Hagfish oral gland cystatin, preparation method and application

Abstract

The invention discloses a lamprey oral gland CystatinF, a preparation method and application. The sequence similarity between the lamprey oral gland CystatinF (Lm-CystatinF) and reported snake poisonprotein cystatin is less than 29%, and the sequence similarity of the lamprey oral gland CystatinF (Lm-CystatinF) and other species are also very low (30%-40%), so that proliferation of human umbilical vein endothelial cells (HUVECs) is significantly restrained in a dose dependent mode, vascular endothelial cells are restrained from differentiating to be of a tubule structure, that is to say, thelamprey oral gland CystatinF (Lm-CystatinF) has anti-angiogenic activity. According to the lamprey oral gland CystatinF, the preparation method and application, the lamprey oral gland CystatinF is prepared through recombination from optimized lamprey oral gland CystatinF genes, the expression cycle is short, the expression rate is high, and obtained protein is soluble protein, so that enzyme digestion is not needed, one-step chromatography is achieved, and the method has the advantages of simple operation and high yield.

Description

technical field [0001] The invention relates to a protein, a preparation method and an application, in particular to a hagfish oral gland Cystatin F with anti-angiogenesis activity, a preparation method and an application. Background technique [0002] Recent studies have pointed out that angiogenesis is closely related to the occurrence and development of various diseases, such as choroidal neovascular disease, chronic inflammation, psoriasis, retinopathy, rheumatoid arthritis, myelodysplastic syndrome, and tumors. [0003] Cysteine ​​protease inhibitors are a class of proteins that inhibit cysteine ​​proteases. According to different molecular structures and functions, cysteine ​​protease inhibitors can be divided into intracellular stefins, secreted cystatins and kininogen family proteins (kininogens). Members of the type II cystatin family are mainly distributed in body fluids or secretory glands, and contain two pairs of highly conserved intrachain disulfide bonds and ...

AI Technical Summary

Problems solved by technology

[0005] 1. Separation and purification from tissues, the steps are cumbersome, and several steps of chromatography are required to be used in combination, and impurities are likely to remain, and the yield is low;

[0006] 2. Using Pichia pastoris for expression, the expression steps are cumbersome and the cycle is long;

[0007] 3. Using prokaryotic expression, the expression amount is relatively small and exists in the form of inclusion bodies, the protein is inactive, and it needs to go through the later denaturation and refolding process to make the protein have biological functions, and the operation steps are cumbersome; some studies also use For prokaryotic expression, the selected carrier needs to contain a large molecular weight solubilizing tag to obtain soluble protein, which increases the step of subsequent restriction enzyme digestion tag, which not only complicates the operation, but also easily causes new foreign proteins and reduces the protein yield.

[0008] So far, there is no research report on the molecular characteristics and biological functions of cystatin F in the oral gland of hagfish

Images