Angiogenesis inhibitant, purification method and medicinal composition therewith

An angiogenin and angiogenesis technology, applied to angiogenesis inhibitors, and its purification method can solve the problems of short half-life of endostatin, insoluble inclusion body protein, unfavorable large-scale production, etc., to overcome short half-life and abundant resources , Inhibition of growth and metastasis

Active Publication Date: 2012-03-14
ZHAOKE GUANGZHOU OPTHALMIC DRUG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, although the Escherichia coli expression system used in this invention has a high expression level, the obtained inclusion body protein is difficult to dissolve and refold, which easily causes inconvenience in application; even if the protein is refolded and soluble, this process will lose a large amount of protein, resulting in the use of rate is not high
In addition, the half-life of endostatin in the body is short, and the preparation process of the angiogenesis-inhibiting polypeptide is complicated and the cost is relatively high, which is not conducive to large-scale production

Method used

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  • Angiogenesis inhibitant, purification method and medicinal composition therewith
  • Angiogenesis inhibitant, purification method and medicinal composition therewith
  • Angiogenesis inhibitant, purification method and medicinal composition therewith

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1 Preparation of high-purity angiostatin

[0048] 1. Treatment of snake venom

[0049] First, accurately weigh 5 g of Agkistrodon halys viper venom purchased from Huizhou Viper Research Institute of Huangshan City, and dissolve it in a 4°C refrigerator with 0.02mol / L Tris-HCl buffer solution at pH 8.0 for 6-12 hours. Then it was centrifuged at 4000r / min, twice for 15min each time, and the supernatant was collected for later use.

[0050] 2. Preliminary purification to obtain antigens for the preparation of monoclonal antibodies

[0051] (1) Anion exchange chromatography

[0052] First, take the obtained supernatant and load it on an equilibrated anion exchange chromatography DEAE column at a flow rate of 3.0ml / min. The miscellaneous protein adsorbed by the medium, the elution time is 360min, and the elution flow rate is 3ml / min; then, add 0.5mol / L NaCl to 0.02mol / L Tris-HCl buffer solution with pH 6.5 for gradient elution, gradient 0~100 %, the elution ti...

Embodiment 2

[0066] Embodiment 2 Quality research of angiostatin according to the present invention

[0067] 1. Purity and structure identification

[0068] Purity identification: Randomly select a group of high-purity angiostatin samples obtained in Example 1 and carry out non-reducing SDS-PAGE electrophoresis (polyacrylamide gel electrophoresis), such as Figure 6 As shown, it is shown as a band with a molecular weight of 30,000 to 35,000 Daltons.

[0069] Structure identification: The third group of samples extracted above were subjected to two-dimensional electrophoresis, that is, isoelectric aggregation electrophoresis and reducing SDS-PAGE electrophoresis, such as Figure 7 As shown, the angiostatin can obtain six bands after two-dimensional electrophoresis, wherein the α chain and the β chain respectively have three bands. ID identification of the six bands by mass spectrometry indicated that they were the isomeric forms of the two subunits, that is, the protein would produce isof...

Embodiment 3

[0077] Example 3 High-purity angiostatin API content detection method

[0078] In order to detect the content of angiopoietin in serum so as to be used in pharmacokinetic test, a set of method for detecting the content of angiopoietin raw material drug by double antibody sandwich method was established, and the detection sensitivity of this method can reach 1ng / ml.

[0079] The specific detection method is as follows:

[0080] 1. Coating a monoclonal antibody: first properly dilute the monoclonal antibody prepared in Example 1 with 0.05mol / L pH9.6 carbonate buffer solution, and put it in the reaction well of each polystyrene microplate plate Add 0.1ml, overnight at 4°C or keep for 18-24h. The next day, the solution in the well was discarded, and washed 3 times with washing buffer for 3 minutes each time (abbreviated as washing, the same below).

[0081] 2. Adding samples: add the sample to be tested or a properly diluted standard reference product into the reaction wells, an...

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Abstract

The invention discloses an angiogenesis inhibitant, which has an amino acid sequence selected from SEQ ID NO.1 and SEQ ID NO.2 and a molecular weight of 25000-35000 Dalton measured by a non-reducing SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) method. The purification method comprises the steps of: first dissolving agkistrodon acutus venom in a buffer solution, conducting centrifugation so as to take supernate; subjecting the obtained supernate to anion exchange chromatography so as to obtain an angiogenesis inhibitant crude solution; then coupling a monoclonal antibody secreted by a hybridoma cell strain 1B9 (CCTCC C200970) with an affinity chromatography vector so as to prepare an immune affinity chromatography column; finally purifying the crude solution with the obtained immune affinity chromatography column, thus obtaining the angiogenesis inhibitant. The angiogenesis inhibitant of the invention can be prepared into powder injections for injection, and has an anti-angiogenesis activity, especially angiogenesis related to tumor growth, and also has an obvious effect in tumor resistance and tumor metastasis resistance.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to an angiogenesis inhibitor, more specifically, the invention relates to an angiostatin, a purification method thereof, and a pharmaceutical composition containing them. Background technique [0002] Tumor angiogenesis refers to the complex biological process in which vascular endothelial cells differentiate and migrate from the existing vascular system to form new microvessels. Adult vascular endothelial cells are basically in a quiescent state, and will generate new blood vessels under physiological stimuli such as wound healing, tissue repair, female fertility and menstrual periods, and fetal development, which belongs to physiological angiogenesis. At this time, angiogenesis is an orderly physiological process within a limited time under the strict control and coordination of stimulators and inhibitors, and the proliferating endothelial cells quickly return to a normal qui...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46C07K1/22C07K1/18A61K38/17A61K9/00A61P9/00A61P35/00
CPCA61K38/00A61P9/00A61P35/00C07K14/46
Inventor 李小羿戴向荣杨中强张国辉方丽钱芳刘娟娟
Owner ZHAOKE GUANGZHOU OPTHALMIC DRUG
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