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Agkistrodon acutus anticoagulant factor XI polypeptide and application thereof

A technology with antithrombotic activity, AVCSQEAMTGPCRAVMPRWYFDMYKKKCIRFIYGGCGGNRNNFESEEYCMAVCKKMI, which is applied in the application field of antithrombotic drugs, can solve bleeding and other problems, and achieve the effect of prolonging the time of carotid artery thrombosis

Active Publication Date: 2021-12-10
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although traditional antithrombotic drugs such as heparin or warfarin have good antithrombotic effects in clinical application, they are often accompanied by adverse reactions such as bleeding

Method used

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  • Agkistrodon acutus anticoagulant factor XI polypeptide and application thereof
  • Agkistrodon acutus anticoagulant factor XI polypeptide and application thereof
  • Agkistrodon acutus anticoagulant factor XI polypeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Discovery of DAKS series polypeptides

[0030] The venom glands of the five-step snake were taken, and the mRNA was enriched with magnetic beads, fragmented, and the first and second strands of cDNA were synthesized with random primers, and purified using the QIAQuickPCR kit. The target fragment was recovered by agarose gel electrophoresis, amplified by PCR, and library construction was completed. Sequencing was performed using an Illumina Hiseq 4000. Trinity (version 2.4.0) was used for de novo analysis of transcriptome without parameters. Blast annotation and Pfam analysis, searched with "Kunitz" as the keyword to get 10 sequences, named DAKS 1-DAKS10. The sequence information is as follows:

[0031]

[0032]

Embodiment 2

[0033] Example 2: Inhibitory effect of DAKS series polypeptides on blood coagulation factor XI

[0034] Construction of recombinant plasmid pPIC9k / DAKS: DAKS sequence was cloned into pPIC9k using seamless cloning (cloning site NotI), linearized with SacI and electroporated into Pichia pastoris GS115, positive colonies were identified by PCR and sequencing, and positive colonies were picked Colony into the growth medium BMGY, 28.5°C, shake culture at 220r / min, when the OD600 of the bacterial solution is around 4-6, centrifuge at 4000r / min for 5min, transfer the bacteria into the expression medium BMMY, add a final concentration of 1 % methanol induction, centrifuged after 72h to collect the supernatant, purified to obtain the DAKS target protein. To test the inhibitory activity of DAKS series peptides on blood coagulation factor XIa, pipette 100 μL FXIa (1 nM) and 50 μL DAKS (1-10) and mix evenly in a 96-well plate, incubate at 37°C for 1 hour, add 50 μL of substrate (FXIa chro...

Embodiment 3

[0040] Example 3: Inhibitory effect of polypeptide DAKS1 provided by the present invention on blood coagulation factor XI

[0041] The polypeptide DAKS1 described in this example is obtained by recombinant expression in Pichia pastoris and nickel column affinity chromatography. The inhibitory effect of the recombinant polypeptide on blood coagulation factor XI was detected by a chromogenic substrate method.

[0042] Pipette 100 μL of FXIa (1 nM) and 50 μL of DAKS1 (0-500 nM) into a 96-well plate and mix evenly, incubate at 37°C for 1 hour, add 50 μL of substrate (FXIa chromogenic substrate is S2366, the final concentration is 0.25 mM, and continue at 405 nm wavelength Detect for 1 hour, scan once every minute. Negative control: 50 μL TBS-BSA buffer + 100 μL HFXIa + 50 μL substrate, blank control: 50 μL sample + 100 μL TBS-BSA buffer + 50 μL substrate (all wells are set as duplicate wells). Draw the absorbance value -Reaction time curve, the slope of the curve is the speed V o...

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Abstract

The invention belongs to the field of polypeptides in biochemistry, and relates to an agkistrodon acutus anticoagulant factor XI polypeptide and application thereof. According to the polypeptide, a Kunitz type polypeptide sequence is screened from an agkistrodon acutus snake venom transcriptome database, the Kunitz type polypeptide sequence is constructed and expressed to obtain the polypeptide with antithrombotic activity, and the polypeptide can be applied to preparation of antithrombotic drugs.

Description

technical field [0001] The invention belongs to the technical field of polypeptide drugs in biochemistry, and relates to a Kunitz-type polypeptide sequence obtained by screening from the venom gland transcriptome database of Agkistrodinacutus (Deinagkistrodonacutus), and constructing and expressing it to obtain a polypeptide with antithrombotic activity, in particular to The preparation method of the polypeptide and its application as an antithrombotic drug. Background technique [0002] With the aging of the global population and people's bad living habits, thrombotic disease has become a common disease that endangers human health, and the morbidity and mortality of cardiovascular and cerebrovascular diseases are increasing year by year. Although traditional antithrombotic drugs such as heparin or warfarin have good antithrombotic effects in clinical application, they are often accompanied by adverse reactions such as bleeding. Therefore, it is of great value to develop an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46C07K14/81A61K38/17A61P7/02
CPCC07K14/46C07K14/8121A61P7/02A61K38/00
Inventor 孔毅郑益政刘云杨李正阳蒋帅贾志萍
Owner CHINA PHARM UNIV
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