Tumor-specific recombinant hirudin and preparation method and application thereof

A recombinant hirudin, tumor-specific technology, applied in the direction of leech inhibitors, antineoplastic drugs, chemical instruments and methods, etc., can solve the problems of time increase, bleeding risk, etc., and achieve the effect of avoiding allergic reactions

Inactive Publication Date: 2017-09-29
新钥(南京)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, hirudin can lead to a significant increase in coagulation-related parameters, such as activated partial throm

Method used

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Examples

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Effect test

preparation example Construction

[0044] Correspondingly, the present invention also provides a method for preparing the above-mentioned tumor-specific recombinant hirudin, comprising the following steps: chemically synthesizing the above-mentioned tumor-specific recombinant hirudin gene fragment, linking a signal peptide at its N-terminus, and adding Restriction sites are added at both ends, the recombinant coding gene is inserted into the gene vector to prepare a recombinant vector, the host cell is transformed or transfected with the recombinant vector, the host cell is cultivated, and the tumor-specific recombinant hirudin is recovered and purified therefrom, The host cells are Escherichia coli, yeast, insect cells or mammalian cells.

[0045] The vector used for recombination is preferably a eukaryotic expression vector, preferably pPIC9K. The host cell is preferably methanolotrophic yeast strain GS115.

[0046] Correspondingly, the present invention also provides the application of the above-mentioned t...

Embodiment 1

[0057] The recombinant hirudin in this example is modified with the oligopeptide PGR at the N-terminal of the hirudin isomer HV1, and together with the N-terminal sequence VV (valine-valine) of the hirudin isomer HV1, it can be synthesized by uPA The recognized and cleaved physiological substrate peptide sequence PGRVV, the recombinant hirudin amino acid sequence is: SEQ ID NO.1, and the gene sequence is: SEQ ID NO.2.

[0058] Among them, PGRVV can be replaced by other uPA enzyme digestion substrate peptide sequences, and its common feature is that it contains RV sequence, such as: GSGRVV, GGSGRVV, LGGSGRVV. To ensure that after uPA digestion, the two amino acids at the amino terminal of the released hirudin are VV. The hirudin isoform HV1 can be replaced by other hirudin isoforms with VV at the N-terminus.

[0059] The application of the tumor-specific recombinant hirudin in the preparation of anticoagulant, antithrombotic and antitumor drugs, especially the application of t...

Embodiment 2

[0061] The recombinant hirudin of this embodiment is to replace the Ser32-Asn33-Gly34-Glu35 of the wild-type hirudin HV1 peptide chain with Arg32-Gly33-Asp34-Ser35, and then modify the PGR oligopeptide at its N-terminus. The amino acid sequence of the recombinant hirudin is : SEQ ID NO.3, the gene sequence is: SEQ ID NO.4.

[0062] The application of the tumor-specific recombinant hirudin in the preparation of anticoagulant, antithrombotic and antitumor drugs, especially the application of the drug in the prevention and treatment of tumor accompanied by hypercoagulable state and thrombotic lesions.

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Abstract

The invention discloses a tumor-specific recombinant hirudin. A tail end N of the recombinant hirudin contains an oligopeptide sequence Pro-Gly-Arg-Val-Val that can be identified and split by uPA, wherein the uPA can be highly expressed by a tumor tissue; and the hirudin for recombination is hirudin isomer, hirudin mutant, hirudin chimera, truncated hirudin peptide, and gene modified hirudin or hirudin fusion protein. Correspondingly, the invention further provides a preparation method of the tumor-specific recombinant hirudin and application thereof in preparation of anti-tumor medicines, and provides a preparation method and application of adoptive immune cells expressing the tumor-specific recombinant hirudin. The hirudin anticoagulant activity of the recombinant hirudin is featured by tumor targeted release, and the recombinant hirudin creates a micro-environment where thrombus is difficult to form, and even dissolves formed small thrombus so as to realize functions of anticoagulation, thrombolysis and prevention of metastasis of the tumor cell, so that the risk of systemic bleeding caused by application of wild hirudin to the whole body is prevented.

Description

technical field [0001] The invention relates to the fields of biotherapy and biomedicine, in particular to a tumor-specific recombinant hirudin and its preparation method and application. Background technique [0002] Hirudin is the most active natural thrombin-specific inhibitor found so far. It was originally isolated from the salivary gland of medical leeches. It consists of 65-66 amino acids and can directly bind thrombin in a 1:1 (molar ratio) manner. Combined to form a non-covalent complex, so that thrombin loses the ability to crack fibrinogen and inhibits the formation of thrombus. There are more than ten kinds of variants of natural hirudin, mainly three kinds of highly homologous isomers (Hirudin Variant) referred to as HV1, HV2 and HV3. [0003] At present, two recombinant hirudin products have been approved for marketing abroad: 1. Desirudin (trade name: Revasc, product of Novartis, Switzerland), 2. Lepirudin (trade name: Refludan, product of British Pharmion an...

Claims

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Application Information

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IPC IPC(8): C07K14/815C12N5/10A61K38/58A61P7/02A61P35/00A23L33/17
CPCC07K14/815A23L33/17A23V2002/00A61K38/00A23V2200/308A23V2200/326
Inventor 何向锋施文
Owner 新钥(南京)生物科技有限公司
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