A kind of forest leech antithrombotic polypeptide sylvestin and its in vitro expression preparation method and application

An anti-thrombotic and leech technology, applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve problems such as instability, low activity, and few types of polypeptides, and achieve high stability, uniform conformation, The effect of high enterokinase cleavage efficiency

Active Publication Date: 2020-05-15
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the currently studied peptides have the problems of few types, low activity and instability.

Method used

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  • A kind of forest leech antithrombotic polypeptide sylvestin and its in vitro expression preparation method and application
  • A kind of forest leech antithrombotic polypeptide sylvestin and its in vitro expression preparation method and application
  • A kind of forest leech antithrombotic polypeptide sylvestin and its in vitro expression preparation method and application

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preparation example Construction

[0044] The present invention also provides a preparation method for the in vitro expression of the polypeptide Sylvestin, comprising the following steps: 1) constructing the recombinant expression strain according to claim 6; 2) inducing the expression of the recombinant strain to obtain a bacterial solution containing the recombinant polypeptide Sylvestin; The induction temperature is 16-30°C, the induced IPTG concentration is 0.05-0.55mmol / L, and the induction time is 1-18h; 3) Separating and purifying the recombinant polypeptide Sylvestin from the bacterial liquid; 4 ) cutting the recombinant polypeptide Sylvestin with enterokinase to obtain the polypeptide Sylvestin.

[0045] In the present invention, the method for constructing the recombinant expression strain comprises the following steps: a, linking the enterokinase cleavage site with the nucleotide sequence encoding the polypeptide Sylvestin to obtain the nucleotide sequence encoding the recombinant polypeptide Sylvest...

Embodiment 1

[0068] Obtaining the Antithrombotic Polypeptide Sylvestin from Natural Forest Leech

[0069] Polypeptide Sylvestin was isolated and purified from the whole homogenate of Leech leech by molecular sieve gel filtration and reversed-phase high-pressure liquid chromatography. The N-terminal 12 amino acid sequences of the polypeptide Sylvestin were determined by the Edman degradation method, and the homologous sequence encoding the polypeptide was screened from the Yunnan forest leech transcriptome, and the homologous nucleotide sequence was used as a template to design a specific Sexual primers, the forward primer is AAACCGAACCGTCGGTATGT, and the reverse primer sequence is GTTCGGTTTGGTGGCTCATT. Then the nucleotide sequence encoding polypeptide Sylvestin was amplified from the constructed leech head cDNA library.

[0070] The mature peptide encoded by the nucleotide sequence is composed of 43 amino acids, contains six cysteines, and has a molecular weight of 4795.4 Da, which is 6 D...

Embodiment 2

[0072] 一种森林山蛭抗血栓重组多肽Sylvestin,包括多肽Sylvestin和肠激酶酶切位点片段,序列如Seq IDNo.3所示,具体为MIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSTSEPVCACPKMLFWVCGKDGETYTHPCIAKCHNVEVEHDGKCK*。 The recombinant polypeptide Sylvestin described in the present invention is connected with an enterokinase enzyme cleavage site on the sequence of the polypeptide Sylvestin, which facilitates the connection of the polypeptide Sylvestin to the carrier and the separation and purification of the polypeptide Sylvestin during the preparation process of in vitro expression.

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Abstract

The invention provides haemadipsa sylvestris antithrombosis polypeptide Sylvestin as well as an in-vitro expression preparation method and application thereof and belongs to the technical field of polypeptide synthesis expression. The amino acid sequence of the polypeptide Sylvestin is shown in Seq ID No.1. The invention further provides an improved peptide of the polypeptide Sylvestin. The aminoacid sequence of the improved peptide is shown in Seq ID No.5, Seq ID No.6, Seq ID No.7 or Seq ID No.8. The polypeptide Sylvestin and the improved peptide thereof have FXIIa inhibition activity and effects of antithrombosis and stroke treatment. By adopting the in-vitro expression preparation method provided by the invention, on-scale low-cost production of the Sylvestin polypeptide in a prokaryote biological body is achieved, and the Sylvestin polypeptide can be applied to preparation of biological functional medicines through purification.

Description

technical field [0001] The invention belongs to the technical field of polypeptide synthesis and expression, and in particular relates to a leech antithrombotic polypeptide Sylvestin and its in vitro expression preparation method and application. Background technique [0002] Thrombosis is the main cause of cardiovascular disease morbidity and death, and the incidence 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. Therefore, mining new and specific antithrombotic drugs has always been a research hotspot. [0003] In recent years, rat experimental models and clinical studies...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/70C12N1/21A61K38/17A61P7/02A61P9/10C12R1/19
CPCA61K38/00C07K14/43504C12N15/70
Inventor 赖仞方鸣谦韩亚君
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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