提高树突状细胞腺病毒感染效率的融合蛋白的表达及活性鉴定  

Expression and activity identification of a fusion protein for promoting adenovirus infection efficiency of dendritic cells

作　　者：田仁礼[1,2] 殷小涛[1,2] 王伟[1,2] 林晓亮[1,2] 朱晓明[2] 徐元基[2] 阎瑾琦[2] 张巍[2] 高江平[1] 于继云[2] 

机构地区：[1]解放军总医院泌尿外科,北京100853 [2]军事医学科学院基础医学研究所,北京100850

出　　处：《细胞与分子免疫学杂志》2014年第5期476-479,484,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(30840094);国家高技术研究发展计划(863)(2007AA02Z451)

摘　　要：目的构建表达靶向树突状细胞融合蛋白CT40L的原核表达载体,在大肠杆菌中表达,并对CT40L融合蛋白进行纯化鉴定及功能验证。方法从GenBank上查找柯萨奇病毒和腺病毒受体(CAR)、T4噬菌体fibritin和小鼠CD40L的基因序列,并将其主要功能区域连接形成拼接序列;序列进行原核表达密码子优化后将其克隆至原核表达载体pET42a(+),构建重组表达载体pET42a-CT40L,并将该载体转化大肠杆菌BL21(DE3),经IPTG诱导表达CT40L/GST融合蛋白,并采用GST琼脂糖凝胶纯化重组蛋白。纯化后的重组蛋白经Western blot法、间接ELISA鉴定其免疫活性。结果重组表达载体经NcoⅠ和EcoRⅠ酶切鉴定正确;IPTG诱导后经SDS-PAGE分析表明获得了相对分子质量(Mr)78 000大小的重组蛋白;纯化后的蛋白纯度达到90%。Western blot法和ELISA检测证实纯化的CT40L分子能够与特异性抗体及相应受体发生反应,表明该融合蛋白具有良好的免疫学活性。结论成功构建了原核表达载体pET42a-CT40L,利用大肠杆菌表达系统实现了融合分子的可溶性表达,纯化后CT40L融合蛋白经检测具备较高的免疫学活性。Objective To construct a prokaryotic expression plasmid for CT40L, express the target protein in E. coli, purify the CT40L fusion protein and verify its antigenicity. Methods Gene sequences of Coxsackie and adenovirus receptor （ CAR）, bacteriophage T4 fibritin and mouse CD40L were found out in GenBank. Then functional domains of three molecules were linked to form a fusion sequence which was then optimized for prokaryotic expression. The optimized sequence was cloned into prokaryotic expression vector pET42a（ ＋ ） to construct the recombinant expression vector pET42a-CT40L. The recombinant vector was transformed into BL21 （DE3） and the fusion protein CT40L/GST was induced by IPTG. The fusion protein was then subjected to purification using GST affinity chromatography and to identification of the immune activity using Western blotting and ELISA. Results The recombinant expression vector was verified correct by double digestion with Nco I and EcoR ]. After IPTG induction, SDS-PAGE showed that the relative molecular mass of the fusion protein was about 78 kDa and that the purity of the purified protein reached 90%. Western blotting and ELISA demonstrated that the purified fusion protein had a valid antigenicity. Conclusion The prokaryotic expression plasmid pET42a-CT40L was successfully constructed and expressed in E. coil, and the purified fusion protein was proved to have a good antigenicity.

关 键 词：CD40L 靶向治疗 原核表达 腺病毒感染效率 

分 类 号：R392.11[医药卫生—免疫学] R392.33[医药卫生—基础医学]