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
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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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