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Serine protease isolated from the venom of bombus ignitus as fibrinogenolytic and fibrinolytic enzymes

A serine protease, fibrin technology, applied in the field of serine protease, can solve the problem of unclear function of serine protease

Inactive Publication Date: 2012-12-19
陈炳来
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the gene of the serine protease and its role in hemostasis and thrombosis are unknown

Method used

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  • Serine protease isolated from the venom of bombus ignitus as fibrinogenolytic and fibrinolytic enzymes
  • Serine protease isolated from the venom of bombus ignitus as fibrinogenolytic and fibrinolytic enzymes
  • Serine protease isolated from the venom of bombus ignitus as fibrinogenolytic and fibrinolytic enzymes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1. Cloning of the gene of the serine protease present in the venom of Bombus rufica

[0027]Total RNA was extracted from the venom glands of Bombus rufica worker bees provided by Dept.of Agricultural Biology in National Academy of Agricultural Science of Rural Development Administration (Dept. of Agricultural Biology in National Academy of Agricultural Science of Rural Development Administration) and extracted using SV Total RNA Isolation System Kit (SV total RNA Isolation System kit) (Promega, USA). Poly(A)+mRNA was then extracted from the total RNA using the PolyATtract mRNA Isolation System kit (Promega, USA). Finally, a cDNA library was constructed using poly(A)+mRNA with Uni-ZAP XP vector and Gigapack III Gold Packing Extract kit (Stratagene, USA), and expressed sequence tags (ESTs) were analyzed. DNA was extracted using Wizard mini-preparation kit (Promega, USA), and its sequence was read using an automatic DNA sequence analyzer (Applied Biosystems, USA)....

Embodiment 2

[0032] Example 2. Venom gland-specific expression, cleavage and O-glycosylation of the serine protease (Bi-VSP) present in the venom of Bombus rufica

[0033] RNA was extracted from the fat body, midgut, muscle and venom gland of Bombus rufica using the Total RNA Isolation kit (Promega, USA). After adding 5 μg of the extracted RNA to each well, electrophoresis was performed on a 1% formaldehyde agarose gel, and the gel was transferred to a nylon blot membrane (Schleicher & Schuell, Germany), and mixed with [α- 32 P] dCTP (Amersham, USA) labeled cDNA probe hybridization of a serine protease (Bi-VSP) present in the venom of Bombus rubra. As a result, it was found that the mRNA of the serine protease (Bi-VSP) present in the venom of Bombus rufica was specifically present only in the venom gland.

[0034] In order to prepare anti-Bombus venom serine protease (Bi-VSP) antibody, the cDNA of Bombus rufica venom serine protease (Bi-VSP) was inserted into the insect alfalfa inchworm (...

Embodiment 3

[0037] Example 3. Comparison of the amino acid sequences of the serine protease in the venom of Bombus rufica and the serine protease in the snake venom

[0038] The nucleic acid sequences of the serine proteases in the venom of Bombus rubra and the serine proteases in snake venom were aligned using the BLAST program of NCBI (http: / / www.ncbi.nlm.nih.gov / BLAST). When comparing the amino acid sequences of the above two serine proteases, it was found that Bombus vulgaris serine protease (Bi-VSP) and Oscutarin C (GenBank No.AY940204) as a prothrombin activator in the blood coagulation mechanism; Batroxobin (GenBank No.AAA48553) with similar activity; TSV-PA (GenBank No.Q91516) that activates plasmin precursor; PA-BJ (GenBank No.P81824); Halytase (GenBank No.P81176) and RVV-V (GenBank No.P18964) have a certain degree of homology, and the histidine, aspartic acid and serine residues in the serine protease region are highly conserved ( Figure 8 ).

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Abstract

Disclosed is serine protease isolated from Bombus ignitus, a bumble bee, capable of activating prothrombins and degrading fibrinogens and fibrins. Since the serine protease of the present invention enables to activate the prothrombin and directly degrade fibrinogens and fibrins it can be used in the development of a therapeutic agent for the treatment of thrombosis.

Description

technical field [0001] The invention relates to a serine protease isolated from a bumble bee, Bombus rufica, which can activate prothrombin and directly degrade fibrinogen and fibrin. Background technique [0002] Bees are able to use the venom they carry inside their bodies as a powerful defense to protect their territory from intruders such as other insects and animals. Bee venom includes a variety of different melittin proteins or peptides, for example, melittin [Gauldie et al., Eur. J. Biochem., 61:369-376 (1976)], phospholipase A 2 (PLA 2 ) Six & Dennis, Biochim.Biophys.Acta 1488: 1-19 (2000)], alittin [Banks et al., Nature 282: 415-417 (1979)], hyaluronidase [Kreil, Protein Sci ., 4: 1666-1669 (1995)], serine protease [Winningham KM et al. J Allergy Clin Immunol 2004; 114: 928-33], etc. [0003] In Eastern countries, various constituents of bee venom have been studied for their use in the field of medicine [Mirshafiey A. Neuropharmacology 2007;53:353-61]. Especiall...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/64A61K38/48A61P7/02A61K35/64C12N15/09
CPCC12N9/6408A61K38/00A61P7/02
Inventor 陈炳来秋英武李光植诸连镐尹馨珠孙兴大
Owner 陈炳来
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