOLLS kit, and the integrity of total RNA was detected by 1% formaldehyde denaturing agarose gel electrophoresis; total RNA was used as template and Oligo (dT) was used as primer , according to the kit instructions to synthesize the first strand of cDNA; using the reverse transcription reaction products as templates, the designed and synthesized FVII, GGCX and VKORC primers were used to amplify the target fragment. The PCR reaction system is as follows: cDNA template 10 ul, 25mmol / L 10×PCRBuffer 5 ul, 25mmol / L Mg 2＋ 1.5 ul, primer (10mmol / L) ...
Embodiment 2
[0073] Example 2: Transfection, screening and cloning of CHO mammalian cells
[0074] (1) Linearization of 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 with 1 / 10 volume of 3 M sodium acetate and 2.5 times the volume of absolute ethanol, incubated at -20 °C for 2 h, and centrifuged at 14,000 rpm for 10 min. Discard the supernatant, add 500 uL of 70% ethanol, and centrifuge at 14,000 rpm for 1 min. Repeat the above steps, discard the supernatant and add 10 uL sterile water.
[0076] (2) Plasmid transfection
[0077] CHO cells were transfected with lipofectin. DHFR-deficient CHO cells were routinely cultured in DMEM medium (HT selection medium) containing 4 g / L hypoxanthine (H) and thymine (T). 5 2 ml / L was inoculated into a 6-well plate, and the culture medium was discarded when the cells grew into a monolayer of 50% to 70%. The cells were washed twice with serum-free medium,...
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 subjected to serum-free acclimation and expanded under MTX pressure, and the culture supernatant was collected. After centrifugation at 1000 rpm at 4°C, the supernatant was filtered through a 0.22 µM filter 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, then apply the filtered supernatant to the column. After loading, wash to baseline with equilibration buffer, then elute impurities with elution buffer (20 mM Tris, 500 mM NaCl, 50 mM imidazole pH 7.8), and finally with elution buffer (20 mM Tris, 50 mM imidazole, pH 7.8). 500 mM sodium chloride, 250 mM imidazole pH 7.8) to elute the peak of the protein of interes...
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