cxcr4特异性拮抗剂sdf-1-54r可溶性表达载体的构建与表达.doc

文档介绍：
CXCR4 特异性拮抗剂 SDF-1/54R 可溶性表
达载体的构建与表达#
曹园芝,杨飞华,马伟峰**
5
10
15
(南方医科大学公共卫生与热带医学学院,广州 510515)
摘要: 目的:构建趋化因子受体 CXCR4 的特异性拮抗剂 SDF-1/54R 的原核可溶表达载体,
并大量表达和纯化 SDF-1/54R 蛋白,为其后续体内生物活性研究奠定基础。方法:以课题
组前期构建的 SDF-1/54R/pET-30a+质粒为模板,PCR 扩增 SDF-1/54R 基因,PCR 产物经 KpnI
和 EcoRI 双酶切后连接至带有 GST 标签的可溶表达载体 pET-41a+,构建重组质粒
SDF-1/54R/pET-41a+。将鉴定正确的重组质粒转化入大肠杆菌 BL21(DE3),经 IPTG 诱导
表达后,表达产物通过 SDS-PAGE 和 Western blotting 进行检测。利用 GST 亲和层析纯化系
统纯化融合蛋白,纯化产物经肠激酶酶切,酶切产物纯化后得到纯度较高的目的蛋白
SDF-1/54R。结果:经 PCR,双酶切和测序鉴定,所构建的重组质粒 SDF-1/54R/pET-41a+
序列正确。表达产物存在于裂解菌液的上清中,经 SDS-PAGE 和 Western blotting 证实在约
30KD 处有 GST-SDF-1/54R 表达条带,用肠激酶酶切 GST 标签,最后得到约 6KD 的
SDF-1/54R 蛋白。结论:成功构建了 SDF-1/54R/pET-41a+原核表达载体,并可溶表达了
GST-SDF-1/54R 融合蛋白,经酶切、纯化,最终获得纯度较高的 SDF-1/54R 蛋白。
关键词:CXCR4 拮抗剂;SDF-1/54R;可溶性表达
中图分类号:R915
20
Soluble Expression of CXCR4 Specific Antagonist
SDF-1/54R
CAO Yuanzhi, YANG Feihua, MA Weifeng
(Pubic Health and Tropicai Medicine School,Southern Medical University,Guangzhou 510515)
25
30
35
40
Abstract: Objective: We aimed to construct the soluble prokaryotic expression vector of CXCR4
specific antagonist SDF-1/54R, obtain adequate amount of purified SDF-1/54R protein for further
study of its biological activity in vivo. Methods: The SDF-1/54R gene was amplified from the
SDF-1/54R/pET-30a+ plasmid template which was previously constructed. The PCR products
were subjected to Kpn I and