Screening Of Conjugated Peptides Of Endoglin And Assessment Of The Binding Function

BACKGROUND: Ovarian cancer is one of three common malignant tumors of the female genital system. Because it is short of effective and earlier diagnostic technique, at the same time it has characters of infiltrative growth and rapid metastasis, the death rate of ovarian cancer has already been the first one among the female genital malignant tumors. Ovarian cancer is a kind of highly vascularize tumour. Its growth depends on abundant of angiogenesis and nutritive substance. The studies demonstrated that even in the earlier stage of the ovarian cancer there is distinguished angiogenesis and the number of angiogenesis is more than that of benign or low potential malignant tumors.Endoglin (CD105) is a kind of membrane antigen on the endothelial cell, which is relative to cellular proliferation. Endoglin expresses highly on vascular endothelial cell of angiogenesis, neonatal tumor organization and the tumor tissue marginal part. On the other hand, it does not express on normal tissue. And Endoglin is relative intimately to tumorous angiogenesis, infiltration and metastasis. Therefore the Endoglin is a latent target for early diagnosis and therapy of ovarian cancer.OBJECTIVE: The fused-peptide which has a high affinity to Endoglin by measuring the binding function was obtained from the freedom 12-peptide library through the technology of phage display. It provides a theoretical basis for early diagnosis of ovarian cancer.METHODS and RESULTS: The rhEndoglin was used as a target protein for biopanning of phage-displayed 12-peptide library. After three rounds of screening, the enriched phage clones were identified by ELISA. Sixteen phage clones were randomly selected and identified by sandwich ELISA. The positive phage clones were sequenced. After translating with software program DNAstar, we got five different inserted peptides sequences. Two amino acid sequence is AHKHVHHVPVRL among these five. Preponderance phage clones showed higher affinity in competitive ELISA. According to the result of competitive inhibition test we concluded that the positive phage clones could imitate the binding epitope of Endoglin. And we assessed the functional affinity of peptide fused- phageⅠ,Ⅳvia non-competitive ELISA method(Beatty way). The affinity ratio that is between the fused peptideⅠandⅣwas 143.1:23.9. The peptideⅠwas confirmed to have more binding function, which demonstrated the prediction of the competitive ELISA analysis.PeptideⅠ(AHKHVHHVPVRL)andⅣ(HYHTHHTKVRTP)were synthesized through the solid-phase way. They were assessed the functional affinity after combining with glutaral. We got the result that is respectively (2.956±0.749)×106M-1 and (8.782±0.189)×104M-1. The consequence indicates the peptideⅠhas more binding ability than peptideⅣ. The conjugation peptide of its binding capability with Endoglin out of body was detected through the indirect stain with immunofluorescence technique which is on the basis of synthesis biotin-labeled peptide(Biotin-AHKHVHHVPVRL).The stain result indicates that peptideⅠcould bind specifically with Endoglin in cell level on the basis of cultivating the human umbilical vein endothelial cell (HUVEC) in vitro.CONCLUSION: The rhEndoglin-binding peptide can be obtained by screening phage random peptide library. The peptide(AHKHVHHVPVRL)showed the high binding ability in the ELISA examination, competitive inhibition test and immunofluorescence. The result indicates that the peptide could be a potent carrier targeted to Endoglin for early diagnosis of ovarian cancer.