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Thrombolysis-targeting fusion protein mAnxB1ScuPA, structure and application thereof

A fusion protein, targeting technology, applied in the field of medicine and biology, can solve the problems of low thrombolysis efficiency, serious side effects such as bleeding, and re-embolization.

Inactive Publication Date: 2012-10-17
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The purpose of the present invention is to aim at the defects of current thrombolytic drugs such as low thrombolytic efficiency, serious side effects such as bleeding, and re-embolization, and utilize mAnxB1's affinity for activated platelets to construct a new type of anticoagulant-guided thrombolytic therapy with dual functions of anticoagulation and thrombolysis. Suppository protein and construction of expression vector to achieve the purpose of targeted thrombolysis

Method used

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  • Thrombolysis-targeting fusion protein mAnxB1ScuPA, structure and application thereof
  • Thrombolysis-targeting fusion protein mAnxB1ScuPA, structure and application thereof
  • Thrombolysis-targeting fusion protein mAnxB1ScuPA, structure and application thereof

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Experimental program
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Embodiment 1

[0028] Embodiment 1: Construction of gene mutant mAnxB1

[0029] The mAnxB1 gene is based on the Annexin B1 gene (GenBank No.AF147955.1) and obtained by site-directed mutagenesis and PCR. The "S-G-D" sequence in the third domain of the Annexin B1 gene is mutated into an "R-G-D" sequence, and the serine (S) is mutated into arginine (R); the "S-G-D" sequence in the fourth domain is mutated into an "R-G-D" sequence, Serine (S) was mutated to arginine (R). The designed PCR primers were:

[0030] PI: 5′-TGATGCGATCAAGGGGGACACGCGTGGCGACTACCGCGAGGCCCTTCT-3′,

[0031] P2: 5'-AGAAGGGCCTCGCGGTAGTCGCCACGCGTGTCCCCCTTGATCGCATCA-3',

[0032] P3: 5'-AAGCGGCAATCAAGGGTGATACTCGTGGTGACTATGAGGCTCTCT-3',

[0033] P4: 5'-AGAGAGCCTCATAGTCACCACGAGTATCACCCTTGATTGCCGCTT-3'.

[0034] First, use the plasmid pJLA503-AnxB1 containing the Annexin B1 gene as a template, PI and P2 as primers to amplify, and the obtained sequence is called AnxB1-S160R; then use AnxB1-S160R as a template, P3 and P4 as prime...

Embodiment 2

[0036] Embodiment 2: the acquisition of fusion gene mAnxB1-ScuPA

[0037] The constructed mutant protein mAnxB1 and the gene encoding amino acids 144-411 of single-chain urokinase were respectively amplified by PCR, and then connected by overlapping region extension method to obtain fusion gene mAnxB1ScuPA(144-411). Specific steps are as follows:

[0038] (1) Use primer A and primer B to amplify the entire coding gene of mAnxB1 gene. Wherein primer A is: 5′-GGAATTC GCCTACTGTCGCTCCCTGGT-3', this primer is consistent with the 5' end of the mAnxB1 coding sequence, and at the same time introduces an NdeI restriction endonuclease site. Primer B: 5-TCATGCACCATGCACTCTTGTCCTCCGCTACTGCCACCTGCAGGGCCGATGAGTTTCAAGCA-3′, including GGSSGG linker and partial fragments of mAnxB1 and ScuPA. PCR reactions were performed using high-fidelity pfu DNA polymerase with a dNTP concentration of 20 μM, Mg 2+ The concentration was 1.5 mM, the temperatures of denaturation, annealing, and extension we...

Embodiment 3

[0041] Embodiment 3: Construction of expression plasmid mAnxB1ScuPA

[0042] The fusion gene mAnxB1ScuPA (144-411) amplified by the above method was sequenced using primers A and D, and the sequencing reaction was completed by Shanghai Jikang Company. After the sequencing is correct, use NdeI, XhoI double enzyme digestion, and use NdeI, XhoI double enzyme digestion vector pET28a (such as figure 1 shown). Restriction endonuclease and T4 ligase for digestion and ligation reactions were purchased from TaKaRa Company, and the reaction was carried out using the system recommended by the enzyme manual. The expression vector pET28a used in the present invention has a full length of 5.3Kb (kilobase pairs), and contains a phage T7 promoter, a plasmid replication origin (ori) and a Kana resistance gene (Kana). Concrete reaction process is as follows:

[0043] The above PCR product and vector were digested with NdeI and XhoI in a high-salt (H) buffer system, and digested at 37°C for 3...

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Abstract

The invention relates to the technical field of medicinal biology, in particular to a thrombolysis-targeting fusion protein mAnxB1ScuPA, a coding gene and a constructing method thereof. The invention provides the fusion gene mAnxB1SeuPA consisting of a gene coding annexin B1 and a gene coding amino acids from 144th to 411th bit of single-chain urokinase, the inductive expression plasmid pET28a-mAnxB1ScuPA of the fusion gene which is an annular double-strand, and engineering bacteria BL21(pET28a-mAnxB1ScuPA) prepared by converting the plasmid into escherichia coli strain BL21-CP-RIL. The fusion gene can express the fusion protein of mutant annexin B1 (mAnxB1) and amino acids from 144th to 411th bit of the single-chain urokinase. The fusion protein has double functions of anticoagulation and thrombolysis-targeting, not only has high thrombolysis efficiency, but also can prevent bleeding and other side effects.

Description

technical field [0001] The invention relates to the field of medical biotechnology, in particular to the preparation of thrombus-targeted thrombolytic protein mAnxB1ScuPA by a genetic engineering method and the construction of an expression vector. Background technique [0002] Thrombotic diseases, such as acute myocardial infarction (AMI), cerebral thrombosis, deep vein thrombosis, etc., are dangerous, with extremely high mortality and disability rates. Thrombolytic therapy is the mainstay of treatment for thrombotic diseases. There are two main types of thrombolytic agents currently used clinically. The first-generation thrombolytic agents are represented by streptokinase (SK) and urokinase (UK). Activation leads to severe bleeding, and SK has strong antigenicity, which is easy to cause allergic reactions in some patients; second-generation thrombolytics mainly include single-chain urokinase (Scu-PA), tissue plasminogen activator ( t-PA), methoxybenzoyl plasminogen activ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/21A61K38/49A61K47/48A61P7/02A61P9/10A61K47/64
Inventor 孙树汉丁飞翔贾音郭瀛军刘音颜宏利
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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