Ancylostoma caninum anticoagulant peptide and its preparation and application

A technology of anticoagulant peptide and hookworm canis, which can be applied in the fields of medicine and biology, and can solve problems such as the need for special monitoring

Inactive Publication Date: 2008-09-10
GUANGDONG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

How to prevent and treat such diseases is currently a hot spot in clinical and medical science research. Although there are many years of application history of drugs such as heparin, oral coumarin, streptokinase and urokinase, the side effects of these drugs such as bleeding and thrombocytopenia limit their application. , and some medications require special monitoring

Method used

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  • Ancylostoma caninum anticoagulant peptide and its preparation and application
  • Ancylostoma caninum anticoagulant peptide and its preparation and application
  • Ancylostoma caninum anticoagulant peptide and its preparation and application

Examples

Experimental program
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Effect test

Embodiment 1

[0059] Example 1: Cloning of hookworm anticoagulant peptide gene

[0060] 1. Primer Design

[0061] According to the nucleotide sequences of AcaNAP7 (GenBank entry number: DQ435781), AcAPc4 (GenBank entry number: AY253915), AcAPc3 (GenBank entry number: AY232998) and AcAPc2 (GenBank entry number: U30793), the downstream primer N2 was designed; The guide sequence design upstream primer NSL1, the sequence is as follows:

[0062] NSL1: 5'-GGTTTAATTACCCAAGTTTGAG-3'

[0063] N2: 5'-TTTGGTCATTTTTCTGTTAGG-3'

[0064] 2. RNA isolation and reverse transcription

[0065] Take 20 adult hookworms of hookworm (the host dogs are from Zhanjiang and Guangxi, Guangdong), and use TRIzol Reagent (Invitrogen) to extract total RNA according to the operating instructions. cDNA was obtained by reverse-transcribing the extracted total RNA of H. caninum with 3'-Full RACECore Set (Takara, Dalian) according to the operating instructions.

[0066] 3. Amplification, cloning and sequencing of anticoag...

Embodiment 2

[0080] Example 2: Sequence information and homology analysis of the anticoagulant peptide AcaNAP9 of hookworm hookworm

[0081] The full-length cDNA sequence of the isolated hookworm anticoagulant peptide AcaNAP9 is 443 bases (including 13 adenosine A at the 3' end) (SEQ ID NO.11), and its open reading frame (26-310) encodes Peptides (SEQ ID NO.1) consisting of 94 amino acids were obtained, of which the mature peptide consisted of 81 amino acid residues (SEQ ID NO.4), and there was also a signal peptide consisting of 13 amino acid residues. ( figure 1 ).

[0082] The full-length cDNA sequence of hookworm anticoagulant peptide AcaNAP9 and its encoded protein were searched for nucleic acid and protein homology in the Genbank+EMBL+DDBJ+PD database with the BLAST program, and it was found that the amino acid sequence homology with AcaNAP9 in the database was the largest Canine hookworm anticoagulant peptide AcAPc4 (GenBank entry number: AAP82926), their identity is 65% (62 / 94); ...

Embodiment 3

[0083] Example 3: Sequence information and homology analysis of the anticoagulant peptide AcaNAP10 of Ancylostoma canis

[0084] The cDNA sequence of the isolated hookworm anticoagulant peptide AcaNAP10 is 243 bases (including stop codon) (SEQ ID NO.12), and the polynucleotide sequence 1-240 encodes 80 amino acids (SEQ ID NO.2). ( figure 2 ).

[0085] Using the BLAST program to search for nucleic acid and protein homology in the Genbank+EMBL+DDBJ+PD database, it shows that the most homologous amino acid sequence of AcaNAP10 in the database is the anticoagulant peptide AcAPc4 (GenBank entry number: AAP82926). The concordance of the gene was 87%, followed by AcAPc3 (GenBank accession number: AAP57305) (76% concordance), AcaNAP7 (70% concordance) and AcAPc2 (66% concordance). The largest homology with AcaNAP10 nucleotide sequence is the anticoagulant peptide AcAPc4 gene of hookworm canis (GenBank entry number: AY253915), and their identity is 93%. The AcaNAP10 nucleotide seque...

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Abstract

The invention discloses a novel ancylostoma caninum anticoagulation peptide and an encoded sequence thereof and a preparation method for the anticoagulation peptide. The anticoagulation peptide acquired by the method possesses of anticoagulation activity, can markedly prolong the plasma prothrombin time (PT) and the activation part thrombozyme time(aPTT) of people and has obvious anti thrombosis effects. The invention also relates to applications of the anticoagulation peptide on aspects of anticoagulation drugs, antithrombotic drugs or anticoagulation preparations.

Description

technical field [0001] The invention relates to the fields of medicine and biotechnology. Specifically, the present invention relates to a new gene of anticoagulant peptide of hookworm isolated from hookworm and its encoded protein. The present invention also relates to the preparation and application of these new hookworm anticoagulant peptides. technical background [0002] Thrombotic disease is a common disease, often manifested as myocardial infarction, ischemic cerebral infarction, and venous thromboembolism. Every year, 1 to 3 people have different forms of thrombotic diseases in every thousand people. Among the current human diseases, thromboembolic diseases have become the main factors that endanger human health. The total number of deaths caused by thromboembolism, such as myocardial infarction, cerebral infarction, and pulmonary infarction, ranks first among various causes. How to prevent and treat such diseases is currently a hot spot in clinical and medical sc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/63A61K38/17A61P7/02
Inventor 彭礼飞吴亚敏邓莉胡晶晶杨陈甘伟琼
Owner GUANGDONG MEDICAL UNIV
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