摘要
目的:对噬菌体随机12肽库进行筛选,寻求可与Endoglin结合的小分子短肽并测定其亲和常数。方法:经过3轮“吸附-洗脱-扩增”后,随机挑取16个克隆,双夹心ELISA法鉴定其亲和性,测定阳性克隆的DNA序列,竞争抑制实验检测优势克隆的特异性,非竞争法ELISA固相法计算阳性噬菌体融合肽的亲和常数。结果:三轮生物筛选,噬菌体得到有效富集,竞争性ELISA显示6个阳性噬菌体克隆与rhEndoglin有较强的结合活性,测序获得5种多肽序列,优势序列为:AHKHVHHVPVRL,竞争抑制实验显示阳性噬菌体克隆与Endoglin有良好的亲和性,其亲和常数为:(1.431±0.293)×107mol/L。结论:利用噬菌体肽库技术可以获得亲和力较强的Endoglin的结合肽,为今后进一步研究Endoglin在卵巢癌的早期诊断方面的作用奠定基础。
Objective：To do screening in phage displayed 12-peptide library to seek short peptides capable of binding Endoglin detect their affinity constants.Methods：After three rounds of screening, 16 phage clones were randomly selected and their affinity was identified by sandwich ELISA.The DNA sequence of positive phage clones was determined,and their speciality was identified by competitive inhibition test.We finally calculated out the affinity constants of those positive phage binding peptides by non-competitive ELISA method.Result：After three rounds of screening,the enriched phage clones were identified. 6 of 16 phage clones were identified positive by competitive ELISA,which had comparatively strong binding activity to rhEndoghn.Five sequences were obtained,and the predominant sequence was AHKHVHHVPVRL.Competitive inhibition test showed that positive phage clones had good affinity to Endoglin,with the affinity constant as （1.431 ±0.293）×10^7 mol/L.Conclusion：The rhEndoglin-binding peptides with high affinity to Endoglin can be obtained through the screening of phage random peptide library, which is beneficial to the further studies on the function of Endoglin in the early diagnosis of ovarian cancer.
出处
《重庆医科大学学报》
CAS
CSCD
2007年第3期232-235,共4页
Journal of Chongqing Medical University
基金
国家自然科学基金(编号:30271360)。
关键词
ENDOGLIN
噬菌体随机肽库
结合肽
筛选
Endoglin
Phage-displayed random peptide library
Binding peptide
Screening
