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A kind of medicine with anti-angiogenesis activity and preparation method thereof

A drug, anti-melanin technology, applied in the directions of biochemical equipment and methods, microorganism-based methods, medical preparations of non-active ingredients, etc., to achieve the effects of low molar concentration, long half-life and high purity

Active Publication Date: 2022-06-14
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] The present invention has discovered a new angiogenesis endostatin ME (amino acid sequence: PIVRRADRAAVPGGGG) with angiogenesis inhibitory activity, and constructed angiogenesis endostatin The fusion protein of ME not only solves the problem of reduced drug efficacy and unsatisfactory expression level caused by the strategy of HSA fusion in the prior art, but also improves the problem of low activity of the existing angiogenesis endostatin

Method used

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  • A kind of medicine with anti-angiogenesis activity and preparation method thereof
  • A kind of medicine with anti-angiogenesis activity and preparation method thereof
  • A kind of medicine with anti-angiogenesis activity and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] 1. Construction of pPINKα-HC / fusion protein vector

[0046] ① Whole-gene synthesis of fusion protein gene fragments, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 1, and the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO: 2;

[0047] ② Kpn I and Stu I double enzyme digestion pPINKα-HC (Invitrogen company product) plasmid and fusion protein gene fragments, gel recovery to obtain pPINKα-HC (Kpn I / Stu I) vector and protein gene fragments, pPINKα-HC recovered from the above gel The (Kpn I / Stu I) carrier fragment and the fusion protein gene fragment are subjected to a recombination reaction to obtain a recombinant yeast expression vector containing the DNA sequence encoding the fusion protein. Transform Escherichia coli competent TOP10, spread on ampicillin-resistant LB plates and culture overnight at 37°C, and screen positive clones. The resulting clones were sent to the company for sequencing;

[0048] ③ Recover the sequ...

Embodiment 2

[0050] Purification of embodiment 2 fusion protein

[0051] According to the properties of the fusion protein, DEAE Sepharose Fast Flow was first selected for purification, and the experimental process was as follows: ①sample pretreatment: the expression supernatant was dialyzed with 20mM PB buffer; ②column balance: the DEAE Sepharosefast flow purification filler was loaded into the purification column, Equilibrate with 20mM PB buffer; ③Sample loading: Load an appropriate amount of sample, collect the flow-through sample, and name it LC; ④Rinse the packing with an appropriate amount of 20mM PB buffer, collect the flow-through liquid, and name it W; Elute with M NaCl, 0.3M NaCl, 0.5M NaCl, 1M NaCl, collect the eluate, and name it E1, E2, E3, E4; ⑥ wash the packing with 0.1M NaOH, collect the flow-through liquid, and name it N; ⑦ Rinse the packing with plenty of distilled water and save the packing with 20% ethanol. Protein samples were analyzed by SDS-PAGE.

[0052] Select Ph...

Embodiment 3

[0062] Example 3 Fusion protein inhibits HUVEC cell proliferation

[0063]It has been reported in the literature that the inhibition rate of HUVEC proliferation and migration rate of bevacizumab (Avastin) at 0-5 mg / mL is gradually increasing (references: Han YS, Lee JE, Jun JW, Lee JS. Inhibitory effects of bevacizumab on angiogenesis and corneal neovascularization.Graefes Arch ClinExp Ophthalmol,2009,247:541–548.), so the inhibition rate at 64μg / mL should theoretically be greater than 16μg / mL, so the maximum concentration of 64μg / mL was uniformly used as a comparison in the experiment , namely the Bv group; HM-3 had the highest inhibition rate at 8 μg / mL, so 8 μg / mL was selected as the positive control.

[0064] Bevacizumab (Avastin) used in the experiment was purchased from Gansu Provincial Cancer Hospital. The manufacturer is Roche Diagnostics GmbH. 4ml; HM-3 was synthesized by Shanghai Jier Polypeptide Co., Ltd. (HM-3 amino acid sequence: IVRRADRAAVPGGGGRGD), with a purit...

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Abstract

The invention discloses an angiogenesis inhibitor and a preparation method thereof, belongs to the field of genetic engineering pharmacy, and in particular relates to a fusion protein which can be used as an angiogenesis inhibitor. The fusion protein has a long-term inhibitory effect on angiogenesis. On the one hand, the technical solution disclosed in the present invention improves the anti-angiogenesis activity of angiogenesis endostatin and the inhibitory effect on melanoma, and on the other hand prolongs its half-life, further enhancing stability.

Description

technical field [0001] The invention belongs to the field of biotechnology and pharmacy, and more specifically relates to a fusion protein with activity of inhibiting angiogenesis, which can be applied in the field of antitumor drugs. Background technique [0002] According to the latest national research report in 2018, the number of new cases of malignant tumors in my country is as high as 3.804 million, and the number of cancer deaths nationwide has reached 2.296 million. Therefore, malignant tumors are currently one of the major diseases that seriously affect human health and threaten human life. Cerebrovascular diseases and accidents constitute the top three causes of death in all countries of the world today. With the continuous development of social economy, the prevalence of tumors is increasing year by year. Therefore, the World Health Organization and the health departments of governments of various countries have listed cancer as a top priority. [0003] The curre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/81C12N1/19A61K38/39A61K47/64A61P35/00C12R1/84
CPCC07K14/78C12N15/815C12N15/62A61P35/00C07K2319/31C07K2319/33A61K38/00A61K38/39A61K47/64C07K19/00C12N15/81C07K14/765A61K38/38C12R2001/84C12N1/165
Inventor 李红玉李婷支德娟李洋王欣史彦斌马兴铭朱红梅王梅竹刘燕段建功张涵姿葛广飞赵陇和岳昭蓉何玉红刘怡瑶宋美娟谢飞王如玥谢亲建宋鹏裴月娟海洋王宁波
Owner LANZHOU UNIVERSITY
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