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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: The establishment of a high-throughput anti-inflammatory drug screening system can provide an ideal technical platform for the research of related drugs. OBJECTIVE: To construct an eukaryotic expression vector p4CCL20-ZsGreen1-DR with SV40 as a promoter and ZsGreen1-DR as a reporter gene using 4 × CCL20 motif as a cis-acting element of nuclear factor κB. Methods: The target fragment SV40 was amplified by PCR using the PGL2-control plasmid as a template. KpnⅠ / BamHⅠ restriction sites were introduced into both sides of the vector and cloned into the Kpn Ⅰ / BamH Ⅰ restriction site of pZsGreen1-DR plasmid to construct pSV40- ZsGreen1-DR vector. The 4 × CCL20 motif double-stranded DNA was cloned into the pSV40-ZsGreen1-DR vector between the BglⅡ and EcoRⅠ restriction sites to construct p4CCL20-ZsGreen1-DR recombinant plasmid. RESULTS AND CONCLUSION: The p4CCL20-ZsGreen1-DR recombinant plasmid was successfully constructed after DNA sequencing analysis. The recombinant plasmid can be used as a high-throughput screening of anti-inflammatory drugs based on the system.