Screening And Preliminary Identification Of Breast Cancer Targeting Peptides

Background Breast cancer is one of the most common malignant diseases in women,and it is threatening female health.International agency for research on cancer statistics reported that about 460,000 women died from breast cancer annually in the world,which is ratio of 13.7%to all female malignant tumor death,and accounted as 1.7%of all female deaths.In recent years in our country,especially in some big cities and coastal economic rapid development areas,there is an upward trend in the incidence of breast cancer.In terms of treatment,the 5-year survival rate of breast cancer is about 50%-60%.With the deepening of the people for breast cancer awareness and research,the treatment of breast cancer have also made certain progress in recent years,has developed from a single surgical treatment to the multi-disciplinary comprehensive treatment.Breast cancer markers research helps screening and diagnosis of tumor high-risk groups,particularly with nano-reagents,chemotherapy drugs,such as tumor targeting therapy after decoupling even,have huge high-efficiency advantages.Phage displayed peptide library has been developed as a best way to select specific peptide.This technique allows fragments of foreign peptides or proteins to be expressed as fusion proteins displayed on the phage surface.These fusion proteins can keep their relatively independent spatial structure and biological activity,establish direct contact between random peptides and DNA coding sequences,and make all kinds of peptide ligand to some molecular targets(antibody,enzyme,cell surface receptor,etc)by in vitro affinity panning procedures to be rapidly identified.Phage Display Technology has been widely exploited to construct tumor-associated antibody and peptide libraries,identify molecule markers for cancer diagnosis and leading peptides with anti-tumor activities,and has been used for anti-tumor bio-product targeted transportation research.Objective In this research paper,phage display peptide library was used to screen the breast cancer cells specific peptides.Breast cancer MCF-7 cell line was used as target cells,for four rounds subtraction biopanning from phage-displayed random peptide library,in order to identify peptide motif sequences that can bind to breast cancer cells with high affinity and specificity.This paper was for further research on breast cancer early diagnosis agents and targeted drugs.Methods Breast cancer MCF-7 cells were used as the target cells,and human embryo kidney HEK293 cells as negative absorber cells for 4 rounds whole-cell subtraction biopanning from phage display-12 random peptide library.Thirty phage clones were picked up randomly,amplified and titered.Positive phage clones with high affinity and specificity were identified by enzyme-linked immunosorbent assay(ELISA).The affinity of positive phage clones with MCF-7 cells was determined in order to exclude false positive clones.The ssDNA of positive phage clones,unrelated phage and some negative phage clones were extracted and sequenced full automatic to identify the conservative sequence.The characteristic of amino acid sequence was analyzed by BioEdit software.The homology between peptides was analyzed by Alignment,searching for the similar amino acid sites contained in several peptides and motif sequences.Searching for proteins data bank,homologous sequence and potentially binding ligand or cell surface receptor were identified by BLAST method.The specificity of positive phage clones which can target to breast cancer MCF-7 cells was detected by immunofluorescence assay.Results After four rounds of subtraction biopanning in vitro,phage clones that could bind to breast cancer cells were enriched and 60 phage clones were picked up from a LB/IPTG/Xgal agrose/agar plate on that about 100 plaques colonized up.The affinity of 60 phage clones was identified by ELISA.Whole Cell-ELISA results suggested that there are 10 positive clones36 clones showed higher binding to MCF-7 cells compared with UPRs and PBS control groups.After DNA sequencing,seven consensus sequences were obtained.With ELISA,cell immunofluorescence further positive cloning specific affinity for the identification of the Consensus sequence,Q35 is the best positive clones.Conclusions The current study screened the peptides binding to breast cancer cells,MCF-7 specifically and sensitively by using the phage displayed peptide library biopanning technology.The preliminary positive clones were identified by an ELISA method,the selected positive clones were sequenced,and the 7 consensus sequences were figured out.Finally,the specificity and sensitivity of the clones of consensus sequences were identified using cyto-immuno-fluorescense staining,and it indicated that all positive clones can bind to breast cancer MCF-7 cells specifically,and the clone,Q35 is the best one.These peptides have the obvious ability to bind specially to breast cancer cell surface receptors and can be further identified for targeted research and development of breast cancer early diagnosis reagent and high-efficiency targeted therapy drugs.