Recombinant hirudin fusion protein with targeting and long-acting functions and coding gene and application thereof

A technology of recombinant hirudin and fusion protein, which is applied in the biological field and can solve problems such as hindering the long-term cycle characteristics of albumin and large molecular weight

Active Publication Date: 2021-09-03
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

We speculate that the size of the fusion protein hAvHA is 50.4kd, and the size of albumin is 66.5kd. The molecular weight of the binary complex combined by the two is relatively large, which probably affects the combination of albumin and FcR to form a ternary complex. This prevents albumin from exerting its long-circulating properties

Method used

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  • Recombinant hirudin fusion protein with targeting and long-acting functions and coding gene and application thereof
  • Recombinant hirudin fusion protein with targeting and long-acting functions and coding gene and application thereof
  • Recombinant hirudin fusion protein with targeting and long-acting functions and coding gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1, the design of hirudin prodrug fusion protein

[0057] The purpose of the present invention is to solve the problems of high bleeding risk and short half-life of hirudin. Based on this, the present invention designs a "targeted long-acting hirudin prodrug" composed of three functional domains - hirudin fusion protein, which has the functions of targeted enrichment, long-acting circulation, local activation, precise anti- The characteristics of condensation ( figure 2 ).

[0058] 1. The inventors of the present invention obtained the sequence of the hirudin variant 2 (hirudin variant-2, HV2) polypeptide (UniProtKB / Swiss-Prot: P09945.1, sequence SEQ ID NO.13). R824 peptide, composed of 6 amino acids (PGDLSR, sequence SEQ ID NO.5), can specifically bind to PS with high affinity, IC 50 1.38×10 -9 M, as the target of "procoagulant platelets", the fusion protein containing R824 can be enriched by targeting "procoagulant platelets". The sequence of albumin...

Embodiment 2

[0061] Embodiment 2, cDNA molecular synthesis of coding R824-HV-ABD and ABD-HV-R824 protein

[0062] 1. Design the gene sequence encoding hirudin fusion protein according to Escherichia coli preferred codon, the cDNA sequence encoding R824-HV-ABD is SEQ ID NO.3, and the cDNA sequence encoding ABD-HV-R824 is SEQ ID NO. 4.

[0063] 2. Sequence synthesis and expression vector construction

[0064] Entrusted Shanghai Jierui Bioengineering Co., Ltd. to synthesize the above two coding sequences, and construct and insert them into the pET30a vector (with NdeI and XhoI as insertion sites), and obtain the recombinant plasmid pET30a / R824-HV-ABD ( Figure 5 ) and pET30a / ABD-HV-R824 ( Figure 6 ), and then transformed Escherichia coli clone strain E.coli JM101. The recombinant plasmid was extracted and verified to be correct by DNA sequencing.

[0065] Transform the constructed expression plasmids pET30a / R824-HV-ABD and pET30a / ABD-HV-R824 into competent E.coli BL21(DE3) strains to obtai...

Embodiment 3

[0066] Embodiment 3, the expression acquisition of target protein

[0067] 1. According to the "Guidelines for Molecular Cloning", first inoculate the glycerol bacteria (constructed in Example 2) frozen at -20°C at a ratio of 1:1000 in the culture medium containing kan + (50 μg / mL) LB medium, shake overnight at 37°C.

[0068] 2. Inoculate the 1:100 ratio of the above-mentioned bacterial solution shaken overnight into the + (50μg / mL) in LB medium (1% NaCl, 1% tryptone, 0.5% yeast extract, autoclaved for later use), shake rapidly at 37°C for 4-5h, until OD 600nm ≈0.6 or so, the bacteria solution can be extracted every corresponding time for OD 600nm Determination, and drawn into OD curve to predict OD 600nm ≈0.6 time.

[0069] 3. Before the induction, the temperature was pre-cooled for 30 minutes. After cooling down to the induction temperature, IPTG (0.5M) was added to the final concentration for induction for 8 hours.

[0070] 4. Pre-weigh the centrifuge bottle, centrifug...

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Abstract

The invention discloses a recombinant hirudin fusion protein with targeting and long-acting functions and a coding gene and application thereof. The fusion protein is formed by fusing a 'procoagulant platelet' targeting binding peptide, hirudin and an albumin binding domain. The fusion protein has the property of a prodrug, can be quickly activated in vivo and exerts an antithrombotic effect; the effect of the fusion protein is equivalent to that of the hirudin, but the duration of the anticoagulant effect is significantly prolonged compared with that of the hirudin, the bleeding time is significantly reduced compared with that of the hirudin and is close to the level of normal saline, the systemic bleeding risk is very low, and the fusion protein has a broad application prospect.

Description

technical field [0001] The invention relates to a recombinant hirudin fusion protein with targeting and long-acting functions and its coding gene, and its application in anticoagulation and prevention of thrombosis, and belongs to the field of biotechnology. Background technique [0002] Cardiovascular and cerebrovascular diseases are the most common diseases that seriously endanger human life and health. They rank first among the causes of death in the population, and are characterized by high morbidity, disability, mortality, recurrence and complications. In May 2018, the World Health Organization announced the top 10 causes of death in the world in 2016. Among the 56.9 million deaths that year, about 1 / 3 died of cardiovascular disease and stroke (Global Health Estimates 2016: Deaths by Cause, Age, Sex, by Country and by Region, 2000-2016. Geneva, World Health Organization; 2018). Therefore, how to reduce the morbidity and mortality of cardiovascular and cerebrovascular d...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K38/58A61K47/42A61P7/02
CPCC07K14/815A61K38/58A61K47/42A61P7/02C07K2319/31
Inventor 王坚成朱元军韩壶壶
Owner PEKING UNIV
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