人巨细胞病毒编码蛋白pUL23在E.coli BL21(DE3)中的表达和纯化.docVIP

  人巨细胞病毒编码蛋白 pUL23 在 E.coli BL21(DE3)中的表达和纯化# 李婧惠，刘明亮，李继东，李弘剑** 5 10 15 20 25 30 35 40 （暨南大学生命科学技术学院，广州 510632） 摘要：目的：在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，发酵 后并进行蛋白纯化。方法：将感染 HCMV Towne 病毒株的 HFF 细胞的总 RNA，逆转录为 cDNA 作为模板，经 PCR 获得 pUL23 的基因片段。将此片段插入携带组氨酸标签的表达载体 pET28a(+)，构建克隆后，经酶切与测序确认后转化至大肠杆菌 BL21(DE3)，进行 IPTG 诱导 表达。表达产物经 Western blotting 分析后用 5L 发酵罐进行发酵，再用 Ni sepharose 亲 和层析纯化，纯化产物进行 SDS和 Western blotting 检测。结果：电泳分析及测序证 明成功构建了携带了 pUL23 的融合表达载体，融合蛋白产物经纯化和鉴定后，表达产物约为 33KD 的融合蛋白，与预期一致。结论：带有组氨酸标签的 pUL23 基因在大肠杆菌中成功表 达，获得纯化的融合蛋白。建立了该蛋白的简便亲和纯化方法，为进一步研究和应用奠定基 础。 关键词：人巨细胞病毒 pUL23；原核表达；纯化；组氨酸标签 中图分类号：Q74 Expression and purification of Human Cytomegalovirus –coding Protein LI Jinghui, LIU Mingliang, LI Jidong, LI Hongjian (College of Life Science and Technology,Jinan University, GuangZhou 510632) Abstract: Objective：To express and purify Human Cytomegalovirus-coding Protein pUL23 with His-tag in E. coli BL21(DE3). Methods：pET28a gene was obtained from the cDNA which after reverse transcription of RNA by PCR.The RNA was collected from HFF cells infected HCMV Towne strain. After digested by restriction enzyme ,pUL23 gene was then inserted into prokaryotic expression vector pET28a(+) with HIS-tag .Then the expression vector was transformed into E. coli BL21（DE3）and the expressive target protein was induced with IPTG. After fermentation the expressed product was purified by affinity chromatography using Ni-NTA column and it was analyzed by Western blotting analysis. Results：Western blot result showed that the about 33 KD of fusion protein band was consistent with the expected one. We constructed the prokaryotic expression vector of pET28a- UL23. Conclusion: The fusion protein of pUL23 with His-tag was successfully expressed and purified in E. coli BL21(DE3). These provide experimental basis for further research on its biological function. Keywords: HCMV pUL23; Prokaryotic expression; purification; HIS-tag 0 引言 人巨细胞病毒（human cytomegalovirus，HCMV）是疱疹病毒科β亚科中基因组最大的 DNA 病毒，其基因组为 235kb，所