Agkistrodon halys ussuriensis disintegrin site-directed mutants cDNA clone, expression and protein preparation method and application thereof

Abstract

The invention relates to the biological field of clone and expression of disintegrin site-directed mutant cDNA of gloydius ussuriensis and a protein preparation method and application thereof. A145, T146 and G148 in the disintegrin cDNA series of the gloydius ussuriensisis are mutated into C145, C146 and A148 by cDNA series of the snake venom mutant protein and then the cDNA containing 219 base pairs, corresponding protein series and the expression in escherichia coli and pichia are obtained. The preparing method is simple and easy to be operated. The invention separates and extracts general RNA from gloydius ussuriensis venom and then general cDNA is reversely transcribed through a RT-PCR method and then is mutated into polynucleotide series with a length of 219bp in a site-directed way.The invention also provides a small molecular-weight protein with a molecular weight of about 8,000 successfully expressed in escherichia coli BL21 and pichia. The protein has remarkable effect on resisting the form of blood vessels while no effect on inhibiting platelet aggregation. And the application of the invention in antitumor drugs has comparatively higher security and fewer side effects and has higher application value.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to disintegrin of Agkistrodon halys. Background technique: [0002] Inhibition of angiogenesis in malignant tumors is a key step in inhibiting tumor growth and tumor metastasis. Tumor within 1mm 3 In the above cases, the source of nutrition cannot rely solely on the penetration of surrounding body fluids, but must rely on the supply of blood vessels, otherwise the tumor will degenerate. If drugs are used to inhibit tumor angiogenesis, the tumor will no longer grow, and further undergo apoptosis, which cannot cause harm to the body. Anti-tumor angiogenesis therapy has many advantages, mainly ① when tumor occurs, angiogenesis has been started, so it has high specificity; ② vascular endothelial cells are exposed to the blood flow, the drug can directly play a role, the dose is small, and the curative effect is high. High, less adverse reactions; ③ endothelial cell gene expr...

AI Technical Summary

Problems solved by technology

Chinese patent CN1487081 discloses that a disintegrin with anti-tumor angiogenesis has been cloned from the venom gland of Agkistrodon halys. This protein contains the motif RGD, which can bind to the adhesion factor integrin αvβ3 on the vascular endothelial cell membrane and inhibit its activity. , but also inhibits platelet aggregation

The anti-platelet aggregation effect can cause bleeding tendency, which is a side effect of Agkistrodon halys disintegrin used for anti-tumor angiogenesis. This side effect has a great impact on the human body. It is necessary to try to remove its anti-platelet aggregation effect and make it specifically anti-tumor blood vessels. Generative effect, increasing the safety of this protein as a drug therapy, but difficult to achieve with current proteins

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