Construction and identification of p4CCL20-ZsGreen1-DR eukaryotic expression vector / 中国组织工程研究

ABSTRACT

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.