人巨细胞病毒编码蛋白pUL23在E.coliBL21(DE3)中的表达和纯化  

Expression and Purification of Human Cytomegalovirus Encoding Protein pUL23 in E.coli BL21(DE3)

作　　者：李婧惠[1] 刘明亮[1] 李继东[1] 张忠明[1] 冉艳红[1] 周天鸿[1] 李弘剑[1] 

机构地区：[1]暨南大学生命科学技术学院,广州510632

出　　处：《生物技术通报》2012年第10期119-123,共5页Biotechnology Bulletin

基　　金：国家自然科学基金项目(30770106);国家自然科学基金重大研究计划面上项目(90608024);广东省自然科学基金项目(06025162)

摘　　要：大肠杆菌中高效表达携带组氨酸标签的人巨细胞病毒皮层蛋白pUL23,并进行纯化以及鉴定。提取感染HCMVTowne病毒株的HFF细胞的总RNA,逆转录为cDNA作为模板,经PCR获得UL23的基因片段,将此片段插入表达载体pET-28a(+),构建pET28a(+)-UL23重组质粒。将pET28a(+)-UL23转化至大肠杆菌BL21(DE3),进行IPTG诱导表达。表达产物经Western blotting分析后进行发酵,再用Ni sepharose亲和层析纯化,纯化产物进行SDS-PAGE和Western blotting检测。结果表明,成功构建pET28a(+)-UL23原核表达载体,表达及纯化了His-pUL23融合蛋白,为进一步研究pUL23奠定基础。To express and purify human cytomegalovirus encoding protein pUL23 with His-tag in E.coli BL21（DE3）.Extracting the RNA of HFF cells which infected HCMV Towne virus strains and reverse transcribed it into cDNA.Using PCR and recombinant DNA technology,UL23 gene sequence was amplified from cDNA and inserted into the prokaryotic expression vector pET-28a（＋）.The recombinant plasmid was then transformed into E.coli BL21（DE3） and induced with IPTG.After fermentation the His-pUL23 was purified by affinity chromatography using Ni-NTA column and it was analyzed by SDS-PAGE and Western blotting.The result shows that we successfully constructed the pET28a（＋）-UL23 recombinant plasmid,produced and purified the His-pUL23 fusion protein,which would lay a foundation for further research of pUL23 protein.

关 键 词：人巨细胞病毒 pUL23 原核表达 纯化 组氨酸标签 

分 类 号：R373[医药卫生—病原生物学]