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.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2006年第11期1154-1156,共3页
Journal of Third Military Medical University
基金
国家"863"课题(2002AA214111)~~
