MUC1黏蛋白模拟表位的筛选和原核表达  

Selection and prokaryotic expression of MUC1 mimic epitope

作　　者：崔志刚[1] 宋波[1] 张立新[2] 路浩军[2] 李春海[2] 

机构地区：[1]第三军医大学西南医院全军泌尿外科中心,重庆400038 [2]军事医学科学院附属医院肿瘤分子生物学实验室,北京100850

出　　处：《第三军医大学学报》2006年第11期1154-1156,共3页Journal of Third Military Medical University

基　　金：国家"863"课题(2002AA214111)~~

摘　　要：目的从噬菌体随机12肽库中筛选出MUC1抗原的模拟表位,构建MUC1模拟表位的原核表达载体。方法通过生物淘洗、基因测序和氨基酸序列比较,筛选出模拟表位,构建重组表达质粒PET-31b(+),用IPTG诱导表达,亲和层析纯化,W estern b lot鉴定其抗原性。结果筛选到的模拟表位与MUC1单克隆抗体的亲和力较强。成功构建了重组表达质粒,重组蛋白在BL21(DE3)p lysS中得到诱导表达,纯化的目的蛋白在SDS-PAGE上呈现特异的单一条带,W estern印迹也检测到目的蛋白特异条带。结论筛选到了MUC1的模拟表位,成功表达和纯化了MUC1模拟表位蛋白,为研究其在肿瘤疫苗方面的应用奠定基础。Objective To screen MUC1 antigen mimic epitope by phage display peptide library technology and to construct a recombinant plasmid expressing MUC1 antigen mimic epitope. Methods MUC1 mimic epitope was screened by Biopanning, DNA sequencing and amino acid sequence comparison. The gene was constructed into PET-31b（ ＋ ） expression vector and expressed in Escherichia coli BL21 （DE3） after transformation and induction by IPTG. The complete protein of the host bacteria was extracted for SDS-PAGE. The recombinant protein was purified by affinity chromatography on a Ni^2＋ -sepharose column and detected by Western blotting. Results The selected MUC1 mimic epitope could specifically combine with MUC1 monoclonal antibody. The recombinant expression vector PET-31b （ ＋ ）-MUC1 was constructed successfully after the fusion protein was induced with IPTG. A specific protein band was shown on SDS-PAGE profile and detected by Western blotting. Conclusion An MUC1 mimic epitope was screened out. The epitope fusion protein was successfully expressed for the development of tumor vaccines targeting MUC1.

关 键 词：MUC1 噬菌体表面展示 模拟表位 克隆 表达 

分 类 号：Q503[生物学—生物化学] Q513.2