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Preparation for specificity anticoagulant substance and application thereof

An anticoagulant and thrombin technology, applied to medical preparations containing active ingredients, using carriers to introduce foreign genetic material, specific peptides, etc., can solve problems such as no anticoagulant activity

Inactive Publication Date: 2012-05-09
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Of course, the above-mentioned substances have no anticoagulant activity on the part of the body where there is no thrombus

Method used

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  • Preparation for specificity anticoagulant substance and application thereof
  • Preparation for specificity anticoagulant substance and application thereof
  • Preparation for specificity anticoagulant substance and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Preparation of IEGR-HV2(FH) and LGPR-HV2(TH) and their function of preventing and treating thrombosis

[0022] 1. Acquisition of FH and TH proteins

[0023] By PCR, the restriction site Xho I and the base sequence corresponding to the coagulation factor Xa recognition sequence IEGR or the thrombin recognition sequence LGPR are introduced into the upstream of the HV2 gene, and the EcoR I restriction site is introduced downstream of the HV2 gene, and the gene is Ligated to pPIC9 plasmids digested with the same restriction enzymes to obtain recombinant plasmids pPIC9-FH and pPIC9-TH. pPIC9-FH, pPIC9-TH, and pPIC9K were digested with BamH I and Sal I and ligated to obtain pPIC9K-FH and pPIC9K-TH. The two recombinant plasmids were electroporated and recombined into the yeast genome, and the expression was induced by methanol. The proteins FH and TH were obtained by separation and purification.

[0024] 2. Analysis of antithrombotic effect and bleeding side effec...

Embodiment 2

[0059] Example 2 Preparation of HSA-EFLGPR-HV2 fusion protein (ATH) and HSA-EFIEGR-HV2 fusion protein (AFH) and their antithrombotic and anticoagulant activities

[0060] Add Xho I and EcoR I restriction sites to the upstream and downstream of the human albumin HSA gene, respectively, to construct the HSA gene without a stop codon into the pPIC9 vector, and use BamH I and EcoR I to double-digest the above vector and the pPIC9K vector. HSA is connected to the pPIC9K carrier; the encoding base of EcoR I restriction site and thrombin / coagulation factor Xa recognition sequence is added at the upstream of hirudin gene by PCR method, and the Not I restriction site is introduced at the downstream of hirudin; EcoR I and Not I double-digest the hirudin gene with thrombin / coagulation factor Xa recognition sequence and connect it to the downstream of HSA of the pPIC9K vector with HSA gene constructed above to form the fusion gene ATH / AFH to obtain the plasmid pPIC9K-ATH / AFH. Linearize ...

Embodiment 3

[0069] Example 3 Preparation of P-IEGR-HV2 fusion protein (PFH) and its antithrombotic and anticoagulant activities

[0070] Use the PCR method to add the coding base of the EcoR I restriction site and the polypeptide (EFDAEAYVIEGR) containing coagulation factor Xa recognition sequence in the upstream of the hirudin gene, introduce the Not I restriction site in the downstream of the hirudin, and connect EcoR I and Not. I double digested the pPIC9K plasmid to obtain the pPIC9K-FH recombinant plasmid. The pPIC9K-FH plasmid was linearized, electroporated and recombined into the yeast genome, and methanol-induced expression. The fusion target protein contains three functional regions of oligopeptide P (the amino acid sequence is DAEAYV), blood coagulation factor Xa recognition sequence and hirudin amino acid sequence.

[0071] In vivo antithrombotic activity of the fusion protein (PFH): The in vivo antithrombotic activity of the fusion protein (PFH) was determined using the Carra...

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Abstract

The invention relates to an anticoagulant protein containing an oligopeptide which is identified and cracked by thrombin or blood coagulation factor Xa; more particularly, the invention relates to a new anticoagulant substance connected by an anticoagulant substance and an amino acid sequence which can be identified and cracked by thrombin or blood coagulation factor Xa, or a new substance connected by an anticoagulant substance and other substances through taking an amino acid sequence which can be identified and cracked by thrombin or blood coagulation factor Xa as oligopeptide, as well as medicine science applications of these new anticoagulant substances.

Description

[0001] The present invention is a divisional application of a patent with the same name whose application number is 200610092323.3 and whose application date is June 1, 2006. technical field [0002] The invention belongs to the field of biotechnology, and relates to a protein structure of an anticoagulant substance, a preparation method thereof and an application in preventing and treating thrombosis-related diseases. Specifically, the present invention relates to a new substance formed by linking an anticoagulant substance with an amino acid sequence that can be recognized and cleaved by thrombin or coagulation factor Xa, or an anticoagulant substance and other substances that can be recognized and cleaved by thrombin or coagulation factor Xa New substances formed by linking identified and cleaved amino acid sequences, and the medical use of these new substances. Background technique [0003] Cardiovascular disease is the number one killer that threatens human health and l...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N15/81C12N15/85A61K38/58A61P7/02
Inventor 吴祖泽于爱平靳继德牛晋阳苑宾石炳兴董春娜唐仲雄
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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