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Whitmania pigra anticoagulant factor XIa polypeptide and application thereof

An antithrombotic drug and amino acid technology, applied in the field of peptides, can solve problems such as undiscovered, and achieve the effect of strong activity in vivo

Active Publication Date: 2022-01-21
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no FXI / FXIa-inhibiting peptides derived from the broad-bodied leeches have been found

Method used

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  • Whitmania pigra anticoagulant factor XIa polypeptide and application thereof
  • Whitmania pigra anticoagulant factor XIa polypeptide and application thereof
  • Whitmania pigra anticoagulant factor XIa polypeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Discovery of WPK series polypeptides

[0033] Take the wide-bodied leech salivary gland, use magnetic beads to enrich mRNA, fragment it, synthesize the first strand and the second strand of cDNA with random primers, use QIAQuickPCR kit to purify, recover the target fragment by agarose gel electrophoresis, and amplify by PCR. Complete library construction. Illumina Hiseq 4000 was used for sequencing, and Trinity (version 2.4.0) was used for de novo analysis of transcriptome without reference transcriptome, Blast annotation and Pfam analysis, and 5 sequences were searched with "Kunitz" as the keyword, named WPK1-5. The sequence information is as follows:

[0034]

Embodiment 2

[0035] Example 2: Inhibitory effect of WPK series polypeptides on blood coagulation factor XIa

[0036] Construction of recombinant plasmid pPIC9k / WPK: Cloning the WPK1-5 sequence into pPIC9k using seamless cloning (cloning site NotI), linearized with SacI and electroporated into Pichia pastoris GS115, positive transformant colonies were identified by PCR and sequencing , Pick positive transformant colonies into the growth medium BMGY, 28.5 ° C, 220r / min shaking culture, when the OD600 of the bacterial solution is around 4-6, centrifuge at 4000r / min for 5min, and transfer the bacteria into the expression medium BMMY , add a final concentration of 1% methanol to induce, centrifuge to collect the supernatant after 72h, and obtain the WPK1-5 target protein through purification ( figure 1 ). To test the inhibitory activity of WPK series peptides on blood coagulation factor XIa, pipette 100 μL FXIa (1nM) and 50 μL WPK1-5 (10 μM) and mix evenly in a 96-well plate, incubate at 37°C ...

Embodiment 3

[0039] Example 3: The polypeptide mutant WPK5-mut sequence provided by the present invention and its comparison with the PN2KPI sequence

[0040] PN2KPI through Loop1( 11 TGPCRAMISR 20 ) and Loop2 ( 34FYGGC 38 ) extensively interacts with the catalytic domain of XIa and exhibits good inhibitory activity against coagulation factor XIa. In this embodiment, in order to further improve the inhibitory activity of WPK5 on blood coagulation factor XIa, the method of loop replacement is adopted, based on the new Kunitz skeleton provided by WPK5, two loops of PN2KPI ( 11 TGPCRAMISR 20 , 34 FYGGC 38 ) for WPK-5 two loops ( 11 TGPCRSNLER 20 , 34 wxya 38 ) to replace. And the amino acid sequence analysis of WPK5-mut and PN2KPI showed that the similarity was 60.78%.

[0041]

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Abstract

The invention belongs to the field of polypeptides, and relates to an application of a whitmania pigra anticoagulant factor XIa polypeptide and a mutant thereof. A Kunitz type polypeptide sequence is screened from a salivary gland transcriptome database of whitmania Pigra, construction expression, screening and amino acid replacement are performed on the Kunitz type polypeptide sequence to obtain a series of polypeptides, and the polypeptides serve as prodrugs or are used for preventing and treating brain injury (such as paralysis and stroke) and other diseases. The polypeptide has the characteristics of safety, effectiveness and the like and is suitable for research and development of antithrombotic drugs.

Description

technical field [0001] The invention belongs to the field of polypeptides, and relates to the application of the broad-bodied leech anticoagulant factor XIa polypeptide and its mutants. Background technique [0002] Thrombotic disease is a common disease that endangers human health and is the leading cause of death worldwide. Although traditional antithrombotic drugs such as heparin or warfarin have good antithrombotic effects in clinical applications, they interfere with the delicate balance of procoagulation and anticoagulation in the body, often accompanied by complications such as bleeding. Therefore, it is of great value to develop antithrombotic drugs with low bleeding risk. [0003] Clinical data show that patients with congenital deficiency of coagulation factor XI (factor XI, FXI) have significantly lower rates of ischemic stroke and deep vein thrombosis, and generally have no spontaneous bleeding. Studies have found that FXI can enhance the generation of thrombin...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K14/815A61K38/17A61P7/02A61P9/10
CPCC07K14/435C07K14/815C07K14/8121A61P7/02A61P9/10A61K38/00
Inventor 孔毅李正阳冀晓茹郑益政贾志萍余香颖
Owner CHINA PHARM UNIV
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