Method for purifying blood coagulation factor VII
A blood coagulation factor and cation chromatography technology, applied in the field of protein purification, can solve the problems of high purification cost and inability to remove impurity proteins, and achieve the effect of simple operation, high success rate and short time consumption
Pending Publication Date: 2022-05-06
北京双鹭立生医药科技有限公司 +2
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Problems solved by technology
[0007] For this reason, the embodiment of the present invention provides a method for purifying blood coagulation factor VII to solve the problem of inability to effectively remove impurity proteins from rabbit milk in the prior art, or obtain high-purity blood coagulation factor VII, but the purification cost is too high question
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Embodiment 1
[0029] Embodiment 1, the purification of coagulation factor VII of the present invention
[0030] In the present invention, the preparation method of transgenic rabbits refers specifically to the technical scheme recorded in the patent application number: 2017800817940, using microinjection technology to obtain FVII transgenic breeding rabbits, and finally obtaining homozygous FVII transgenic female rabbits through mating, breeding, identification, and screening , for the acquisition of FVII transgenic rabbit milk.
[0031] 1. Rabbit milk defatted from coagulation factor VII transgenic rabbits
[0032] Use a filter with a filter membrane Supra cap 50 P080 (Pall) to defat the diluted rabbit milk, wherein, after the rabbit milk melts at room temperature, mix the rabbit milk with the rabbit milk dilution in a ratio of 1:9 (v / v) Uniform, wherein, the rabbit milk dilution composition is pH 5.0, 20mM acetate buffer. Connect the peristaltic pump, pressure gauge, etc.; flush the pip...
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The embodiment of the invention discloses a method for purifying a blood coagulation factor VII. The method comprises the following steps: degreasing rabbit milk of a blood coagulation factor VII transgenic rabbit to obtain degreased rabbit milk; the degreased rabbit milk is subjected to SP Fast Flow cation chromatography, and a first purified solution containing the blood coagulation factor VII is obtained; carrying out chromatography on the first purified liquid by virtue of a Heparin Bestarose HP chromatographic column, so as to obtain a second purified liquid containing the blood coagulation factor VII; and carrying out Butyl-650M chromatography on the second purified liquid to obtain a third purified liquid containing the blood coagulation factor VII. According to the method for purifying the blood coagulation factor VII, the blood coagulation factor VII is subjected to crude purification by using an SP Fast Flow filler so as to remove most of impure proteins in rabbit milk, then an SP Fast Flow elution sample is purified by using a Heparin Bestarose HP chromatographic column, and finally the Heparin Bestarose HP elution sample is purified by using Butyl-650M chromatography, so that the blood coagulation factor VII is purified. Therefore, the influence of a large amount of casein in the rabbit milk on the affinity filler loading capacity and the purification effect is reduced, the blood coagulation factor purity can be effectively improved, the cost is reduced, and the economic benefit is improved.
Description
technical field [0001] The invention relates to the technical field of protein purification, in particular to a method for purifying coagulation factor VII. Background technique [0002] Hemophilia is an inherited blood clotting disorder and the most common bleeding disorder in humans. It is characterized by disorders of active thromboplastin generation, prolongation of coagulation time, and a lifelong tendency to hemorrhage after minor trauma. Bleeding can occur in severe patients without obvious trauma, and hemophilia is lifelong. Hemophilia is caused by the absence of certain functional clotting factors in the blood. Usually the most rapid and effective treatment is injection of clotting factor. The most common therapeutic clotting factor today is factor VIII. However, clinical studies have shown that about 5% of hemophiliacs may experience side effects including chills, nausea, vomiting, dizziness and headache after injection of coagulation factor VIII, and long-term ...
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IPC IPC(8): C12N9/64
CPCC12N9/6437C12Y304/21021
Inventor 周凯强朱红雨岳永峰杨仲璠王红丽徐明波
Owner 北京双鹭立生医药科技有限公司