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Antithrombotic polypeptide cystatin-T and preparation method and application thereof

An anti-thrombotic, cardiovascular and cerebrovascular technology, applied in the field of biomedicine, can solve the problems of systemic side effects such as hemorrhagic activity, low blood pressure, etc., and achieve the effect of significant anti-thrombotic effect, small molecular weight, and broad application prospects.

Active Publication Date: 2020-03-03
CANCER CENT OF GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many antithrombotic drugs are limited due to systemic side effects such as hemorrhagic activity and hypotension
[0003] At present, there are many kinds of drugs for cardiovascular and cerebrovascular diseases, but biomolecules from food sources, especially peptide drugs obtained through biotechnology, have high purity, good curative effect, less toxic side effects and dependence are still research hotspots

Method used

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  • Antithrombotic polypeptide cystatin-T and preparation method and application thereof
  • Antithrombotic polypeptide cystatin-T and preparation method and application thereof
  • Antithrombotic polypeptide cystatin-T and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1. Cloning of cystatin-T gene.

[0024] Extraction of tea tree total mRNA: mRNA was extracted from tea tree buds, and high-quality mRNA was extracted by simple CTAB-LiCl method and modified.

[0025] The first strand of cDNA was synthesized using the BioTeKe supermoⅢ RT Kit from Beijing BioTeKe Biotechnology Co., Ltd.

[0026] In an RNAase-free PCR tube, add the following reagents on ice:

[0027]

[0028] Perform reverse transcription on the PCR machine according to the following conditions:

[0029] 50℃, 45min; 70℃, 10min;

[0030] Construction of the target fragment cloned into the vector: the double restriction sites of KPn1 and BamH1 were introduced, and protective bases were added at both ends. Before expressing the mature polypeptide, a chemical cleavage site: a formic acid cleavage site GACCCG (encoded amino acid is DP) was added to ensure that the tagged protein was chemically excised after expression from the vector. Insert the target sequence o...

Embodiment 2

[0036] Example 2 Preparation of antithrombotic polypeptide cystatin-T.

[0037] The prokaryotic expression method of the antithrombotic polypeptide cystatin-T: According to the coding gene sequence, add different restriction endonuclease sequences at both ends of the gene, connect with the expression vector PET-32a by double enzyme digestion, and then transfer into the expression host cell Expressed in BL21DE3 Escherichia coli. The supernatant was obtained by ultrasonic crushing and centrifugation, its purity was identified by Ni affinity chromatography, FPLC, and other purification means, the isoelectric point was determined by isoelectric focusing electrophoresis, and the amino acid sequence structure was determined by an automatic amino acid sequencer.

Embodiment 3

[0038] Example 3: Detection of anticoagulant activity of antithrombotic polypeptide cystatin-T in whole blood.

[0039] Platelets are the key factor of the anticoagulant system. In this method, the platelet aggregation experiment was carried out by using the Beijing Prism Platelet Aggregator, and the partial thromboplastin time APTT and plasma recalcification time of the whole blood of rats in each group were detected by the automatic coagulation instrument method. Clotting time of PRT.

[0040] The specific operation is as follows: get 40 wistar male rats (200g-250g), divide them into 4 groups at random, 10 in each group, respectively normal saline (control group), administration group (1mg / kg, 5mg / kg, 10mg / kg). The rats were fasted for 12 hours before the administration, and the rats were normally fasted for 1 hour after the tail vein administration. After 4 hours of the administration, the rats were anesthetized with pentobarbital sodium 60 mg / kg, and blood was collected ...

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Abstract

The invention discloses an antithrombotic polypeptide cystatin-T. The antithrombotic polypeptide cystatin-T is composed of 101 amino acid residues, has a molecular weight of 11,063.48 Da and an isoelectric point of 6.72, and has an amino acid sequence as shown in SEQ ID NO. 1 and a nucleic acid sequence as shown in SEQ ID NO. 2. The invention also discloses a preparation method and application ofthe above antithrombotic polypeptide cystatin-T. The antithrombotic polypeptide cystatin-T of the invention has the advantages of good stability, simple artificial expression, remarkable antithrombotic and cardio-cerebral vascular protection, and low hemolytic activity, and has a wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to an antithrombotic polypeptide cystatin-T and its preparation method and application. Background technique [0002] Thrombosis is the main cause of cardiovascular disease morbidity and death, and it is positively correlated with age. my country is entering an aging country, and cardiovascular disease has become a heavy social burden. For a long time, a large number of antithrombotic drugs have been developed for the physiological mechanisms of blood coagulation, platelet activation and aggregation, and thrombus dissolution. However, many antithrombotic drugs are limited due to systemic side effects such as hemorrhagic activity and hypotension. [0003] At present, there are many kinds of drugs for cardiovascular and cerebrovascular diseases, but biomolecules from food sources, especially peptide drugs obtained through biotechnology, have high purity, good curativ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C07K1/16C07K1/22C12N15/29A61K38/16A61P7/02A61P9/00A61P9/10
CPCC07K14/415A61P7/02A61P9/00A61P9/10A61K38/00
Inventor 严秀文杨文豪方鸣谦曹能琦
Owner CANCER CENT OF GUANGZHOU MEDICAL UNIV
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