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Sj12 polypeptide and application thereof in preparation of anticoagulation medicine

A technology of sj12 and polypeptide, which is applied in the field of preparation of anticoagulant drugs, can solve problems such as unsatisfactory anticoagulant effects, and achieve the effect of inhibiting blood coagulation, important anticoagulation, and excellent activity

Active Publication Date: 2018-07-24
HUBEI UNIVERSITY OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In 2015, Australian scholar SL Ranasinghe et al. discovered the first anticoagulant polypeptide SjKI-1 in the cDNA of Schistosoma japonicum, but its anticoagulant effect was not satisfactory

Method used

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  • Sj12 polypeptide and application thereof in preparation of anticoagulation medicine
  • Sj12 polypeptide and application thereof in preparation of anticoagulation medicine
  • Sj12 polypeptide and application thereof in preparation of anticoagulation medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: Preparation of expression vector pET-28a (+) plasmid

[0027] 1. Expansion of strains

[0028] Add 1 μl of the Trans 5α strain containing the pET-28a(+) plasmid (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) stored in our laboratory into 10ml of LB medium (containing 30μg / ml kanamycin) and mix Mix evenly, place on a constant temperature shaker, 37°C, <180rpm, cultivate for 11-12 hours, OD600=0.8.

[0029] 2. Extraction of expression vector pET-28a(+) plasmid

[0030] (1) Take 1-2ml of the overnight cultured bacterial solution and add it to a 1.5ml Eppendorf centrifuge tube, centrifuge at 8000rpm for 5 minutes, discard the supernatant as much as possible, and collect the bacterial precipitate.

[0031] (2) Add 250 μl Buffer P1 (check to see if RNaseA has been detected) into the centrifuge tube with the bacterial pellet, and mix well.

[0032] (3) Add 250 μl Buffer P2 to the centrifuge tube, gently invert up and down for 6-8 times, and mix th...

Embodiment 2

[0037] Embodiment 2: Construction of Schistosoma japonicum polypeptide vector

[0038] 1. Sj12PCR primer design

[0039] (1) Through the combination of structural biology and bioinformatics, a new Schistosoma japonicum gene was identified from the Schistosoma japonicum gene bank, named Sj12, and the amino acid sequence of the encoded polypeptide was as follows:

[0040] ARLRTSDDCLRPMKKGYGLRQRTRYYYDLNMNSCLEFTYKGRGGSNRRFHSREDCEKVCLPEAINNNTN (SEQ ID NO. 1)

[0041] (2) Obtain the cDNA sequence of Sj12 through the sequence reverse translation website, as follows:

[0042] GCGCGCCTGCGCACCAGCGATGATTGCCTGCGCCCGATGAAAAAAGGCTATGGCCTGCGCCAGCGCACCCGCTATTATTATGATCTGAACATGAACAGCTGCCTGGAATTTACCTATAAAGGCCGCGGCGGCAGCCGCAACCGCTTTCATAGCCGCGAAGATTGCGAAAAAGTGTGCCTGCCGGAAGCGATTAACAACAACACCAAC (SEQ ID NO. 2)

[0043] (3) Use primer design software to design primers, as follows:

[0044] Sj12-FP1: AGCGCACCCGCTATTATTATGATCTGAACATGAACAGCTGCCTGGAATTT (SEQ ID NO. 3)

[0045] Sj12-RP1: AAGCGGTTGCGGCTGC...

Embodiment 3

[0082] Embodiment 3: Expression and purification of Schistosoma japonicum polypeptide

[0083] 1. Preparation of recombinant plasmid pET-28a-Sj12

[0084] (1) Inoculate the positive clones with successful sequencing into 10ml of liquid LB medium (containing 30μg / ml kanamycin), culture at 37°C, 600 = 0.8.

[0085] (2) Use a high-purity plasmid extraction kit (Beijing Kangwei Century Biotechnology Co., Ltd.) to extract the plasmid. For specific steps, refer to 1.2.1.2 Preparation of expression vector pET-28a(+) plasmid. Store the extracted plasmids in a -20°C refrigerator.

[0086] 2. Induced expression of Sj12 protein

[0087] (1) Slowly add 2 μl of the extracted recombinant plasmid pET-28a-Sj12 with correct sequencing to 100 μl of E.coli Transetta (DE3) competent cells that have just been melted for transformation.

[0088] (2) Pick a single positive colony, place it in an Eppendorf centrifuge tube filled with 0.5ml LB liquid medium (containing 30μg / ml kanamycin) and mark ...

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Abstract

The invention discloses an Sj12 polypeptide from schistosoma japonicum katsurada. The polypeptide has no influence on an extrinsic coagulation system and a coagulation common path, but can interfere an endogenic coagulation system, so that the coagulation time is prolonged to a value being 10 seconds or more longer than the normal detection range; the effect of inhibiting the blood coagulation isachieved; the coagulation activity is superior to that of anticoagulant peptide in the prior art; the important anticoagulant and antithrombotic medicine development and application values are realized.

Description

technical field [0001] The invention belongs to the field of biomedicine and medical treatment, and specifically relates to Sj12 polypeptide derived from Schistosoma japonicum and its application in the preparation of anticoagulant drugs. Background technique [0002] For blood to circulate and flow normally in the human body, in addition to the normal work of the heart, it is also necessary for the place where the blood flows to remain unblocked at all times. In order to maintain the unimpeded operation of blood in the vascular system, the vascular endothelium must be intact; the body's coagulation, anticoagulation and fibrinolysis systems must be in a balanced state, which play an important role in the body's normal physiological functions. When the coagulation, anticoagulation or fibrinolytic system of the body is dysfunctional, the balance between coagulation and anticoagulation is broken, followed by bleeding or thrombosis. A small amount of long-term hemorrhage may ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N5/10C12N15/11A61K38/17A61P7/02
CPCA61K38/00C07K14/43559Y02A50/30
Inventor 陈宗运丁莉罗旭东阮绪芝胡扬根
Owner HUBEI UNIVERSITY OF MEDICINE
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