| Heparin-binding epidermal growth factor-like growth factor(HB-EGF), a member of epidermal growth factor, can strongly bind to heparin. It exhibits activity after binding to its receptors in autocrine, paracrine, and juxtacrine interactions. HB-EGF plays important roles in several biological and pathological processes, such as wound healing, blastocyst implantation, atherosclerosis, and heart development. Clinical studies have shown that HB-EGF is highly expressed in breast cancer and ovarian cancer. Targeted inhibition of HB-EGF or suppression on its downstream signal pathways leads to cell apoptosis and cell proliferation, tumorigenesis and metastasis inhibition. Furthermore, combined with HB-EGF inhibitor, the efficacy of anti-cancer drugs on drug resistant mutant cells is significantly improved. Taken together, HB-EGF is closely related with tumorigenesis, metastasis, and drug resistance in breast cancer and ovarian carcinoma and is a therapeutic target for mammary carcinoma and ovarian cancer treatment. Therefore, anti-HB-EGF drugs have the potential value to overcome certain cancers.Phage-displayed random peptide library is constructed by fusing exogenous random peptides with phage coat protein and displaying them on the surface of phage, thus establishing a direct link between large number peptides and their encoding DNA sequences. For biopanning in vitro, a display library is firstly incubated with an immobilized target, followed by extensive washing to remove nonbinding phages. Binders are usually eluted and enriched by amplification in the appropriate host cells. Three to five rounds of biopanning are usually performed in order to obtain peptides that bind target with high affinity. The primary structure of the peptide can then be determined by sequencing the DNA of individual.Peptide drugs have many advantages over large molecular drugs, like low immunogenicity, small and easy absorption. In this work, we aim to obtain bioactive peptides specifically binding to and inhibiting HB-EGF through phage display, thus laying foundation to the development of anti-cancer drugs.In this work, we acquired sHB-EGF by PCR firstly. After enzyme digestion, the PCR product was inserted into prokaryotic expression vector pET-30 a and the recombinant plasmid was transformed into BL21(ED3). Induced with IPTG, the recombinant plasmid successfully produced massive protein(His)6-sHB-EGF. Pure protein(His)6-sHB-EGF was gained by Ni-NTA and heparin affinity chromatography. To avoid false positive outcomes caused by tag protein(His)6 which accounts for 53% of(His)6-sHB-EGF, the full length protein was digested by enterokinase. Finally, we acquired pure protein sHB-EGF.We performed phage display in vitro using sHB-EGF as target, taking heparin sodium and sHB-EGF as elution buffer respectively. Three candidate peptides TUZG1, 2 and 3 were obtained and ELISA test verified that all peptides bind to sHB-EGF specifically.Both wound healing assay and transwell invasion assay have demonstrated that sHB-EGF promotes the migration and metastasis of ovarian cancer cell line SKOV3. Through biological effect validation experiments, we found TUZG3 could significantly inhibit the tumor migration and metastasis induced by sHB-EGF, whereas TUZG1 and TUZG2 could not. Since TUZG1 and TUZG2 were eluted by heparin sodium solution, we speculated they may have anticoagulation effect similar to heparin sodium. This hypothesis was subsequently confirmed by APTT test, tube method and cutting-tail method.In conclusion, we have successfully obtained target protein sHB-EGF of high purity through molecular cloning, prokaryotic expression, affinity chromatography, and gained three bioactive peptides that specifically bind to sHB-EGF through phage display. Biological effect validation experiments showed TUZG3 could inhibit sHB-EGF induced cancer cell migration and metastasis, thus laying foundation to the development of anti-cancer drugs. |
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