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Polypeptides with anticoagulation activity screened by phage display technique

An anticoagulant activity and anticoagulant technology, applied in the fields of peptides, blood diseases, peptide/protein components, etc., can solve the problems of low efficiency and large workload, achieve easy transportation, improve the success rate, and broad clinical application value and foreground effects

Active Publication Date: 2015-12-23
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The current screening methods for anticoagulant peptides mainly include biochemical separation techniques, bioinformatics methods based on sequence homology, modification and synthesis of existing natural peptides, etc., which often have the disadvantages of heavy workload and low efficiency.

Method used

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  • Polypeptides with anticoagulation activity screened by phage display technique
  • Polypeptides with anticoagulation activity screened by phage display technique
  • Polypeptides with anticoagulation activity screened by phage display technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1. pET-30a / (His) 6 Construction of / HB-EGF protein expression vector

[0025] Extract the fragment encoding HB-EGF (aa63–149, GenBank registration number NM_001945) from GeneBank, and design PCR primers: Forwardprimer5’-CG GGATCC GACTTGCAAGAGGCAGAT-3' (SEQ.ID.NO.3) and Reverseprimer5'-CC AAGCTT TCATGGGAGGCTCAGCCC-3' (SEQ.ID.NO.4), the underlined parts are restriction enzyme cutting sites BamHI and HindIII respectively. After the PCR product and vector pET-30a (Novagen, #69909-3) were digested with BamHI (NEWENGLANDBioLabs, #R0136S) and HindIII (NEWENGLANDBioLabs, #R0104S) at 37°C for 3 h, T4 DNA ligase (NEWENGLANDBioLabs, #M0202S) was used at 16 ℃ connection for 12h. The ligated product was transformed into DH5α competent cells (full type gold, CD201), and then the transformed product was spread on a kanamycin-resistant (50 μg / ml) LB plate and cultured until a single colony grew out, and a single colony was picked, Extract the plasmid for enzyme digestion ...

Embodiment 2

[0027] Example 2. (His) 6 -Induced expression, purification, digestion and verification of HB-EGF

[0028] (His) 6 -Induced expression of HB-EGF: the recombinant plasmid pET-30a / (His) 6 / HB-EGF transforms the host strain BL21 (DE3) (full gold, CD601), utilizes the kanamycin-resistant LB plate to screen the recombinant, picks a single colony and cultures it in the LB liquid medium containing kanamycin until OD 600 = 0.5 to 0.8. The culture was inoculated in LB liquid medium at a volume ratio of 1:50, and cultured to OD at 37°C with vigorous shaking 600 =0.5-0.8, add IPTG (Amresco, #0478) at a final concentration of 0.8mM and induce at 25°C for 12h.

[0029] Nickel column affinity chromatography purification (His) 6 -HB-EGF: Centrifuge the bacterial solution at 6,000rpm for 5min, remove the supernatant, and resuspend the bacterial pellet in the lysate (50mM Tris–HCl, 20mM imidazole, 100mM NaCl, 10 % glycerol, 1% Triton, 1 mM protease inhibitor PMSF, 1 mg / ml lysozyme, pH 8...

Embodiment 3

[0034] Example 3. Phage display panning for biologically active peptides specifically binding to HB-EGF

[0035] The phage panning process is as follows: Figure 5 shown.

[0036] (1) Target molecule immobilization: 600 μl of target molecule solution (dissolved in 0.1M NaHCO 3 PH8.6) was added to a six-well plate, placed on a shaker and shaken slightly, and incubated overnight at 4°C. The target molecule solution was removed and washed 6 times with TBST (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% [v / v] Tween-20). Finally, the blocking solution (0.1M NaHCO 3 PH8.6, 5mg / mlBSA, 0.02%NaN 3 ) closed for 1h.

[0037] (2) Binding of the phage random peptide library to the target molecule: remove the blocking solution, and wash with TBST (0.1% [v / v] Tween-20) for 10 times. After the phage library or the amplified phage is diluted with TBST (0.1% [v / v] Tween-20), the theoretical value of the copy number of the phage is 10 9 ~10 11 In between, the diluted phage was added to the si...

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Abstract

The invention relates to polypeptides with anticoagulation activity and a method for screening polypeptides with anticoagulation activity by a phage display technique, belonging to the technical field of development, research and application of anticoagulant drugs. The screening method comprises the following steps: construction of protein expression vector, expression and purification of target protein, verification of target protein, elutriation of bioactive peptide capable of specifically binding target protein by phage display, polypeptide anticoagulation action in-vivo / in-vitro detection, toxicity experimentation and the like, thereby finally obtaining the polypeptides with anticoagulation activity. The polypeptides are prepared by the following steps: by using a heparin-binding epidermal growth factor as a target molecule, carrying out elutriation three times by using a phage display technique, and eluting the specific binding target molecule phage by using heparin sodium as an effective component. The polypeptides can be used for preparing anticoagulant drugs. The polypeptides have shorter sequence, are easy for synthesis, can easily implement large-scale production, and have no obvious short-term and long-term toxicity for mice in vivo, thereby having important application value in the aspect of development and research of anticoagulant drugs.

Description

technical field [0001] The invention relates to a polypeptide with anticoagulant activity screened by phage display technology, and belongs to the technical field of research and development and application of anticoagulant drugs. Background technique [0002] Blood coagulation is an important physiological defense process of the body, but pathological thrombus seriously endangers human health. Thromboembolism has become one of the important causes of clinical disability and death, and it mainly occurs in patients with cardiovascular and cerebrovascular diseases, postoperative patients and pregnant women. In addition, in modern clinical tests, blood samples for many test items require anticoagulation for detection, and blood is easy to coagulate in vitro. This makes the study of anticoagulant drugs or preparations with high efficiency, stability, and small side effects a hot spot with great clinical application value and prospects. [0003] Chemical agents or substances th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08A61K38/10A61P7/02G01N33/68
Inventor 屠志刚廉彩霞鲁永金刘晗青陈克平张春霞尚东胜阮玲玲侍海娇吴燕芳徐莉莉丹增曲吉
Owner JIANGSU UNIV
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