真核表达载体p4CCL20-ZsGreen1-DR的构建与鉴定(英文)

Construction and identification of p4CCL20-ZsGreen1-DR eukaryotic expression vector

背景:抗炎药物高通量筛选体系的建立,可为相关药物的研究提供一个理想的技术平台。目的:构建以核因子κB顺式作用元件4×CCL20基序为增强子,以SV40为启动子,以ZsGreen1-DR为报告基因的真核表达载体p4CCL20-ZsGreen1-DR。方法:以PGL2-control质粒为模板,PCR扩增目的片段SV40,两侧引入KpnⅠ/BamHⅠ酶切位点,克隆至pZsGreen1-DR质粒的Kpn Ⅰ/BamH Ⅰ酶切位点中,构建成pSV40-ZsGreen1-DR载体。将4×CCL20基序双链DNA克隆到pSV40-ZsGreen1-DR载体的BglⅡ和EcoRⅠ酶切位点之间,构建p4CCL20-ZsGreen1-DR重组质粒。结果与结论:经过DNA测序分析证实p4CCL20-ZsGreen1-DR重组质粒构建成功。该重组质粒可作为抗炎药物高通量筛选体系的基础。

BACKGROUND：It is necessary to establish a high throughput screening system for anti-inflammatory drugs for rheumatoid arthritis.OBJECTIVE：To construct an eukaryotic expression vector p4CCL20-ZsGreen1-DR with the NF-？B cis-acting element 4×CCL20 motif as an enhancer,SV40 as a promoter,and ZsGreen1-DR as a reporter gene.METHODS：The target fragment SV40 was PCR amplified using PGL2-control plasmid as a template.KpnⅠ/Bam HⅠ restriction sites were introduced into the flank of the target fragment.Then,pSV40-ZsGreen1-DR vector was constructed by cloning the target fragment into pZsGreen1-DR plasmid.Finally,p4CCL20-ZsGreen1-DR plasmid was constructed by cloning the double strand DNA of 4×CCL20 motif（with BglⅡ and EcoRⅠ sticky ends at the 5＇ and 3＇ terminus,respectively） into the corresponding restriction sites of pSV40-ZsGreen1-DR vector（upstream of SV40 promoter）.RESULTS AND CONCLUSION：DNA sequencing demonstrated successful construction of p4CCL20-ZsGreen1-DR plasmid.The construction of p4CCL20-ZsGreen1-DR plasmid might be useful to establish a high throughput screening system for anti-inflammatory drugs.