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sj12 polypeptide and its application in the preparation of anticoagulant drugs

An anti-coagulation and anti-thrombotic technology, applied in applications, drug combinations, and resistance to vector-borne diseases, etc., can solve problems such as unsatisfactory anti-coagulation effects, achieve the development and application value of anti-thrombosis, inhibit blood coagulation, and be important. anticoagulant effect

Active Publication Date: 2021-12-07
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 its application in the preparation of anticoagulant drugs
  • sj12 polypeptide and its application in the preparation of anticoagulant drugs
  • sj12 polypeptide and its application in the preparation of anticoagulant drugs

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 a Sj12 polypeptide derived from Schistosoma japonicum. The polypeptide has no influence on the exogenous blood coagulation system and the common pathway of blood coagulation, but can interfere with the endogenous blood coagulation system, prolonging the blood coagulation time beyond the normal detection range by more than 10 seconds , plays the role of inhibiting blood coagulation, its coagulation activity is better than the anticoagulant polypeptide in the prior art, and has important development and application value of anticoagulant and antithrombotic drugs.

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 Patents(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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