MUC1黏蛋白模拟表位的筛选和原核表达

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,Western blot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)plysS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,Western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.

Selection and prokaryotic expression of MUC1 mimic epitope

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.