真核表达载体p4CCL20-ZsGreen1-DR 的构建与鉴定

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B 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/BamH Ⅰ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.

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

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-kB cis-acting element 4×CCL20motif 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-ZsGreeR plasmid might be useful to establish a high throughput screening system for anti -inflammatory drugs.