体内噬菌体展示技术筛选人髓样乳腺癌Bcap-37细胞特异性结合肽及其性质、结合效果研究  

Peptide specifically binding to human medullary breast carcinoma Bcap-37 cells selected by phage display in vivo and its property as well as combination effect

作　　者：郭超[1] 左琪 杨晓峰[2] 张帆[1] Guo Chao;Zuo Qi;Yang Xiaofeng;Zhang Fan(Department of Microbiology and Immunology,Shanxi Medical University,Taiyuan 030001,China;Department of Urology,the First Affiliated Hospital of Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学微生物学与免疫学教研室,太原030001 [2]山西医科大学第一医院泌尿外科,太原030001

出　　处：《肿瘤研究与临床》2020年第2期79-84,共6页Cancer Research and Clinic

基　　金：山西转型综合改革示范区晋中开发区科技重点研发计划。

摘　　要：目的探讨体内噬菌体展示技术筛选的人髓样乳腺癌Bcap-37细胞特异性结合肽的性质和结合效果,为乳腺癌早期诊断提供分子靶向探针。方法制备人髓样乳腺癌Bcap-37细胞荷瘤裸鼠模型,采用噬菌体环七肽库进行3轮体内筛选。免疫组织化学法检测筛选的噬菌体在肿瘤及正常组织中的分布情况。酶联免疫吸附试验(ELISA)鉴定单克隆噬菌体对Bcap-37细胞的亲合力。提取阳性单克隆噬菌体DNA并测序,选取重复率高的序列合成多肽,制备光学分子探针,应用荧光分子成像验证合成的多肽在荷瘤小鼠体内对乳腺移植瘤的特异性和靶向性。结果第3轮体内筛选的噬菌体回收率为第1轮的107.2倍。免疫组织化学结果显示,随筛选轮次增加,肿瘤组织中结合的噬菌体依次增加;肿瘤组织结合的噬菌体多于正常组织(肺脏、骨骼肌、肝脏、肾脏),肿瘤组织切片扫描图像的吸光度(A)值均较正常组织高,差异均有统计学意义(均P<0.05)。ELISA结果显示,随机选取的50个单克隆噬菌体中,22个为阳性(亲合力≥2)。阳性单克隆噬菌体DNA测序分析后,得到4条有重复的氨基酸序列,选择重复率最高的氨基酸序列CSPLNTRFC,化学合成异硫氰酸荧光素(FITC)标记的CSPLNTRFC多肽。Bcap-37细胞荷瘤裸鼠模型体内验证实验显示,FITC-CSPLNTRFC多肽能明显富集在乳腺移植瘤组织。结论利用体内噬菌体展示技术能够筛选出可与人髓样乳腺癌Bcap-37细胞特异性结合的多肽CSPLNTRFC,有助于进行乳腺癌早期诊断的体外研究。Objective To investigate the property and the combination effect of the peptide specifically binding to human medullary breast cancer Bcap-37 cells by using phage display in vivo and to provide molecular targeting probe for early diagnosis of breast cancer.Methods The human medullary breast carcinoma Bcap-37 cells tumor-bearing nude mice model was prepared and three rounds in vivo were performed by using Ph.D.-C7CTM phage display peptide library.The distribution of screened phages in tumors and normal tissues was detected by using immunohistochemistry.The affinity of monoclonal phage to Bcap-37 cells was identified by using enzyme linked immunosorbent assay(ELISA).The positive monoclonal phage DNA was taken and sequenced,and the sequence with high repetition rate was selected to synthesize peptide by using chemical methods.Optical molecular probe was prepared and fluorescence molecular imaging was used to test its specificity and targeting ability for breast transplantation tumor of tumor-bearing nude mice in vivo.Results The recovery rate of phage in the third round screening in vivo was 107.2 times than that in the first round.Immunohistochemical results showed that the phages binding to the tumor tissues were increased successively with the increasing screening rounds in vivo.The number of phages binding to tumor tissues was more than that binding to normal tissues(lung,skeletal muscle,liver and kidney).The absorbancy(A)value of section scanning image in the tumor tissues was higher than that in the normal tissues,and the difference was statistically significant different(P<0.05).ELISA results showed that 22 phages(affinity≥2)among the 50 randomly selected monoclonal phage were positive.After DNA sequencing analysis of the positive monoclonal phage,4 repeat amino acid sequences were obtained.The fluorescein isothiocyanate-labelled(FITC)-CSPLNTRFC with the highest repetition rate was synthesized for FITC-CSPLNTRFC peptide.Bcap-37 cells tumor-bearing nude mice model assay in vivo showed that FITC-CSPLNTRFC pep

关 键 词：乳腺肿瘤 肽库 噬菌体展示 特异性结合肽 

分 类 号：R737[医药卫生—肿瘤]