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sj13 polypeptide and its application in the preparation of antithrombotic drugs

A technology for inhibiting coagulation and preparations, applied in the application of antithrombotic drugs, in the field of preparation of anticoagulant, Sj13 polypeptide and its mutants, can solve the problems of weak anticoagulant activity, no anticoagulant polypeptide report, etc., and achieve inhibition of thrombosis. Formation, the development and application value of important anticoagulant/antithrombotic drugs, and the effect of prolonging the detection time

Active Publication Date: 2021-12-21
HUBEI UNIVERSITY OF MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the research on the anticoagulant active substances in Schistosoma mansoni, which is widely prevalent in Africa, South America and Asia, is relatively clear. So far, SmAP, SmPDE, sK1, SmSP, Sm22.6, SHW4-2 and annexin have been found. Anticoagulant proteins, and anticoagulant small molecules such as eicosanoids, but no reports of anticoagulant polypeptides
In 2015, SL Ranasinghe et al. reported the first schistosome anticoagulant polypeptide SjKI-1, which comes from Schistosoma japonicum. SjKI-1 at 7.5 μM can prolong the partial thromboplastin time (APTT) by 2 times and has weak anticoagulant activity , there is no report on the follow-up study of this polypeptide

Method used

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  • sj13 polypeptide and its application in the preparation of antithrombotic drugs
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  • sj13 polypeptide and its application in the preparation of antithrombotic drugs

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Experimental program
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Effect test

Embodiment 1

[0049] Embodiment 1: Construction of Schistosoma japonicum polypeptide vector

[0050] 1. Sj13PCR primer design

[0051] (1) Through the combination of structural biology and bioinformatics, a new Schistosoma japonicum gene / protein was determined from the Schistosoma japonicum gene / protein library, named Sj13, and its polypeptide amino acid sequence is as follows:

[0052] ETLKRYCNLPSDEGICRGYFRRYFYNVTSGECEVFYYGGCLGNRNRFSTIEKCWWYCKGL (SEQ ID NO. 1)

[0053] The protein sequence contains 6 cysteines, which can form 3 pairs of disulfide bonds, which are CysⅠ-CysVI, CysII-CysIV and CysIII-CysⅤ. This pairing mode is a typical feature of protein structure.

[0054] (2) Obtain the cDNA sequence of Sj13 through the sequence reverse translation website, as follows:

[0055] gaaaccctgaaacgctattgcaacctgccgagcgatgaaggcatttgccgcggctattttcgccgctatttttataacgtgaccagcggcgaatgcgaagtgttttattatggcggctgcctgggcaaccgcaaccgctttagcaccatgaaaaatgctggtggtattgcaaaggcctg (SEQ ID NO. 2)

[0056] (3) Use ...

Embodiment 2

[0079] Embodiment 2: Expression and purification of Schistosoma japonicum polypeptide

[0080] The constructed pET-28a plasmid was transformed into E.coli Transetta (DE3) expression strains for strain expansion culture to obtain protein inclusion bodies. After the inclusion body was washed, it was denatured, diluted and refolded, concentrated by ultrafiltration and high-performance liquid chromatography (HPLC) to obtain a purified protein solution of the recombinant Schistosoma japonicum protein Sj13. The separation results of high performance liquid chromatography are as follows: figure 2 shown. High performance liquid chromatograph, obvious Sj13 single peak appears, and the protein liquid flowing out under its peak time is all collected, and the purified protein liquid collected is frozen into dry powder with a lyophilizer.

Embodiment 3

[0081] Embodiment 3: BCA quantification of Sj13 protein purification solution

[0082] The BCA protein concentration determination kit (Shanghai Biyuntian Biotechnology Co., Ltd.) was used to quantify the BCA of the purified Sj13 protein solution, and the detected protein concentration was calculated. 10 μg of protein powder was taken out and sent to the Institute of Chemistry, Chinese Academy of Sciences for mass spectrometry detection to further identify whether the protein was recombined successfully. For mass spectrometry results, see image 3 . The mass spectrometry test results provided by the Institute of Chemistry, Chinese Academy of Sciences showed that the molecular weight of the recombinant Sj13 was 9375.3Da; the theoretical molecular weight of Sj13 predicted by the ExPASy-ProtParam tool (http: / / web.expasy.org / protparam / ) website was 9380.52Da. The actual detected value of Sj13 mass spectrometry is consistent with the theoretical value.

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Abstract

The invention discloses a Sj13 polypeptide derived from Schistosoma japonicum and its mutants. The recombinant polypeptide Sj13 and its mutants are obtained by using genetic engineering technology. Through the detection of APTT, PT and TT coagulation functions, it is identified that Sj13 and its mutants have Good in vitro anticoagulant function; detected by enzyme kinetics experiments, it was found that Sj13 polypeptide and its mutants have the function of inhibiting blood coagulation-related factors XIa, Xa and Plasmin; tested by mouse FeCl 3 Injury to the common carotid artery thrombosis model, it was found that Sj13 inhibits common carotid artery thrombosis, has antithrombotic function, and has a lower risk of bleeding. Sj13 polypeptide and its mutants have important anticoagulant / antithrombotic drug development and application value.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and biomedicine, and specifically relates to Sj13 polypeptide derived from Schistosoma japonicum and its mutant, and its application in the preparation of anticoagulant and antithrombotic drugs. Background technique [0002] Thrombosis can cause ischemia, hypoxia, necrosis (arterial thrombosis) and congestion, edema (venous thrombosis) of corresponding tissues and organs, which seriously threaten people's life and health. Arterial thrombosis is the main cause of more than 90% of myocardial infarction and 80% of cerebral apoplexy, and arterial thromboembolic cardiovascular and cerebrovascular diseases are the leading cause of human death. Venous thromboembolism (VTE), including deep vein thrombosis (DVT) and pulmonary embolism (PE), is the third leading cause of death from cardiovascular-related diseases after myocardial infarction and stroke. There are millions of VTE patients diagnosed every ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/11A61K38/17A61P7/02
CPCA61P7/02C07K14/43559A61K38/00Y02A50/30
Inventor 陈宗运丁莉罗旭东阮绪芝胡扬根
Owner HUBEI UNIVERSITY OF MEDICINE
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