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Albumin variant having anti-angiogenesis activity and preparation method thereof

A technology of albumin and variants, which is applied in the field of protein variants and their preparation, can solve the problems of large dosage and short half-life, and achieve the effect of improving functional effects and strengthening the anti-angiogenesis effect in vivo

Active Publication Date: 2012-07-18
CHENGDU ZEN BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] What the present invention aims to solve is the problem that the existing ATWLPPR peptide has a short half-life in vivo and a large dosage

Method used

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  • Albumin variant having anti-angiogenesis activity and preparation method thereof
  • Albumin variant having anti-angiogenesis activity and preparation method thereof
  • Albumin variant having anti-angiogenesis activity and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of ATWLPPR-albumin variant expression vector and protein synthesis and purification of the present invention

[0042] The mRNA was isolated from human hepatocytes, then reverse-transcribed to synthesize cDNA, and then human albumin cDNA was synthesized by PCR method under the guidance of human albumin-specific primers. The PCR product was cloned into a PCRScript plasmid.

[0043] The full-length sequence of the inserted human albumin cDNA was determined, and the sequence is shown in SEQ ID NO.1. Further in vitro mutagenesis was performed. oligonucleotide primer

[0044] 5'GAGCTTGTGAAACACAAGCCCAAGGCAACAtggctgccaccgcgaGCTGTTATGGATGATTTCGCAGCTTTTGTAGAG 3' (SEQ ID NO: 5).

[0045] Simultaneously synthesize a paired complementary sequence primer as a primer pair. Using the above-mentioned PCRScript plasmid cloned with human albumin cDNA as a template, site-directed mutagenesis was carried out under the mediation of these two primers. As indicated ...

Embodiment 2

[0052] Example 2. Efficacy verification experiment of the ATWLPPR-albumin variant of the present invention

[0053] 1. Experimental design

[0054] (1) ATWLPPR polypeptide was synthesized by Syma-Genosys (Woodland_TX). The ATWLPPR-albumin variant was prepared from Example 1.

[0055] (2) HUVEC cell proliferation assay: Huvec cells were purchased from Cell Applications Inc. (San Diego, CA). Seed Huvec cells at 1500 cells / well in a 96-well culture plate, and place the cells in 37°C CO 2 After culturing in the incubator for 16 hours, different concentrations of synthetic ATWLPPR polypeptides or ATWLPPR albumin variants were added, followed by 2 ng / ml VEGF. After further culturing for 48 hours, 3H-TdR was added. After 6 hours, the cells were harvested, and the amount of 3 H-TdR incorporated was measured with a liquid scintillation counter. N1H-3T3 cells served as controls.

[0056] (3) Polypeptide competition inhibition binding experiment: HEK293 cells were transfected with ...

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Abstract

The invention belongs to the technical field of biomedicine, in particular to the technical field of an albumin medicament. In order to solve the technical problems of short half-life period in vivo and high use level of the existing ATWLPPR peptide, the invention provides an albumin variant. The albumin variant is obtained by mutating a KEQLK peptide section at the tail end of albumin C to a WLPPR peptide section. The albumin variant completely reserves the functions of human albumin and simultaneously has a new ATWLPPR sequence function. Experiments in vitro prove that the albumin variant can obviously inhibit the proliferation and migration of vascular endothelial cells induced by VEGF (Vascular Endothelial Growth Factor) in vitro, and the efficiency of the albumin variant corresponds to that of small peptide. Moreover, when the albumin variant is injected into animals, the efficiency of inhibiting the cornea angiogenesis functions of rabbits is 40-60 times higher than that of the small peptide with the same concentration. The albumin variant can be used as a novel anti-angiogenesis medicament with high efficiency.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a protein variant and a preparation method thereof. Background technique [0002] The term "angiogenesis" was first proposed by the British surgeon John Hunter in 1787. From this, it was discovered that "angiogenesis" is related to embryonic development, tissue regeneration, and chronic inflammation [1]. Until 1971, Folkmani proved that "angiogenesis" was related to tumor growth [2]. Since then, "anti-angiogenesis" has become a new anti-tumor treatment strategy. [0003] Regulation of growth and differentiation of vascular endothelial cells is a key step in angiogenesis. The former has been shown to be regulated by tyrosine protein kinases. Vascular endothelial cell growth factor (VEGF) receptor-2 (KDR) binds to VEGF, leading to the activation of its receptor tyrosine protein kinase activity, thereby initiating a series of subsequent cell signal transduction p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/765C12N15/14C12N15/63C12N1/19A61K38/38A61K48/00A61P35/00C12R1/84
Inventor 吴炯白敏易兴旺
Owner CHENGDU ZEN BIOSCI
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