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Polypeptides with anticoagulant activity screened by phage display technology

An anticoagulant activity and anticoagulant technology, applied in the fields of peptides, blood diseases, material inspection products, etc., can solve the problems of heavy workload and low efficiency, and achieve improved success rate, easy transportation, broad clinical application value and foreground effect

Active Publication Date: 2018-06-01
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 anticoagulant activity screened by phage display technology
  • Polypeptides with anticoagulant activity screened by phage display technology
  • Polypeptides with anticoagulant activity screened by phage display technology

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: Forward primer 5'-CG GGATCC GACTTGCAAGAGGCAGAT-3'(SEQ.ID.NO.3) and Reverse primer 5'-CC AAGCTT TCATGGGAGGCTCAGCCC-3' (SEQ.ID.NO.4), the underlined parts are restriction sites BamHI and Hind III respectively. The PCR product and vector pET-30a (Novagen, #69909-3) were digested with BamHI (NEWENGLAND BioLabs, #R0136S) and Hind III (NEW ENGLAND BioLabs, #R0104S) at 37°C for 3h, and then digested with T4 DNA ligase (NEW ENGLAND BioLabs , #M0202S) at 16°C 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 verifica...

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 PH 8.6, 5mg / ml BSA, 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 t...

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Abstract

The invention relates to a polypeptide with anticoagulant activity and a method for screening the polypeptide with anticoagulant activity through phage display technology, which belongs to the technical field of research and development and application of anticoagulant drugs; the invention adopts the construction of protein expression vector, expression and purification of target protein , verification of the target protein, phage display panning for bioactive peptides specifically binding to the target protein, in vivo and in vitro detection of the anticoagulant effect of the polypeptide, and toxicity experiments, and finally screened the polypeptides with anticoagulant activity; these polypeptides are based on Heparin-binding epidermal growth factor is used as the target molecule, and after three rounds of panning by phage display technology, heparin sodium is used as the active ingredient to elute the phage specifically binding to the target molecule. The polypeptide can be used to prepare anticoagulant drugs; the polypeptide of the present invention The sequence is short, easy to synthesize and achieve large-scale production, and has no significant short-term and long-term toxicity to mice in vivo, showing that it has important application value in the development 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 Patents(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吕鹏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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